Cell Motility and the Cytoskeleton via MedWorm.com

Isolation and partial purification of the saccharomyces cerevisiae cytokinetic apparatus

Cell Motility and the Cytoskeleton — Tue, 29 Sep 2009 23:00:00 +0100

Cytokinesis is the process by which a cell physically divides in two at the conclusion of a cell cycle. In animal and fungal cells, this process is mediated by a conserved set of proteins including actin, type II myosin, IQGAP proteins, F-BAR proteins, and the septins. To facilitate biochemical and ultrastructural analysis of cytokinesis, we have isolated and partially purified the Saccharomyces cerevisiae cytokinetic apparatus. The isolated apparatus contains all components of the actomyosin ring for which we tested - actin, myosin heavy and light chain, and IQGAP - as well as septins and the cytokinetic F-BAR protein, Hof1p. We also present evidence indicating that the actomyosin rings associated with isolated cytokinetic apparati may be contractile in vitro, and show preliminary electro...

Hyperpolarization of the plasma membrane potential provokes reorganization of the actin cytoskeleton and increases the stability of adherens junctions in bovine corneal endothelial cells in culture

Cell Motility and the Cytoskeleton — Mon, 14 Sep 2009 23:00:00 +0100

In previous works we showed that the depolarization of the plasma membrane potential (PMP) determines a reorganization of the cytoskeleton of diverse epithelia in culture, consisting mainly of a reallocation of peripheral actin toward the cell center, ultimately provoking intercellular disruption. In view of this evidence, we explored in this study the possible effects of membrane potential hyperpolarization on the cytoskeletal organization and adherens junction (AJ) morphology and the stability of confluent bovine corneal endothelial cells in culture. For this purpose, hyperpolarization was achieved by substitution of extracellular sodium by nondiffusible cations or via the incorporation of valinomycin to the control solution. Actin compactness at the cell periphery was assessed by quanti...

Centriole symmetry: A big tale from small organisms

Cell Motility and the Cytoskeleton — Thu, 10 Sep 2009 23:00:00 +0100

Centrioles are microtubule-based cylindrical organelles with a 9-fold symmetry. They are essential for axoneme formation in cilia and flagella and for centrosome organization. In the basal hexapods Acerentomon microrhinus, we discovered unusually large centrioles composed of 14 doublet microtubules that serve as templates for cilia and flagella and organize mitotic and meiotic spindles. These observations challenge the long-standing view that centriole symmetry is highly conserved among eukaryotes. Strikingly, daughter centrioles contain a transient cartwheel that is lost after maturation. The length of radial spokes is like that found in 9-fold cartwheels, whereas the diameter of the hub varies according to the dimensions of the centriole cylinder. This suggests that the hub may dictate t...

Fluorescence-based quantitative scratch wound healing assay demonstrating the role of MAPKAPK-2/3 in fibroblast migration

Cell Motility and the Cytoskeleton — Wed, 09 Sep 2009 23:00:00 +0100

The scratch wound healing assay is a sensitive method to characterize cell proliferation and migration, but it is difficult to be quantitatively evaluated. Therefore, we developed an infrared fluorescence detection-based real-time assay for sensitive and accurate quantification of cell migration in vitro. The method offers sensitivity, simplicity, and the potential for integration into automated large-scale screening studies. A live cell staining lipophilic tracer - 1,1[prime]-dioctadecyl-3,3,3[prime],3[prime]-tetramethyl indotricarbocyanine iodide (DiR) - is used for accurate imaging of wound closure in a simple 96-well scratch assay. Scratches are made on prestained confluent cell monolayers using a pipette tip and scanned at different time intervals using a fluorescent scanner. Images a...

Myosin IIB isoform plays an essential role in the formation of two distinct types of macropinosomes

Cell Motility and the Cytoskeleton — Tue, 08 Sep 2009 23:00:00 +0100

The function and mechanism of macropinocytosis in cells outside of the immune system remain poorly understood. We used a neuroblastoma cell line, Neuro-2a, to study macropinocytosis in neuronal cells. We found that phorbol 12-myristate 13-acetate (PMA) and insulin-like growth factor 1 (IGF-1) induced two dinstinct types of macropinocytosis in the Neuro-2a cells. IGF-1-induced macropinocytosis occurs mostly around the cell bodies and requires phosphoinositide 3-kinase (PI3K), while PMA-induced macropinocytosis occurs predominantly in the neurites and is independent of PI3K. Both types of macropinocytosis were inhibited by a specific inhibitor of nonmuscle myosin II, blebbistatin. siRNA knock-down of nonmuscle myosin II isoforms, -IIA and -IIB, resulted in opposite effects on macropinocytosi...

Actin-like protein 1 (ALP1) is a component of dynamic, high molecular weight complexes in Toxoplasma gondii

Cell Motility and the Cytoskeleton — Sat, 22 Aug 2009 23:00:00 +0100

Apicomplexan parasites, such as Toxoplasma gondii, rely on actin-based motility for cell invasion, yet conventional actin does not appear to be required for cell division in these parasites. Apicomplexans also contain a variety of actin-related proteins (Arps); however, most of these not directly orthologous to Arps in well-studied systems. We recently identified an apicomplexan-specific member of this family called Actin-Like Protein 1, (ALP1), which plays a role in the assembly of vesicular components recruited to the inner membrane complex (IMC) of daughter cells during cell division. In addition to its enrichment at daughter cell membranes, ALP1 is localized throughout the cytoplasm both diffusely distributed and concentrated in clusters that are detected by fluorescence microscopy, su...

Effect of GFP tags on the localization of EB1 and EB1 fragments in vivo

Cell Motility and the Cytoskeleton — Thu, 20 Aug 2009 23:00:00 +0100

EB1 is a microtubule plus-end tracking protein that plays a central role in the regulation of microtubule (MT) dynamics. GFP-tagged EB1 constructs are commonly used to study EB1 itself and also as markers of dynamic MT plus ends. To properly interpret these studies, it is important to understand the impact of tags on the behavior of EB1 and other proteins in vivo. To address this problem and improve understanding of EB1 function, we surveyed the localization of expressed EB1 fragments and investigated whether GFP tags alter these localizations. We found that neither N-terminal nor C-terminal tags are benign: tagged EB1 and EB1 fragments generally behave differently from their untagged counterparts. N-terminal tags significantly compromise the ability of expressed EB1 proteins to bind MTs a...

Migration of Dictyostelium slugs: Anterior-like cells may provide the motive force for the prespore zone

Cell Motility and the Cytoskeleton — Wed, 19 Aug 2009 23:00:00 +0100

The collective motion of cells in a biological tissue originates from their individual responses to chemical and mechanical signals. The Dictyostelium slug moves as a collective of up to 100,000 cells with prestalk cells in the anterior 10-30% and prespore cells, intermingled with anterior-like cells (AL cells), in the posterior. We used traction force microscopy to measure the forces exerted by migrating slugs. Wild-type slugs exert frictional forces on their substratum in the direction of motion in their anterior, balanced by motive forces dispersed down their length. StlB- mutants lack the signal molecule DIF-1 and hence a subpopulation of AL cells. They produce little if any motive force in their rear and immediately break up. This argues that AL cells, but not prespore cells, are the ...

Cysteine protease-mediated cytoskeleton interactions with LFA-1 promote T-cell morphological changes

Cell Motility and the Cytoskeleton — Fri, 07 Aug 2009 23:00:00 +0100

T cells migrate through restrictive barriers in a protease-independent, amoeboid fashion that is characterized by morphological cell polarization. The interaction of cysteine-dependent carboxypeptidase cathepsin X with [beta]2 integrin LFA-1 (lymphocyte function associated antigen 1) induces T-cell morphological changes, displaying into a 3D extracellular matrix a cytoplasmic projection termed a uropod. In the present study we show that inhibition of cathepsin X and a cysteine-dependent endopeptidase, cathepsin L, markedly inhibits T-cell actin polymerization, shape polarization, and chemotaxis. We propose that cathepsin L promotes T-cell migration associated processes by activating procathepsin X in the endolysosomal vesicles near the cell membrane and at the peak of the uropod, where bot...

Myosin Vb localises to nucleoli and associates with the RNA polymerase I transcription complex

Cell Motility and the Cytoskeleton — Thu, 16 Jul 2009 23:00:00 +0100

It is becoming increasingly clear that the mammalian class V myosins are involved in a wide range of cellular processes such as receptor trafficking, mRNA transport, myelination in oligodendrocytes and cell division. Using paralog-specific antibodies, we observed significant nuclear localisation for both myosin Va and myosin Vb. Myosin Vb was present in nucleoli where it co-localises with RNA polymerase I, and newly synthesised ribosomal RNA (rRNA), indicating that it may play a role in transcription. Indeed, its nucleolar pattern was altered upon treatment with RNA polymerase I inhibitors. In contrast, myosin Va is largely excluded from nucleoli and is unaffected by these inhibitors. Myosin Vb was also found to physically associate with RNA polymerase I and actin in co-immunoprecipitation...

The heel and toe of the cell's foot: A multifaceted approach for understanding the structure and dynamics of focal adhesions

Cell Motility and the Cytoskeleton — Mon, 13 Jul 2009 23:00:00 +0100

Focal adhesions (FAs) are large clusters of transmembrane receptors of the integrin family and a multitude of associated cytoplasmic "plaque" proteins, which connect the extracellular matrix-bound receptors with the actin cytoskeleton. The formation of nearly stationary FAs defines a boundary between the dense and highly dynamic actin network in lamellipodium and the sparser and more diverse cytoskeletal organization in the lamella proper, creating a template for the organization of the entire actin network. The major "mechanical" and "sensory" functions of FAs; namely, the nucleation and regulation of the contractile, myosin-II-containing stress fibers and the mechanosensing of external surfaces depend, to a major extent, on the dynamics of molecular components within FAs. A central eleme...

Profilin-1 overexpression restores adherence junctions in MDA-MB-231 breast cancer cells in R-cadherin-dependent manner

Cell Motility and the Cytoskeleton — Fri, 10 Jul 2009 23:00:00 +0100

Profilin-1 (Pfn1), a ubiquitously expressed actin-binding protein, is downregulated in several different types of adenocarcinoma and elicits tumor-suppressive effect on breast cancer cell lines. MDA-MB-231 (MDA-231), a breast cancer cell line that displays all the characteristics of post-epithelial-to-mesenchymal transition and does not form cell-cell adhesion, can be reverted to an epithelioid phenotype by Pfn1 overexpression. This morphological transition is caused by restoration of adherence junctions (AJ) requiring Pfn1's interaction with actin. Pfn1 overexpression increases the expression level of R-cadherin (a type of cadherin that is endogenously expressed in the parental cell line) and restores AJ in MDA-231 cells in R-cadherin-dependent manner. These findings highlight important r...

Profilin-1 overexpression restores adherens junctions in MDA-MB-231 breast cancer cells in R-cadherin-dependent manner

Cell Motility and the Cytoskeleton — Fri, 10 Jul 2009 00:00:00 +0100

Profilin-1 (Pfn1), a ubiquitously expressed actin-binding protein, is downregulated in several different types of adenocarcinoma and elicits tumor-suppressive effect on breast cancer cell lines. MDA-MB-231 (MDA-231), a breast cancer cell line that displays all the characteristics of post-epithelial-to-mesenchymal transition and does not form cell-cell adhesion, can be reverted to an epithelioid phenotype by Pfn1 overexpression. This morphological transition is caused by restoration of adherens junctions (AJ) requiring Pfn1's interaction with actin. Pfn1 overexpression increases the expression level of R-cadherin (a type of cadherin that is endogenously expressed in the parental cell line) and restores AJ in MDA-231 cells in R-cadherin-dependent manner. These findings highlight important ro...

Human angiomotin-like 1 associates with an angiomotin protein complex through its coiled-coil domain and induces the remodeling of the actin cytoskeleton

Cell Motility and the Cytoskeleton — Mon, 29 Jun 2009 23:00:00 +0100

We report that angiomotin-like 1 forms part of a protein complex containing p80-angiomotin. Structure-function studies revealed that angiomotin-like 1 associates with this p80-angiomotin-containing complex via its coiled-coil domain. Since p80-angiomotin plays a role in cell migration, a process that involves the remodeling of the actin cytoskeleton, we then addressed the hypothesis that angiomotin-like 1 may interact with the cytoskeleton. Immunofluorescence studies reveal that angiomotin-like 1 not only co-localizes with filamentous actin but also significantly modifies the architecture of the actin cytoskeleton. Regarding migration, angiomotin-like 1 increases the velocity of migration and decreases the persistence of migration directionality. Together these observations strongly sugges...

Dynein-2 and ciliogenesis in Tetrahymena

Cell Motility and the Cytoskeleton — Fri, 26 Jun 2009 23:00:00 +0100

Dynein-2 is the motor responsible for retrograde intraflagellar transport. In situ, dynein-2 comprises four subunits: the dynein-2 heavy chain (DYH2); the dynein-2 intermediate chain; the dynein-2 light-intermediate chain (D2LIC); and dynein light chain 8 (Rompolas et al. 2007. Chlamydomonas FAP133 is a dynein intermediate chain associated with the retrograde intraflagellar transport motor. J Cell Sci 120:3653-3665). In contrast to what has been reported in other model organisms, when the DYH2 gene or the D2LIC gene was disrupted in Tetrahymena, the cells continued to produce motile cilia that were not swollen or filled with material [Rajagopalan et al.. Dynein-2 affects the regulation of ciliary length but is not required for ciliogenesis in Tetrahymena thermophila. Mol Biol Cell 20:708-7...

Actin-dependent dynamics of keratin filament precursors

Cell Motility and the Cytoskeleton — Wed, 24 Jun 2009 23:00:00 +0100

Actin filament and microtubule growth characteristics are defined by their different plus and minus ends. In contrast, intermediate filaments lack this type of polarity. Yet, intermediate filament network growth occurs by selective addition of newly formed and polymerizing keratin particles at peripheral network domains thereby allowing polarized network reorganization. To examine this process at high resolution in living cells, mammary epithelium-derived, immortalized EpH4-cells were infected with retroviral cDNA constructs coding for human keratin 18-fluorescent protein hybrids. Several stable cell lines were established presenting characteristic fluorescent keratin filament (KF) networks. These cells contain particularly large and abundant lamellipodia in which nascent keratin particle ...

Calcium-dependent flagellar motility activation in Chlamydomonas reinhardtii in response to mechanical agitation

Cell Motility and the Cytoskeleton — Wed, 17 Jun 2009 23:00:00 +0100

Flagellar beating in Chlamydomonas was found to be activated by mechanical stimulation. Immediately after a wild-type cell suspension was vortexed, the average swimming velocity of cells increased from 130 [mu]m/second to 150 [mu]m/second, due to an elevation of flagellar beat frequency from [sim]60 Hz to [sim]70 Hz without detectable change in the flagellar waveforms. This response required outer arm dynein. Treatment with EGTA, Ca2+-channel blockers, or mechanosensitive-channel blockers inhibited it. In demembranated and reactivated cell models, a modest increase in Ca2+ concentration elevated the axonemal beat frequency. These data indicate that the mechanical agitation increases beat frequency because it causes Ca2+ influx into flagella, which then activates outer arm dynein. Cell Moti...

Graded actin filament polarity is the organization of oriented actomyosin II filament bundles required for fibroblast polarization

Cell Motility and the Cytoskeleton — Wed, 17 Jun 2009 23:00:00 +0100

Actomyosin II filament assemblies in cells are required for shaping the cell body and forming the cell rear during morphological polarization and triggering of migration. However, precise steps in myosin II-based mechanisms are unknown in this event; one reason is due to lack of information on the organization of the actin filament substrate for myosin II. Whilst muscle sarcomeric-like contraction drives cell tension in stationary nonmuscle cells, alternative nonsarcomeric modes of myosin II force-generation power forwards movement of the cell body in already migrating cells. Which one contributes to initial cell shape change has not previously been experimentally sought in any polarizing cell. Sarcomeric and nonsarcomeric-based force require completely different types of organization and ...

Connexins, cell motility, and the cytoskeleton

Cell Motility and the Cytoskeleton — Wed, 17 Jun 2009 23:00:00 +0100

Connexins (Cx) comprise a family of transmembrane proteins, which form intercellular channels between plasma membranes of two adjoining cells, commonly known as gap junctions. Recent reports revealed that Cx proteins interact with diverse cellular components to form a multiprotein complex, which has been termed "Nexus". Potential interaction partners include proteins such as cytoskeletal proteins, scaffolding proteins, protein kinases and phosphatases. These interactions allow correct subcellular localization of Cxs and functional regulation of gap junction-mediated intercellular communication. Evidence is accruing that Cxs might have channel-independent functions, which potentially include regulation of cell migration, cell polarization and growth control. In the current review, we summar...

Interactions of MAP/microtubule affinity regulating kinases with the adaptor complex AP-2 of clathrin-coated vesicles

Cell Motility and the Cytoskeleton — Tue, 16 Jun 2009 23:00:00 +0100

We report here that MARK copurifies with clathrin-coated vesicles (CCVs) via interaction with the adaptor complex AP-2. The adaptin binding site on MARK includes the regulatory loop of its catalytic domain. Immunofluorescence demonstrates the colocalization of MARK with AP-2 and clathrin, as well as other MARK-interacting proteins such as PAK5. The results are consistent with an observed influence of MARK on the trafficking of CCVs. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. (Source: Cell Motility and the Cytoskeleton)

AKAP-independent localization of type-II protein kinase A to dynamic actin microspikes

Cell Motility and the Cytoskeleton — Tue, 16 Jun 2009 23:00:00 +0100

Regulation of the cyclic AMP-dependent protein kinase (PKA) in subcellular space is required for cytoskeletal dynamics and chemotaxis. Currently, spatial regulation of PKA is thought to require the association of PKA regulatory (R) subunits with A-kinase anchoring proteins (AKAPs). Here, we show that the regulatory RII[alpha] subunit of PKA associates with dynamic actin microspikes in an AKAP-independent manner. Both endogenous RII[alpha] and a GFP-RII[alpha] fusion protein co-localize with F-actin in microspikes within hippocampal neuron growth cones and the leading edge lamellae of NG108-15 cells. Live-cell imaging demonstrates that RII[alpha]-associated microspikes are highly dynamic and that the coupling of RII[alpha] to actin is tight, as the movement of both actin and RII[alpha] are ...

Mechanical properties of the passive sea urchin sperm flagellum

Cell Motility and the Cytoskeleton — Tue, 16 Jun 2009 23:00:00 +0100

In this study we used Triton X-100 extracted sea urchin spermatozoa to investigate the mechanical behavior of the basic 9+2 axoneme. The dynein motors were disabled by vanadate so that the flagellum is rendered a passive structure. We find that when a proximal portion of the flagellum is bent with a glass microprobe, the remainder of the flagellum distal to the probe exhibits a bend in the opposite direction (a counterbend). The counterbend can be understood from the prevailing sliding doublet model of axoneme mechanics, but does require the existence of elastic linkages between the outer doublets. Analysis of the shapes of counterbends provides a consensus value of 0.03-0.08/[mu]m2 for the ratio of the interdoublet shear resistance (ES) to the bending resistance (EB) and we find that the ...

Structures of kinesin motor proteins

Cell Motility and the Cytoskeleton — Sun, 14 Jun 2009 23:00:00 +0100

Almost 25 years of kinesin research have led to the accumulation of a large body of knowledge about this widespread superfamily of motor and nonmotor proteins present in all eukaryotic cells. This review covers developments in kinesin research with an emphasis on structural aspects obtained by X-ray crystallography and cryoelectron microscopy 3-D analysis on kinesin motor domains complexed to microtubules. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. (Source: Cell Motility and the Cytoskeleton)

Interference of amino-terminal desmin fragments with desmin filament formation

Cell Motility and the Cytoskeleton — Sun, 14 Jun 2009 23:00:00 +0100

Short polypeptides from intermediate filament (IF) proteins containing one of the two IF-consensus motifs interfere severely with filament assembly in vitro. We now have systematically investigated a series of larger fragments of the muscle-specific IF protein desmin representing entire functional domains such as coil1 or coil 2. "Half molecules" comprising the amino-terminal portion of desmin, such as Des[Delta]C240 and the "tagged" derivative Des(ESA)[Delta]C244, assembled into large, roundish aggregates already at low ionic strength, Des[Delta]C250 formed extended, relatively uniform filaments, whereas Des[Delta]C265 and Des[Delta]C300 were soluble under these conditions. Surprisingly, all mutant desmin fragments assembled very rapidly into long thick filaments or spacious aggregates wh...

Structure and dynamics of an Arp2/3 complex-independent component of the lamellipodial actin network

Cell Motility and the Cytoskeleton — Sun, 14 Jun 2009 23:00:00 +0100

Sea urchin coelomocytes contain an unusually broad lamellipodial region and have served as a useful model experimental system for studying the process of actin-based retrograde/centripetal flow. In the current study the small molecule drug 2,3-butanedione monoxime (BDM) was employed as a means of delocalizing the Arp2/3 complex from the cell edge in an effort to investigate the Arp2/3 complex-independent aspects of retrograde flow. Digitally-enhanced phase contrast, fluorescence and polarization light microscopy, along with rotary shadow transmission electron microscopy methods demonstrated that BDM treatment resulted in the centripetal displacement of the Arp2/3 complex and the associated dendritic lamellipodial (LP) actin network from the cell edge. In its wake there remained an array of...

A cytoskeletal tropomyosin can compromise the structural integrity of skeletal muscle

Cell Motility and the Cytoskeleton — Sun, 14 Jun 2009 23:00:00 +0100

We have identified a number of extra-sarcomeric actin filaments defined by cytoskeletal tropomyosin (Tm) isoforms. Expression of a cytoskeletal Tm (Tm3) not normally present in skeletal muscle in a transgenic mouse resulted in muscular dystrophy. In the present report we show that muscle pathology in this mouse is late onset (between 2 and 6 months of age) and is predominately in the back and paraspinal muscles. In the Tm3 mice, Evans blue dye uptake in muscle and serum levels of creatine kinase were markedly increased following downhill exercise, and the force drop following a series of lengthening contractions in isolated muscles (extensor digitorum longus) was also significantly increased in these mice. These results demonstrate that expression of an inappropriate Tm in skeletal muscle ...

Inhibition of cytokinesis by Clostridium difficile toxin B and cytotoxic necrotizing factors - reinforcing the critical role of RhoA in cytokinesis

Cell Motility and the Cytoskeleton — Mon, 08 Jun 2009 04:00:00 +0100

This study provides evidence that RhoA-activating as well as RhoA-inactivating toxins cause inhibition of cytokinesis and cell division. The toxins' effects on cytokinesis were analyzed in Hela cells synchronized using the thymidine double block technique. Treatment of G2-phase cells with either the RhoA-activating CNFY or CNF1 or the RhoA-inactivating C3-lim or TcdB resulted in cytokinesis inhibition, as evidenced by the formation of a 4N population on flow cytometry, the inhibition of contractile ring formation, and the formation of bi-nucleated cells. While TcdB and CNF1 modify a broad-spectrum of Rho proteins, C3-lim and CNFY specifically target RhoA. Since C3-lim and CNFY both caused cytokinesis inhibition, our study re-inforces the critical role of RhoA (not Rac1 or Cdc42) in cytokin...

Actin bundling in plants

Cell Motility and the Cytoskeleton — Fri, 05 Jun 2009 04:00:00 +0100

Tight regulation of plant actin cytoskeleton organization and dynamics is crucial for numerous cellular processes including cell division, expansion and intracellular trafficking. Among the various actin regulatory proteins, actin-bundling proteins trigger the formation of bundles composed of several parallel actin filaments closely packed together. Actin bundles are present in virtually all plant cells, but their biological roles have rarely been addressed directly. However, decades of research in the plant cytoskeleton field yielded a bulk of data from which an overall picture of the functions supplied by actin bundles in plant cells emerges. Although plants lack several equivalents of animal actin-bundling proteins, they do possess major bundler classes including fimbrins, villins and f...

Mechanical dynamics of single cells during early apoptosis

Cell Motility and the Cytoskeleton — Fri, 05 Jun 2009 04:00:00 +0100

Dynamic mechanical properties of cells are becoming recognized as indicators and regulators of physiological processes such as differentiation, malignant phenotypes and mitosis. A key process in development and homeostasis is apoptosis and whilst the molecular control over this pathway is well studied, little is known about the mechanical consequences of cell death. Here, we study the caspase-dependent mechanical kinetics of single cells during early apoptosis initiated with the general protein-kinase inhibitor staurosporine. This results in internal remodelling of the cytoskeleton and nucleus which is reflected in dynamic changes in the mechanical properties of the cell. Utilizing simultaneous confocal and atomic force microscopy (AFM), we measured distinct mechanical dynamics in the inst...

Microtubule plus-end and minus-end capture at adherens junctions is involved in the assembly of apico-basal arrays in polarised epithelial cells

Cell Motility and the Cytoskeleton — Sun, 31 May 2009 04:00:00 +0100

Apico-basal polarisation of epithelial cells involves a dramatic reorganisation of the microtubule cytoskeleton. The classic radial array of microtubules focused on a centrally located centrosome typical of many animal cells is lost or greatly reduced and a non-centrosomal apico-basal array develops. The molecules and mechanisms responsible for the assembly and positioning of these non-centrosomal microtubules have not been fully elucidated. Using a Nocodazole induced regrowth assay in invitro culture (MDCK) and in situ epithelial (cochlear Kolliker's) cell models we establish that the apico-basal array originates from the centrosome and that the non-centrosomal microtubule minus-end anchoring sites do not contribute significantly to their nucleation. Confocal and electron microscopy revea...

The hard life of soft cells

Cell Motility and the Cytoskeleton — Thu, 28 May 2009 04:00:00 +0100

Cells are mechanical as well as chemical machines, and much of the energy they consume is used to apply forces to each other and to the extracellular matrix around them. The cytoskeleton, the cell membrane, and the macromolecules composing the extracellular matrix form networks that in concert with the forces generated by the cell create dynamic materials with viscoelastic properties unique to each tissue. Numerous recent studies suggest that the forces that cells create and are subjected to, as well as the mechanical properties of the materials to which they adhere, can have large effects on cell structure and function that can act in concert with or override signals from soluble stimuli. This brief review summarizes recent studies of the effects of substrate mechanics on cell motility, d...

The STE group kinase sepA controls cleavage furrow formation in dictyostelium

Cell Motility and the Cytoskeleton — Thu, 28 May 2009 04:00:00 +0100

During a REMI screen for proteins regulating cytokinesis in Dictyostelium discoideum we isolated a mutant forming multinucleate cells. The gene affected in this mutant encoded a kinase, SepA, which is an ortholog of Cdc7, a serine-threonine kinase essential for septum formation in Schizosaccharomyces pombe. Localization of SepA-GFP in live cells and its presence in isolated centrosomes indicated that SepA, like its upstream regulator Spg1, is associated with centrosomes. Knockout mutants of SepA showed a severe cytokinesis defect and a delay in development. In multinucleate SepA-null cells nuclear division proceeded normally and synchronously. However, often cleavage furrows were either missing or atypical: they were extremely asymmetric and constriction was impaired. Cortexillin-I, a mark...

Cytoskeletal pathologies of Alzheimer disease

Cell Motility and the Cytoskeleton — Thu, 28 May 2009 04:00:00 +0100

The histopathological hallmarks of Alzheimer disease are the extracellular amyloid plaques, composed principally of the amyloid beta peptide, and the intracellular neurofibrillary tangles, composed of paired helical filaments of the microtubule-associated protein, tau. Other histopathological structures involving actin and the actin-binding protein, cofilin, have more recently been recognized. Here we review new findings about these cytoskeletal pathologies, and, emphasize how plaques, tangles, the actin-containing inclusions and their respective building blocks may contribute to Alzheimer pathogenesis and the primary behavioral symptoms of the disease. Cell Motil. Cytoskeleton, 2009. © 2009 Wiley-Liss, Inc. (Source: Cell Motility and the Cytoskeleton)

Protein methylation in full length Chlamydomonas flagella

Cell Motility and the Cytoskeleton — Wed, 27 May 2009 04:00:00 +0100

Post-translational protein modification occurs extensively in eukaryotic flagella. Here we examine protein methylation, a protein modification that has only recently been reported to occur in flagella [Schneider MJ, Ulland M, Sloboda RD.. Mol Biol Cell 19(10):4319-4327.]. The cobalamin (vitamin B12) independent form of the enzyme methionine synthase (MetE), which catalyzes the final step in methionine production, is localized to flagella. Here we demonstrate, using immunogold scanning electron microscopy, that MetE is bound to the outer doublets of the flagellum. Methionine can be converted to S-adenosyl methionine, which then serves as the methyl donor for protein methylation reactions. Using antibodies that recognize symmetrically or asymmetrically methylated arginine residues, we identi...

Cytoplasmic Ig-domain proteins: Cytoskeletal regulators with a role in human disease

Cell Motility and the Cytoskeleton — Sun, 24 May 2009 04:00:00 +0100

Immunoglobulin domains are found in a wide variety of functionally diverse transmembrane proteins, and also in a smaller number of cytoplasmic proteins. Members of this latter group are usually associated with the actin cytoskeleton, and most of them bind directly to either actin or myosin, or both. Recently, studies of inherited human disorders have identified disease-causing mutations in five cytoplasmic Ig-domain proteins: myosin-binding protein C, titin, myotilin, palladin, and myopalladin. Together with results obtained from cultured cells and mouse models, these clinical studies have yielded novel insights into the unexpected roles of Ig domain proteins in mechanotransduction and signaling to the nucleus. An emerging theme in this field is that cytoskeleton-associated Ig domain prote...

[beta]3-Tubulin is induced by estradiol in human breast carcinoma cells through an estrogen-receptor dependent pathway

Cell Motility and the Cytoskeleton — Fri, 22 May 2009 04:00:00 +0100

Microtubules are involved in a variety of essential cell functions. Their role during mitosis has made them a target for anti-cancer drugs. However development of resistance has limited their use. It has been established that enhanced [beta]3-tubulin expression is correlated with reduced response to antimicrotubule agent-based chemotherapy or worse outcome in a variety of tumor settings. However little is known regarding the regulation of [beta]3-tubulin expression. We investigated the regulatory mechanisms of expression of [beta]3-tubulin in the MCF-7 cell line, a model of hormone-dependent breast cancer. Exposure of MCF-7 cells to estradiol was found to induce [beta]3-tubulin mRNA as well as [beta]3-tubulin protein expression. Conversely, we did not observe induction of [beta]3-tubulin m...

Dynamics of bacterial cytoskeletal elements

Cell Motility and the Cytoskeleton — Fri, 22 May 2009 04:00:00 +0100

Bacterial cytoskeletal elements are involved in an astonishing spectrum of cellular functions, from cell shape determination to cell division, plasmid segregation, the positioning of membrane-associated proteins and membrane structures, and other aspects of bacterial physiology. Interestingly, these functions are not necessarily conserved, neither between different bacterial species nor between bacteria and eukaryotic cells. The flexibility of cytoskeletal elements in performing different tasks is amazing and emphasises their very early development during evolution. This review focuses on the dynamics of cytoskeletal elements from bacteria. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. (Source: Cell Motility and the Cytoskeleton)

Identification and cell cycle-dependent localization of nine novel, genuine centrosomal components in Dictyostelium discoideum

Cell Motility and the Cytoskeleton — Fri, 22 May 2009 04:00:00 +0100

The centrosome is the main microtubule-organizing center and constitutes the largest protein complex in a eukaryotic cell. The Dictyostelium centrosome is an established model for acentriolar centrosomes and it consists of a layered core structure surrounded by a so-called corona, which harbors microtubule nucleation complexes. We have identified 34 new centrosomal candidate proteins through mass spectrometrical analysis of the proteome of isolated Dictyostelium centrosomes. Here we present a characterization of 12 centrosomal candidate proteins all featuring coiled coil regions and low expression levels, which are the most common attributes of centrosomal proteins. We used GFP fusion proteins to localize the candidate proteins in whole cells and on microtubule-free, isolated centrosomes. ...

Actin-ADF/cofilin rod formation in Caenorhabditis elegans muscle requires a putative F-actin binding site of ADF/cofilin at the C-terminus

Cell Motility and the Cytoskeleton — Thu, 21 May 2009 04:00:00 +0100

This study suggests that C. elegans can be a new model to study functions of actin-ADF/cofilin rods. Cell Motil. Cytoskeleton, 2009. © 2009 Wiley-Liss, Inc. (Source: Cell Motility and the Cytoskeleton)

Review of the mechanism of processive actin filament elongation by formins

Cell Motility and the Cytoskeleton — Wed, 20 May 2009 04:00:00 +0100

We review recent structural and biophysical studies of the mechanism of action of formins, proteins that direct the assembly of unbranched actin filaments for cytokinetic contractile rings and other cellular structures. Formins use free actin monomers to nucleate filaments and then remain bound to the barbed ends of these filaments as they elongate. In addition to variable regulatory domains, formins typically have formin homology 1 (FH1) and formin homology 2 (FH2) domains. FH1 domains have multiple binding sites for profilin, an abundant actin monomer binding protein. FH2 homodimers encircle the barbed end of a filament. Most FH2 domains inhibit actin filament elongation, but FH1 domains concentrate multiple profilin-actin complexes near the end of the filament. FH1 domains transfer acti...

Regulation of cellular contractile force in response to mechanical stretch by diphosphorylation of myosin regulatory light chain via RhoA signaling cascade

Cell Motility and the Cytoskeleton — Mon, 18 May 2009 04:00:00 +0100

Fibroblasts regulate their contractile force in response to external stretch; however, the detailed mechanism by which the force is regulated is unclear. Here, we show that diphosphorylation and dephosphorylation of myosin regulatory light chain (MRLC) are involved in the stretch-induced force response. Cellular stiffness, which reflects the cellular contractile force, under external stretch was measured by mechanical-scanning probe microscopy. Fibroblasts (NIH-3T3) expressing green fluorescent protein (GFP)-tagged mutant-type MRLC (MRLCT18A-GFP), which cannot be diphosphorylated, did not show any stretch-induced stiffness response, whereas the stiffness in cells expressing GFP-tagged wild-type MRLC (MRLCWT-GFP) increased immediately after the stretch and subsequently decreased after 1-2 h...

Intermediate filaments in Caenorhabditis elegans

Cell Motility and the Cytoskeleton — Tue, 12 May 2009 04:00:00 +0100

Intermediate filaments (IFs) make up one of the three major fibrous cytoskeletal systems in metazoans. Numerous IF polypeptides are synthesized in cell type-specific combinations suggesting specialized functions. The review concentrates on IFs in the model organism Caenorhabditis elegans which carries great promise to elucidate the still unresolved mechanisms of IF assembly into complex networks and to determine IF function in a living organism. In contrast to Drosophila melanogaster, which lacks cytoplasmic IFs altogether, the nematode genome contains 11 genes coding for cytoplasmic IFs and only a single gene for a nuclear lamin. Its cytoplasmic IFs are expressed in developmentally and spatially defined patterns. As an example we present the case of the intestinal IFs which are abundant i...

Helicobacter pylori activates protein kinase C delta to control Raf in MAP kinase signalling: Role in AGS epithelial cell scattering and elongation

Cell Motility and the Cytoskeleton — Tue, 12 May 2009 04:00:00 +0100

Helicobacter pylori is a major etiological agent in the development of chronic gastritis, duodenal ulcer and gastric carcinoma in humans. Virulent H. pylori strains harbor a type IV secretion system (T4SS) encoded by the cag pathogenicity island. This T4SS injects the CagA protein into gastric epithelial cells leading to actin-cytoskeletal rearrangements followed by cell elongation and scattering. Here we report that PMA (4[beta]-phorbol-12-myristate-13-acetate), a well-known cell-permeable activator of protein kinase C (PKC), induces a remarkably similar cellular phenotype as compared to infection with H. pylori. PKCs comprise a large family of serine/threonine kinases which are important for multiple physiological processes of host cells. We therefore investigated the role of individual ...

Cortactin: Coordinating adhesion and the actin cytoskeleton at cellular protrusions

Cell Motility and the Cytoskeleton — Tue, 12 May 2009 04:00:00 +0100

It has long been recognized that adhesion receptors cooperate with the cytoskeleton during morphogenesis, tissue remodeling and homeostasis. But how this occurs is less well-understood. A host of cytoskeletal regulators have been reported to have functional and biochemical linkage with adhesion receptors. The challenge remains to find functionally-coherent patterns within this increasingly large corpus of molecular information. In this review we discuss one approach, to identify distinctive functional modules that contribute to different adhesive processes. We illustrate this by considering Arp2/3-driven surface protrusion, which is utilized at both integrin-based cell-matrix adhesions and cadherin-based cell-cell adhesions. We further argue that regulatory proteins, such as cortactin, ser...

Becoming stable and strong: The interplay between vinculin exchange dynamics and adhesion strength during adhesion site maturation

Cell Motility and the Cytoskeleton — Mon, 11 May 2009 04:00:00 +0100

The coordinated formation and release of focal adhesions is necessary for cell attachment and migration. According to current models, these processes are caused by temporal variations in protein composition. Protein incorporation into focal adhesions is believed to be controlled by phosphorylation. Here, we tested the exchange dynamics of GFP-vinculin as marker protein of focal adhesions using the method of Fluorescence Recovery After Photobleaching. The relevance of the phosphorylation state of the protein, the age of focal adhesions and the acting force were investigated. For stable focal adhesions of stationary keratinocytes, we determined an exchangeable vinculin fraction of 52% and a recovery halftime of 57 s. Nascent focal adhesions of moving cells contained a fraction of exchanging ...

Rabs and cancer cell motility

Cell Motility and the Cytoskeleton — Thu, 07 May 2009 04:00:00 +0100

The Rab family of small GTPases functions in regulating vesicular transport in all eukaryotes. In the past few years, several important reports have linked some members of the Rab family to intriguing mechanistic aspects of cancer cell migration and invasiveness. Rab5 and Rab21 associate with [alpha]-integrin subunits and modulate their endosomal traffic and subcellular localization. Expression of the latter enhances adhesion and migration of certain cancer cell types. Rab25 has been functionally linked to tumor progression and the invasiveness of some epithelial cancers. Rab25 promotes invasive migration of cells in three-dimensional microenvironments by associating with [alpha]5[beta]1 integrin, and directing its recycling to dynamic ruffling protrusions at the migrating cell front. Acti...

The roles of actin cytoskeleton and microtubules for membrane recycling of a food vacuole in Tetrahymena thermophila

Cell Motility and the Cytoskeleton — Tue, 05 May 2009 04:00:00 +0100

Phagocytosis is a fundamental cellular event for the uptake of nutrients from the environment in several kinds of eukaryote. Most ciliates egest waste and undigested materials in food vacuoles (FVs) through a cytoproct, which is a specific organelle for defecation. It is considered that FV egestion is initiated by fusion between the FV membrane and plasma membrane in a cytoproct and completed with retrieval of the membrane into a cytoplasmic space. In addition, electron microscopy indicated that microfilaments might be involved in the recycling process of the FV membrane in ciliates over 30 years ago; however, there is no conclusive evidence. Here we demonstrated actin organization on FV near a cytoproct in Tetrahymena thermophila by using a marker for a cytoproct. Moreover, it was reveale...

The [beta]-thymosins: Intracellular and extracellular activities of a versatile actin binding protein family

Cell Motility and the Cytoskeleton — Thu, 30 Apr 2009 04:00:00 +0100

The [beta]-thymosins are N-terminally acetylated peptides of about 5 kDa molecular mass and composed of about 40-44 amino acid residues. The first member of the family, thymosin [beta]4, was initially isolated from thymosin fraction 5, prepared in five steps from calf thymus. Thymosin [beta]4 was supposed to be specifically produced and released by the thymic gland and to possess hormonal activities modulating the immune response. Various paracrine effects have indeed been reported for these peptides such as cardiac protection, angiogenesis, stimulation of wound healing, and hair growth. Besides these paracrine effects, it was noted that [beta]-thymosins occur in high concentration in the cytoplasm of many eukaryotic cells and bind to the cytoskeletal component actin. Subsequently it becam...

Centriole assembly in CHO cells expressing Plk4/SAS6/SAS4 is similar to centriogenesis in ciliated epithelial cells

Cell Motility and the Cytoskeleton — Wed, 29 Apr 2009 04:00:00 +0100

Centriole assembly is initiated by Plk4, a Polo-like kinase 4, which causes the recruitment of downstream regulators, such as SAS6 and SAS4, to a nascent centriole. Simultaneous expression of Plk4, SAS6 and SAS4 in CHO cells resulted in the formation of massive fibrogranular aggregates of various sizes and shapes. These aggregates were surrounded by dense particles of about 70 nm in diameter, similar to the centriolar satellite that has been observed around the centrosome in normal cycling cells. Within the fibrillar material, ring-like structures appeared and eventually differentiated into centrioles by association with short microtubule bundles. Centrioles were also assembled around a parent centriole in a cluster, a configuration that has been described as a "flower structure" formation...

Function of dynein in budding yeast: Mitotic spindle positioning in a polarized cell

Cell Motility and the Cytoskeleton — Tue, 28 Apr 2009 04:00:00 +0100

Cytoplasmic dynein is a microtubule motor that powers minus-end-directed motility in a variety of biological settings. The budding yeast, Saccharomyces cerevisiae, has been a useful system for the study of dynein, due to its molecular genetics and cell biology capabilities, coupled with the conservation of dynein-pathway proteins. In this review we discuss how budding yeast use dynein to manipulate the position of the mitotic spindle and the nucleus during cell division, using cytoplasmic microtubules, and we describe our current understanding of the genes required for dynein function. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. (Source: Cell Motility and the Cytoskeleton)

Mammalian Navigators are microtubule plus-end tracking proteins that can reorganize the cytoskeleton to induce neurite-like extensions

Cell Motility and the Cytoskeleton — Sat, 25 Apr 2009 04:00:00 +0100

Mammalian microtubule plus-end tracking proteins (+TIPs) specifically associate with the ends of growing microtubules. +TIPs are involved in many cellular processes, including mitosis, cell migration and neurite extension. Navigators are mammalian homologues of the C. elegans unc-53 protein, an ATPase that has been linked to the migration and outgrowth of muscles, axons and excretory canals. Here we show that all three mammalian Navigators are +TIPs, consistent with a previous study on Navigator 1 (NAV1) (Martinez-Lopez et al., Mol Cell Neurosci 2005;28:599-612). Overexpression of GFP-tagged Navigators causes displacement of CAP_GLY-motif containing +TIPs, such as CLIP-170, from microtubule ends, suggesting that the Navigator-binding sites on microtubule ends overlap with those of the CAP_...

The geometry and motion of nematode sperm cells

Cell Motility and the Cytoskeleton — Fri, 24 Apr 2009 04:00:00 +0100

The nematode sperm cell crawls by recycling major sperm protein (MSP) from dimers into subfilaments, filaments, and filament complexes, as a result of thermal writhing in the presence of hydrophobic patches. Polymerization near leading edges of the cell intercolates MSP dimers onto the tips of growing filament complexes, forcing them against the cell boundary, and extending the cytoskeleton in the direction of motion. Strong adhesive forces attach the cell to the substrate in the forward part of the lamellipod, while depolymerization in the rearward part of the cell breaks down the cytoskeleton, contracting the lamellipod and pulling the cell body forward. The movement of these cells, then, is caused by coordinated protrusive, adhesive and contractile forces, spatially separated across the...

HA-tagging of putative flagellar proteins in Chlamydomonas reinhardtii identifies a novel protein of intraflagellar transport complex B

Cell Motility and the Cytoskeleton — Tue, 21 Apr 2009 04:00:00 +0100

Proteomic analysis of flagella from the green alga Chlamydomonas reinhardtii has identified over 600 putative flagellar proteins. The genes encoding nine of these not previously characterized plus the previously described PACRG protein were cloned, inserted into a vector adding a triple-HA tag to the C-terminus of the gene product, and transformed into C. reinhardtii. Expression was confirmed by western blotting. Indirect immunofluorescence located all 10 fusion proteins in the flagellum; PACRG was localized to a subset of outer doublet microtubules. For some proteins, additional signal was observed in the cell body. Among the latter was FAP232-HA, which showed a spotted distribution along the flagella and an accumulation at the basal bodies. This pattern is characteristic for intraflagell...

Myofibrillogenesis in skeletal muscle cells in zebrafish

Cell Motility and the Cytoskeleton — Mon, 20 Apr 2009 04:00:00 +0100

The "premyofibril" model of myofibrillogenesis, based on observations in cultured avian muscle cells, proposes that mature myofibrils are preceded by two intermediary structures: premyofibrils and nascent myofibrils. To determine if this model applies to zebrafish skeletal muscle development, we stained developing embryos with antibodies to sarcomeric alpha-actinin and myosin II. In the youngest muscle cells, sarcomeric alpha-actinin and non-muscle myosin II were each localized in linear arrays of small bands that resembled the premyofibrils in avian myocytes. The distribution of muscle-specific myosin II began as scattered short filaments followed in time by overlapping bundles of filaments and organized A-bands in the older somites. Alpha-actinin organization changed from small z-bodies ...

Neutrophils display biphasic relationship between migration and substrate stiffness

Cell Motility and the Cytoskeleton — Fri, 17 Apr 2009 04:00:00 +0100

This study investigated the effects of substrate stiffness on the morphology, random motility coefficient, track speed (v), spreading area, and distribution of turning angles of neutrophils during chemokinesis. Human neutrophils were plated onto polyacrylamide gels of varying stiffness, ranging from 3 to 13 kPa, and coated with the extracellular matrix protein fibronectin, and timelapse images were taken with phase contrast microscopy. Our results show a biphasic behavior between neutrophil motility and substrate stiffness, with the optimum stiffness for motility depending on the concentration of fibronectin on the surface of the gel. On 100 [mu]g/mL fibronectin, the optimum stiffness is 4 kPa (v = 6.9 ± 0.6 [mu]m/min) while on 10 [mu]g/mL fibronectin, the optimum stiffness increases to 7...

The dynamin related protein Dnm1 fragments mitochondria in a microtubule-dependent manner during the fission yeast cell cycle

Cell Motility and the Cytoskeleton — Thu, 16 Apr 2009 04:00:00 +0100

Mitochondria are dynamic organelles that undergo cycles of fission and fusion. In the fission yeast, Schizosaccharomyces pombe, mitochondria align with microtubules and mitochondrial integrity is dependent upon an intact microtubule cytoskeleton. Here we show that mitochondria re-organize during the cell cycle and that this process is both dynamin- and microtubule-dependent. Microtubule depolymerization results in mitochondrial fragmentation but only when the dynamin-related protein Dnm1 is present. Mitochondrial fusion is, on the other hand, microtubule-independent. dnm1[Delta] cells, besides showing extensively fused mitochondria, are specifically resistant to anti-microtubule drugs. Dnm1-YFP localizes to foci at sites of mitochondrial severing which occupy the interface between adjacent...

TOGp regulates microtubule assembly and density during mitosis and contributes to chromosome directional instability

Cell Motility and the Cytoskeleton — Thu, 16 Apr 2009 04:00:00 +0100

TOGp, a member of the XMAP215 MAP family, is required for bipolar mitotic spindle assembly. To understand how TOGp contributes to spindle assembly, we examined microtubule dynamics after depleting TOGp by siRNA. Fluorescence recovery after photobleaching of GFP-tubulin demonstrated that spindle microtubule turnover is slowed two-fold in the absence of TOGp. Consistent with photobleaching results, microtubule regrowth after washout of the microtubule depolymerizing drug nocodazole was slower at the centrosomes and in the vicinity of mitotic chromatin in cells depleted of TOGp. The slower microtubule turnover is likely due to either nucleation or the transitions of dynamic instability because TOGp depletion did not effect the rate of plus end growth, measured by tracking EB1-GFP at microtubu...

N-WASP and cortactin are involved in invadopodium-dependent chemotaxis to EGF in breast tumor cells

Cell Motility and the Cytoskeleton — Thu, 16 Apr 2009 04:00:00 +0100

Metastatic mammary carcinoma cells, which have previously been observed to form mature, matrix degrading invadopodia on a thick ECM matrix, are able to form invadopodia with similar characteristics on glass without previously applied matrix. They form in response to epidermal growth factor (EGF), and contain the usual invadopodium core proteins N-WASP, Arp2/3, cortactin, cofilin, and F-actin. The study of invadopodia on glass allows for higher resolution analysis including the use of total internal reflection microscopy and analysis of their relationship to other cell motility events, in particular, lamellipodium extension and chemotaxis toward an EGF gradient. Invadopodium formation on glass requires N-WASP and cortactin but not microtubules. In a gradient of EGF more invadopodia form on ...

Microtubule involvement in the deposition of radial fibrillar callose arrays in stomata of the fern Asplenium nidus L

Cell Motility and the Cytoskeleton — Fri, 10 Apr 2009 04:00:00 +0100

Aniline blue staining and callose immunolabeling revealed the deposition of significant callose quantities, in the form of fibrils, in the periclinal walls of guard cells (GCs) of stomata of the fern Asplenium nidus. The stomata that were at an early stage of differentiation displayed short callose fibrils at the junctions of the periclinal walls with the dorsal ones, which converged on the site of the future stomatal pore. In stomata being at an advanced stage of differentiation, callose fibrils were radially arranged around the stomatal pore, while in mature closed ones they were focused on the margins of the wall thickenings lining the stomatal pore. The pattern of the callose fibril organization resembled that of cellulose microfibrils in the same walls. Like the cellulose microfibrils...

The effects of extracellular calcium on motility, pseudopod and uropod formation, chemotaxis, and the cortical localization of myosin II in Dictyostelium discoideum

Cell Motility and the Cytoskeleton — Fri, 10 Apr 2009 04:00:00 +0100

Extracellular Ca++, a ubiquitous cation in the soluble environment of cells both free living and within the human body, regulates most aspects of amoeboid cell motility, including shape, uropod formation, pseudopod formation, velocity, and turning in Dictyostelium discoideum. Hence it affects the efficiency of both basic motile behavior and chemotaxis. Extracellular Ca++ is optimal at 10 mM. A gradient of the chemoattractant cAMP generated in the absence of added Ca++ only affects turning, but in combination with extracellular Ca++, enhances the effects of extracellular Ca++. Potassium, at 40 mM, can partially substitute for Ca++. Mg++, Mn++, Zn++, and Na+ cannot. Extracellular Ca++, or K+, also induce the cortical localization of myosin II in a polar fashion. The effects of Ca++, K+ or a ...

Water flux in cell motility: Expanding the mechanisms of membrane protrusion

Cell Motility and the Cytoskeleton — Sun, 05 Apr 2009 04:00:00 +0100

Transmembrane water fluxes through aquaporins (AQPs) are suggested to play pivotal roles in cell polarization and directional cell motility. Local dilution by water influences the dynamics of the subcortical actin polymerization and directs the formation of nascent membrane protrusions. In this paper, recent evidence is discussed in support of such a central role of AQP in membrane protrusion formation and cell migration as a basis for our understanding of the underlying molecular mechanisms of directional motility. Specifically, AQP9 in a physiological context controls transmembrane water fluxes driving membrane protrusion formation, as an initial cellular response to a chemoattractant or other migratory signals. The importance of AQP-facilitated water fluxes in directional cell motility ...

Central pair apparatus enhances outer-arm dynein activities through regulation of inner-arm dyneins

Cell Motility and the Cytoskeleton — Fri, 03 Apr 2009 04:00:00 +0100

The beating of eukaryotic cilia and flagella is controlled by multiple species of inner-arm and outer-arm dyneins. To clarify the regulation on axonemal beating by nucleotide conditions and central-pair microtubules, microtubule sliding in disintegrating Chlamydomonas axonemes of various mutants and in vitro microtubule gliding by isolated axonemal dyneins were examined. In the in vitro motility assays with outer-arm dyneins ([alpha][beta] and [gamma]), microtubule translocation velocity decreased at high concentrations of ATP, while this inhibition was canceled by the simultaneous presence of ADP or ribose-modified analogues, mantATP/ADP. In contrast, motility of inner-arm dyneins was rather insensitive to these nucleotides. The velocity of sliding disintegration in axonemes lacking the c...

Directional change produced by perpendicularly-oriented microgrooves is microtubule-dependent for fibroblasts and epithelium

Cell Motility and the Cytoskeleton — Thu, 02 Apr 2009 04:00:00 +0100

Anisotropic substrata such as micromachined grooves can control cell shape, orientation, and the direction of cell movement, a phenomena termed topographic guidance. Although many types of cells exhibit topographic guidance, little is known regarding cell responses to conflicting topographic cues. We employed a substratum with intersecting grooves in order to present fibroblasts and epithelial cells with conflicting topographic cues. Using time-lapse and confocal microscopy, we examined cell behavior at groove intersections. Migrating fibroblasts and epithelial cells typically extended a cell process into the intersection ahead of the cell body. After travelling along the "X" groove to enter the intersection, the leading lamellipodia of the cell body encountered the perpendicular "Y" groov...

Fascin1 is dispensable for mouse development but is favorable for neonatal survival

Cell Motility and the Cytoskeleton — Thu, 02 Apr 2009 04:00:00 +0100

Fascin1, an actin-bundling protein, has been demonstrated to be critical for filopodia formation in cultured cells, and thus is believed to be vital in motile activities including neurite extension and cell migration. To test whether fascin1 plays such essential roles within a whole animal, we have generated and characterized fascin1-deficient mice. Unexpectedly, fascin1-deficient mice are viable and fertile with no major developmental defect. Nissl staining of serial coronal brain sections reveals that fascin1-deficient brain is grossly normal except that knockout mouse brain lacks the posterior region of the anterior commissure neuron and has larger lateral ventricle. Fascin1-deficient, dorsal root ganglion neurons are able to extend neurites in vitro as well as those from wild-type mice...

Bending-induced switching of dynein activity in elastase-treated axonemes of sea urchin sperm - Roles of Ca2+ and ADP

Cell Motility and the Cytoskeleton — Thu, 02 Apr 2009 04:00:00 +0100

Flagellar beating is caused by microtubule sliding, driven by the activity of dynein, between adjacent two of the nine doublet microtubules. An essential process in the regulation of dynein is to alternate its activity (switching) between the two sides of the central pair microtubules. The switching of dynein activity can be detected, in an in vitro system using elastase-treated axonemes of sea urchin sperm flagella, as a reversal of the relative direction of ATP-induced sliding between the two bundles of doublets at high Ca2+ (10-4 M) at pH 7.8-8.0. The reversal is triggered by externally applied bending of the doublet bundle. However, the mechanism of this bending-induced reversal (or backward sliding) remains unclear. To understand how the switching of dynein activity in flagella can be...

A novel mechanism of sperm motility in a viscous environment: Corkscrew-shaped spermatozoa cruise by spinning

Cell Motility and the Cytoskeleton — Mon, 30 Mar 2009 04:00:00 +0100

Fertilization of the green tree frog, Rhacophorus arboreus, occurs in the viscous environment of a foam nest, which is laid on vegetation. Their spermatozoa have a characteristic corkscrew-shaped head and a thick tail that extends perpendicularly to its longitudinal axis. However, it is unclear how these corkscrew-shaped spermatozoa move in this highly viscous environment. Here, we found that the spinning of the corkscrew-shaped head, caused by winding and unwinding of the tail, enables the spermatozoa to move through the highly viscous environment of a foam nest, like a corkscrew rotating into a cork. We suggested that dislocations observed in the matrix of satellite microtubules surrounding two axonemes, reflected the planes of sliding of the axonemes, and dyneins on doublets two and six...

Centrioles to basal bodies in the spermiogenesis of Mastotermes darwiniensis (Insecta, Isoptera)

Cell Motility and the Cytoskeleton — Tue, 24 Mar 2009 04:00:00 +0100

We present evidence of a possible de novo pathway for basal body formation in the differentiating germ cell. This cell also contains typical centrosomal proteins, such as centrosomin, pericentrin-like protein, [gamma]-tubulin, that undergo redistribution as spermatid differentiation proceeds. The spermatid centrioles are long structures formed by nine doublet rather than triplet microtubules provided with short projections extending towards the surrounding cytoplasm and with links between doublets. The sperm basal bodies are aligned in parallel beneath the nucleus. They consist of long regions close to the nucleus showing nine doublets in a cartwheel array devoid of any projections; on the contrary, the short region close to the plasma membrane, where the sperm flagella emerge, is characte...

Cytoskeleton rearrangements during Listeria infection: Clathrin and septins as new players in the game

Cell Motility and the Cytoskeleton — Thu, 19 Mar 2009 04:00:00 +0100

The study of an infection process can reveal how microbes exploit the host, and can illuminate unknown host cellular functions. Invasive pathogens have evolved efficient strategies to promote their internalization within normally non-phagocytic host cells. The so-called "zippering" bacteria present to host cell receptors molecules that mimic endogenous ligands, thereby inducing specific intracellular signaling cascades ultimately resulting in actin polymerization and uptake. Here we review how the bacterial pathogen Listeria monocytogenes enters into cells, and present a series of studies revealing that in addition to actin rearrangements this bacterium exploits the clathrin-mediated endocytosis machinery together with septins, a novel cytoskeleton element. The challenge is now to decipher...

Actin isoform expression patterns during mammalian development and in pathology: Insights from mouse models

Cell Motility and the Cytoskeleton — Wed, 18 Mar 2009 04:00:00 +0100

The dynamic actin cytoskeleton, consisting of six actin isoforms in mammals and a variety of actin binding proteins is essential for all developmental processes and for the viability of the adult organism. Actin isoform specific functions have been proposed for muscle contraction, cell migration, endo- and exocytosis and maintaining cell shape. However, these specific functions for each of the actin isoforms during development are not well understood. Based on transgenic mouse models, we will discuss the expression patterns of the six conventional actin isoforms in mammals during development and adult life. Ablation of actin genes usually leads to lethality and affects expression of other actin isoforms at the cell or tissue level. A good knowledge of their expression and functions will co...

Kinesin-5 in Drosophila embryo mitosis: Sliding filament or spindle matrix mechanism?

Cell Motility and the Cytoskeleton — Mon, 16 Mar 2009 04:00:00 +0100

The Drosophila syncytial embryo uses multiple astral mitotic spindles that are specialized for rapid mitosis. The homotetrameric kinesin-5, KLP61F contributes to various aspects of mitosis in this system, all of which are consistent with it exerting outward forces on spindle poles. In principle, kinesin-5 could accomplish this by (i) sliding microtubules (MTs), minus end leading, relative to a static spindle matrix or (ii) crosslinking and sliding apart adjacent pairs of antiparallel interpolar (ip) MTs. Here, I critically review data on the biochemistry of purified KLP61F, its localization and dynamic properties within spindles, and quantitative modeling of KLP61F function. While a matrix-based mechanism may operate in some systems, the work tends to support the latter "sliding filament" ...

GEF1 is a ciliary Sec7 GEF of Tetrahymena thermophila

Cell Motility and the Cytoskeleton — Sun, 08 Mar 2009 05:00:00 +0100

Ciliary guanine nucleotide exchange factors (GEFs) potentially activate G proteins in intraflagellar transport (IFT) cargo release. Several classes of GEFs have been localized to cilia or basal bodies and shown to be functionally important in the prevention of ciliopathies, but ciliary Arl-type Sec 7 related GEFs have not been well characterized. Nair et al. [ ] identified a Paramecium ciliary Sec7 GEF, PSec7. In Tetrahymena, Gef1p (GEF1), tentatively identified by PSec7 antibody, possesses ciliary and nuclear targeting sequences and like PSec7 localizes to cilia and macronuclei. Upregulation of GEF1 RNA followed deciliation and subsequent ciliary regrowth. Corresponding to similar Psec7 domains, GEF1domains contain IQ-like motifs and putative PH domains, in addition to GBF/BIG canonical m...

Characterization of mouse IFT complex B

Cell Motility and the Cytoskeleton — Sat, 28 Feb 2009 05:00:00 +0100

The primary cilium plays a key role in the development of mammals and in the maintenance of health. Primary cilia are assembled and maintained by the process of intraflagellar transport (IFT). In this work, we characterize mouse IFT complex B by identifying all of the mammalian orthologues of complex B and B-associated proteins previously identified in Chlamydomonas and Caenorhabditis and also identify a new component (IFT25/Hspb11) of complex B by database analysis. We tagged each of these proteins with the FLAG epitope and show that all except IFT172 and IFT20 localize to cilia and the peri-basal body or centrosomal region at the base of cilia. All of the proteins except IFT172 immunoprecipitate IFT88 indicating that they are co-assembled into a complex. IFT20 is the only complex B prote...

The evolution of the cilium and the eukaryotic cell

Cell Motility and the Cytoskeleton — Fri, 27 Feb 2009 05:00:00 +0100

The Cilium, the Nucleus and the Mitochondrion are three important organelles whose evolutionary histories are intimately related to the evolution and origin of the eukaryotic cell. The cilium is involved in motility and sensory transduction. The cilium is only found in the eukaryotic cells. Here we show that eight gene duplications prior to the last common ancestor of all extant eukaryotes account for the expansion of the Heavy Chain Dynein family of motor proteins and the evolution of the complexity of the cilium. The ambiguities in the branching of the phylogenetic tree of the HC-Dyneins were resolved by creating well-defined subtrees and using them to create the full tree. Due to the intimate relationship between the nucleus, the division center, mitosis and the basal body/centriole, th...

Bend propagation in the flagella of migrating human sperm, and its modulation by viscosity

Cell Motility and the Cytoskeleton — Thu, 26 Feb 2009 05:00:00 +0100

A pre-requisite for sexual reproduction is successful unification of the male and female gametes; in externally-fertilising echinoderms the male gamete is brought into close proximity to the female gamete through chemotaxis, the associated signalling and flagellar beat changes being elegantly characterised in several species. In the human, sperm traverse a relatively high-viscosity mucus coating the tract surfaces, there being a tantalising possible role for chemotaxis. To understand human sperm migration and guidance, studies must therefore employ similar viscous in vitro environments. High frame rate digital imaging is used for the first time to characterise the flagellar movement of migrating sperm in low and high viscosities. While qualitative features have been reported previously, we...

Gliotoxin reverses age-dependent nuclear morphology phenotypes, ameliorates motility, but fails to affect lifespan of adult Caenorhabditis elegans

Cell Motility and the Cytoskeleton — Sun, 22 Feb 2009 05:00:00 +0100

Specific mutations in human LMNA or loss of ZMPSTE26 activity cause abnormal processing of lamin A and early aging diseases, including Hutchinson Gilford progeria syndrome (HGPS). HGPS fibroblasts in culture undergo age-dependent progressive changes in nuclear architecture. Treating these cells with farnesyl transferase inhibitors (FTIs) reverse these nuclear phenotypes and also extend lifespan of mice HGPS models. Dermal cells derived from healthy old humans also accumulate the abnormally processed lamin A. However, the effect of FTIs on normal aging cells was not tested. Aging adult C. elegans cells show changes in nuclear architecture similar to HGPS fibroblasts and down regulating lamin expression in adult C. elegans reduces their lifespan. Here, we show that nuclei of adult C. elegans...

Compaction of cell shape occurs before decrease of elasticity in CHO-K1 cells treated with actin cytoskeleton disrupting drug cytochalasin D

Cell Motility and the Cytoskeleton — Fri, 20 Feb 2009 05:00:00 +0100

The actin filaments of the cytoskeleton form a highly dynamic polymer scaffold which is actively involved in many essential mechanisms such as cell migration, transport, mitosis, and mechanosensitivity. We treated CHO-K1 cells with different concentrations of the actin cytoskeleton disrupting drug cytochalasin D. Then investigating the cells' elastic behaviour by scanning force microscopy-based rheology we confirmed for high cytochalasin D concentrations ([ge]1.5 [mu]M) a significant decrease of mechanical stability. At lower concentrations we measured no significant softening, but flattening and a horizontal contraction was observable even at low concentrations ([ge]0.3 [mu]M) of cytochalasin D. The observed changes in cell shape resulted in a lower cell volume, showing that there is comp...

Regulation of sperm flagellar motility activation and chemotaxis caused by egg-derived substance(s) in sea cucumber

Cell Motility and the Cytoskeleton — Fri, 20 Feb 2009 05:00:00 +0100

The sea cucumber Holothuria atra is a broadcast spawner. Among broadcast spawners, fertilization occurs by means of an egg-derived substance(s) that induces sperm flagellar motility activation and chemotaxis. Holothuria atra sperm were quiescent in seawater, but exhibited flagellar motility activation near eggs with chorion (intact eggs). In addition, they moved in a helical motion toward intact eggs as well as a capillary filled with the water layer of the egg extracts, suggesting that an egg-derived compound(s) causes motility activation and chemotaxis. Furthermore, demembranated sperm flagella were reactivated in high pH (>7.8) solution without cAMP, and a phosphorylation assay using ([gamma]-32P)ATP showed that axonemal protein phosphorylation and dephosphorylation also occurred in a p...

The Wave complex is intrinsically inactive

Cell Motility and the Cytoskeleton — Wed, 11 Feb 2009 05:00:00 +0100

The Wave proteins activate the Arp2/3 complex at the leading edge of migrating cells. The resulting actin polymerization powers the projection of the plasma membrane in lamellipodia and membrane ruffles. The Wave proteins are always found associated with partner proteins. The canonical Wave complex is a stable complex containing five subunits. Even though it is well admitted that this complex plays an essential regulatory role on Wave function, the mechanisms by which Wave proteins are regulated within the complex are still elusive. Even the constitutive activity or inactivity of the complex is controversial. The major difficulty of these assays resides in the long and difficult purification of the Wave complex by a combination of several chromatography steps, which gives an overall low yi...

Analysis of cell movement by simultaneous quantification of local membrane displacement and fluorescent intensities using Quimp2

Cell Motility and the Cytoskeleton — Tue, 10 Feb 2009 05:00:00 +0100

The use of fluorescent markers in living cells has increased dramatically in the recent years. The quantitative analysis of the images requires specific analysis software. Previously, the program Quimp was launched for quantitating fluorescent intensities at the membrane or the cortex of the cell. However, Quimp is not well suited to quantitate local membrane displacement. Here we present Quimp2 that is capable of tracking membrane subregions in time, which enables the simultaneous quantification of fluorescent intensities and membrane movement. Quimp2 has two new tools, (i) conversion filters to analyze movies obtained with fluorescent, DIC and phase contrast different microscopes, and (ii) a macro that calculates the local membrane displacement and provides various options to display the...

Modulating [alpha]-actinin-4 dynamics in podocytes

Cell Motility and the Cytoskeleton — Tue, 10 Feb 2009 05:00:00 +0100

Podocytes are epithelial cells that line the outer aspect of renal blood vessels and provide a platform for the kidney's filtering apparatus, the slit diaphragm. Mutations in [alpha]-actinin-4, an actin bundling protein highly expressed in podocytes, result in increased affinity for actin and cause a familial form of focal segmental glomerulosclerosis. We hypothesized that such gain-of-affinity mutations would override [alpha]-actinin-4's sensitivity to regulatory factors such as calcium (acting via two EF-hand motifs), and phosphoinositides. We generated calcium- (mutEF) and phosphoinositide- (mutPI) insensitive variants of [alpha]-actinin-4, comparing their properties to a disease-associated mutant (K256E) and to the wildtype (wt) protein. [alpha]-Actinin-4mutPI displayed increased affin...

[alpha]-Skeletal muscle actin mutants causing different congenital myopathies induce similar cytoskeletal defects in cell line cultures

Cell Motility and the Cytoskeleton — Tue, 10 Feb 2009 05:00:00 +0100

Central core disease (CCD), congenital fibre type disproportion (CFTD), and nemaline myopathy (NM) are earlyonset clinically heterogeneous congenital myopathies, characterized by generalized muscle weakness and hypotonia. All three diseases are associated with [alpha]-skeletal muscle actin mutations. We biochemically characterized the CCD and CFTD causing actin mutants and show that all mutants fold correctly and are stable. Expression studies in fibroblasts, myoblasts, and myotubes show that these mutants incorporate in filamentous structures. However they do not intercalate between the nascent z-lines in differentiating muscle cell cultures. We also show that the distribution of mitochondria and of the ryanodine receptors, and calcium release properties from ryanodine receptors, are unch...

Fibroblast elongation and dendritic extensions in constrained versus unconstrained microtissues

Cell Motility and the Cytoskeleton — Sat, 24 Jan 2009 05:00:00 +0100

Cytoskeletal tension is fundamental to many biological processes, including germ layer sorting during embryogenesis [Krieg et al., ]. In vitro, such tension influences cell sorting in self-assembled, 3D microtissues and can be of sufficient magnitude to cause complex-shaped microtissue failure [Dean et al., ]. To examine the process of failure under cell-derived tension, we subjected normal human fibroblasts (NHFs) to directed self-assembly [Dean et al., ] in micro-molds designed to yield self-constraining microtissues. As cells contracted in this assay, the constrained microtissues narrowed, thinned and ultimately failed at their midpoints. By adding small numbers of GFP+ cells, changes in cell movement and morphology were assessed and compared to those of unconstrained microtissues. We f...

Guiding cell migration in 3D: A collagen matrix with graded directional stiffness

Cell Motility and the Cytoskeleton — Fri, 23 Jan 2009 05:00:00 +0100

While matrix stiffness has been implicated in cell adhesion and migration, most studies have focused on the effects of substrate stiffness in 2D. The present work describes a novel continuous stiffness gradient model for studying such processes in 3D. Wedge-shaped collagen scaffolds were compressed to produce sheets of a desired (0.1 mm) uniform thickness, but with increasing collagen density along the length of the sheet. Dynamic mechanical analysis, carried out on 1 mm wide strips obtained from the two ends and the middle of each sheet, showed that the elastic modulus increased from 1057 ± 487 kPa to 2305 ± 693 kPa at the soft and stiff end respectively and was 1835 ± 31 kPa in the middle. In constructs seeded with agarose marker beads prior to compression, mean agarose bead density r...

Microtubule-dependent motility and orientation of the cortical endoplasmic reticulum in elongating characean internodal cells

Cell Motility and the Cytoskeleton — Sat, 10 Jan 2009 05:00:00 +0100

Motility of the endoplasmic reticulum (ER) is predominantly microtubule- dependent in animal cells but thought to be entirely actomyosin-dependent in plant cells. Using live cell imaging and transmission electron microscopy to examine ER motility and structural organization in giant internodal cells of characean algae, we discovered that at the onset of cell elongation, the cortical ER situated near the plasma membrane formed a tight meshwork of predominantly transverse ER tubules that frequently coaligned with microtubules. Microtubule depolymerization increased mesh size and decreased the dynamics of the cortical ER. In contrast, perturbing the cortical actin array with cytochalasins did not affect the transverse orientation but decreased mesh size and increased ER dynamics. Our data sug...

A Remembrance of Harunori Ishikawa (1935-2008)

Cell Motility and the Cytoskeleton — Fri, 09 Jan 2009 05:00:00 +0100

No abstract. (Source: Cell Motility and the Cytoskeleton)

An in vitro model system for cytoskeletal confinement

Cell Motility and the Cytoskeleton — Fri, 09 Jan 2009 05:00:00 +0100

The motility, shape, and functionality of the cell depend sensitively on cytoskeletal mechanics which in turn is governed by the properties of filamentous proteins - mainly actin, microtubules, and intermediate filaments. These biopolymers are confined in the dense cytoplasm and therefore experience strong geometric constraints on their equilibrium thermal fluctuations. To obtain a better understanding of the influence of confinement on cytoskeletal filaments we study the thermal fluctuations of individual actin filaments in a microfluidic in vitro system by fluorescence microscopy and determine the persistence length of the filaments by analyzing the radial distribution function. A unique feature of this method is that we obtain the persistence length without detailed knowledge of the com...

In vivo analysis of MT-based vesicle transport by confocal reflection microscopy

Cell Motility and the Cytoskeleton — Thu, 08 Jan 2009 05:00:00 +0100

The use of confocal reflection microscopy (CRM) for the in vivo analysis of microtubule (MT) mediated transport of lipid droplets in the developing Drosophila egg primordia is described here. The developing Drosophila oocytes are ideal objects to study MT-mediated transport in vivo: transport of e.g. the lipid droplets can be conveniently, selectively and sensitively monitored through CRM and the egg primordia are readily available for physical, chemical and/or genetic manipulations. CRM is a non-destructive way to follow vesicle movement and allows high frame rate image recording. When combined with fluorescence imaging, CRM offers simultaneous visualization of the cargo and the protein(s) of interest, i.e. a motor or a cargo adapter, thus allowing a better understanding of MT-mediated tr...

Assembly properties of the Bacillus subtilis actin, MreB

Cell Motility and the Cytoskeleton — Thu, 01 Jan 2009 05:00:00 +0100

The bacterial actin MreB has been implicated in a variety of cellular roles including cell shape determination, cell wall synthesis, chromosome condensation and segregation, and the establishment and maintenance of cell polarity. Toward elucidating a clearer understanding of how MreB functions inside the bacterial cell, we investigated biochemically the polymerization of MreB from Bacillus subtilis. Light scattering and sedimentation assays revealed pH-, ionic-, cationic-, and temperature-dependent behavior. B. subtilis MreB polymerizes in the presence of millimolar divalent cations in a protein concentration-dependent manner. Polymerization is favored by decreasing pH and inhibited by monovalent salts and low temperatures. Although B. subtilis MreB binds and hydrolyzes both ATP and GTP, i...

Functional implications of membrane modification with semenogelins for inhibition of sperm motility in humans

Cell Motility and the Cytoskeleton — Wed, 17 Dec 2008 03:48:40 +0100

This study investigated the role of Sgs on human sperm motility through regulation of membrane potential and membrane permeability. Fresh semen samples were obtained from normozoospermic volunteers, and studies were conducted using motile cells selected using the swim-up method. Sgs changed the characteristics of sperm motion from circular to straightforward as evaluated by a computer-assisted motility analyzer, and all parameters were decreased more than 2.5 mg/mL. The results demonstrate that Sgs treatment immediately hyperpolarized the membrane potential of swim-up-selected sperm, changed the membrane structure, and time-dependently increased membrane permeability, as determined through flow cytometric analysis. The biphasic effects of Sgs were time- and dose-dependent and partially rev...

The actin-binding domain of cortactin is dynamic and unstructured and affects lateral and longitudinal contacts in F-actin

Cell Motility and the Cytoskeleton — Tue, 16 Dec 2008 05:00:00 +0100

Cortactin is an F-actin- and Arp2/3 complex-binding protein, implicated in the regulation of cytoskeleton dynamics and cortical actin-assembly. The actin-binding domain of cortactin consists of a 6.5 tandem repeat of a 37-amino acid sequence known as the cortactin repeat (residues 80-325). Using a combination of structure prediction, circular dichroism, and cysteine crosslinking, we tested a recently published three-dimensional model of the cortactin molecule in which the cortactin repeat is folded as a globular helical domain [Zhang et al., 2007, Mol Cell 27:197-213]. We show that the cortactin repeat is unstructured in solution. Thus, wild type and mutant constructs of the cortactin repeat, containing pairs of cysteines at positions 112 and 246, 83 and 112, 83 and 246, and 83 and 306, co...

An experimental model for assessing fibroblast migration in 3-D collagen matrices

Cell Motility and the Cytoskeleton — Sat, 06 Dec 2008 05:00:00 +0100

The purpose of this study was to develop and test a novel culture model for studying fibroblast migration in 3-D collagen matrices. Human corneal fibroblasts were seeded within dense, randomly oriented compressed collagen matrices. A 6 mm diameter button of this cell-seeded matrix was placed in the middle of an acellular, less dense outer collagen matrix. These constructs were cultured for 1, 3, 5 or 7 days in serum-free media, 10% fetal bovine serum (FBS), or 50 ng/ml PDGF. Constructs were then fixed and labeled with AlexaFluor 546 phalloidin (for f-actin) and TOTO-3 (for nuclei). Cell-matrix interactions were assessed using a combination of fluorescent and reflected light confocal imaging. Human corneal fibroblasts in serum-free media showed minimal migration into the outer (non-compress...

Transfection-induced defects in dynein-driven transport: evidence that ICs mediate cargo-binding

Cell Motility and the Cytoskeleton — Fri, 05 Dec 2008 05:00:00 +0100

In this study we investigated the mechanisms of transfection-induced disruption of organelle transport. Transfected ICs did not: (1) disrupt the dynein holoenzyme, (2) incorporate into the native dynein complex, (3) dimerize with native dynein ICs or (4) sequester dynein LCs in a phosphorylation-sensitive manner. Consistent with saturation of dynactin as an inhibitory mechanism, truncated ICs containing only the dynactin-binding domain were as effective as full-length IC constructs in disrupting organelle transport, and this effect was influenced by phosphorylation-state. Competition analysis demonstrated that S84D ICs were less capable than dephosphorylated ICs in disrupting the dynein-dynactin interaction. Finally, two-dimensional gel analysis revealed phosphorylation of the wild-type bu...

Correction: Myosin Va phosphorylated on Ser1650 is found in speckles and redistributes to nucleoli upon inhibition of transcription

Cell Motility and the Cytoskeleton — Sat, 22 Nov 2008 05:00:00 +0100

In the published article by Pranchevicius et al. "Myosin Va phosphorylated on Ser1650 is found in speckles and redistributes to nucleoli upon inhibition of transcription" [Cell Motil. Cytoskeleton 65: 441-456, 2008; DOI: 10.1002/cm.20269], there was an error in an author's name.The author's name should be indexed as: Maria C. Roque-Barreira, and not in the form in which it was published: Maria Cristina R. Barreira.This correction is published to address the error. (Source: Cell Motility and the Cytoskeleton)

Polarized MT1-MMP-CD44 interaction and CD44 cleavage during cell retraction reveal an essential role for MT1-MMP in CD44-mediated invasion

Cell Motility and the Cytoskeleton — Thu, 20 Nov 2008 05:00:00 +0100

The adhesion molecule CD44 and the membrane-type matrix metalloproteinase MT1-MMP act coordinately in tumor cells to promote cell invasion through a yet unclear mechanism. We are interested in studying the interplay between CD44 and MT1-MMP in carcinoma cells embedded in HA containing three-dimensional collagen I matrices (3D HA-Col I) by time-lapse confocal microscopy imaging. Here we report the in vivo interaction between CD44 and MT1-MMP, revealed by fluorescence resonance energy transfer (FRET) microscopy. MT1-MMP interacts with CD44 preferentially at the trailing edge of the invading tumor cells during rear retraction and on membrane fragments released during the invasion process. A fluorescent biosensor designed to monitor the proteolytic processing of CD44 by live cell imaging demon...

A novel ankyrin-repeat protein interacts with the regulatory proteins of inner arm dynein f (I1) of Chlamydomonas reinhardtii

Cell Motility and the Cytoskeleton — Thu, 20 Nov 2008 05:00:00 +0100

How ciliary and flagellar motility is regulated is a challenging problem. The flagellar movement in Chlamydomonas reinhardtii is in part regulated by phosphorylation of a 138 kD intermediate chain (IC138) of inner arm dynein f (also called I1). In the present study, we found that the axoneme of mutants lacking dynein f lacks a novel protein having ankyrin repeat motifs, registered as FAP120 in the flagellar proteome database. FAP120 is also missing or decreased in the axonemes of bop5, a mutant that has a mutation in the structural gene of IC138 but assembles the dynein f complex. Intriguingly, the amounts of FAP120 in the axonemes of different alleles of bop5 and several dynein f-lacking mutants roughly parallel their contents of IC138. These results suggest a weak but stoichiometric inte...

WAVE2 is regulated by multiple phosphorylation events within its VCA domain

Cell Motility and the Cytoskeleton — Sun, 16 Nov 2008 05:00:00 +0100

The (Wiskott-Aldrich Syndrome Protein)-family verprolin homologous protein (WAVE) family of proteins occupies a pivotal position in the cell, converting extracellular signals into the formation of branched filamentous (F) actin structures. WAVE proteins contain a verprolin central acidic (VCA) domain at their C-terminus, responsible for binding to and activating the Arp2/3 complex, which in-turn nucleates the formation of new actin filaments. Here we identify five Casein Kinase 2 (CK2) phosphorylation sites within the VCA domain of WAVE2, serines 482, 484, 488, 489, and 497. Phosphorylation of these sites is required for a high affinity interaction with the Arp2/3 complex. Phosphorylation of ser 482 and 484 specifically inhibits the activation of the Arp2/3 complex by the WAVE2 VCA domain,...

Functional studies of an evolutionarily conserved, cytochrome b5 domain protein reveal a specific role in axonemal organisation and the general phenomenon of post-division axonemal growth in trypanosomes

Cell Motility and the Cytoskeleton — Fri, 14 Nov 2008 05:00:00 +0100

Eukaryotic cilia and flagella are highly conserved structures composed of a canonical 9+2 microtubule axoneme. Several recent proteomic studies of cilia and flagella have been published, including a proteome of the flagellum of the protozoan parasite Trypanosoma brucei. Comparing proteomes reveals many novel proteins that appear to be widely conserved in evolution. Amongst these, we found a previously uncharacterised protein which localised to the axoneme in T. brucei, and therefore named it Trypanosome Axonemal protein (TAX)-2. Ablation of the protein using RNA interference in the procyclic form of the parasite has no effect on growth but causes a reduction in motility. Using transmission electron microscopy, various structural defects were seen in some axonemes, most frequently with micr...

Phosphorylation of tropomyosin extends cooperative binding of myosin beyond a single regulatory unit

Cell Motility and the Cytoskeleton — Tue, 04 Nov 2008 05:00:00 +0100

Tropomyosin (Tm) is one of the major phosphoproteins comprising the thin filament of muscle. However, the specific role of Tm phosphorylation in modulating the mechanics of actomyosin interaction has not been determined. Here we show that Tm phosphorylation is necessary for long-range cooperative activation of myosin binding. We used a novel optical trapping assay to measure the isometric stall force of an ensemble of myosin molecules moving actin filaments reconstituted with either natively phosphorylated or dephosphorylated Tm. The data show that the thin filament is cooperatively activated by myosin across regulatory units when Tm is phosphorylated. When Tm is dephosphorylated, this "long-range" cooperative activation is lost and the filament behaves identically to bare actin filaments....

A tropomyosin 1 induced defect in cytokinesis can be rescued by elevated expression of cofilin

Cell Motility and the Cytoskeleton — Wed, 22 Oct 2008 04:00:00 +0100

In this study we find that increased expression of a single tropomyosin isoform, tropomyosin 1, in U373MG astrocytoma cells leads to multinucleated cells and mitotic spindle defects. Furthermore, cells expressing increased levels of tropomyosin 1 usually fail to complete cytokinesis and this is accompanied by reduced accumulation of actin depolymerising factor/cofilin in the contractile ring. Adenovirus mediated expression of cofilin is able to relieve the tropomyosin 1 induced effects on cytokinesis. We conclude that tropomyosin 1 and cofilin play antagonistic roles within the contractile ring and that the balance between tropomyosin 1 and cofilin expression is important for cytokinesis. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. (Source: Cell Motility and the Cytoskeleton)

Disparate effects of different mutations in plakoglobin on cell mechanical behavior

Cell Motility and the Cytoskeleton — Mon, 20 Oct 2008 04:00:00 +0100

In this study, we examined the effects of two different mutations in plakoglobin on cell migration, stiffness, and adhesion. One is a C-terminal mutation causing Naxos disease, a recessive syndrome of arrhythmogenic right ventricular cardiomyopathy (ARVC) and abnormal skin and hair. The other is an N-terminal mutation causing dominant inheritance of ARVC without cutaneous abnormalities. To assess the effects of plakoglobin mutations on a broad range of cell mechanical behavior, we characterized a model system consisting of stably transfected HEK cells which are particularly well suited for analyses of cell migration and adhesion. Both mutations increased the speed of wound healing which appeared to be related to increased cell motility rather than increased cell proliferation. However, the...

Thinking about flagellar oscillation

Cell Motility and the Cytoskeleton — Thu, 02 Oct 2008 04:00:00 +0100

Bending of cilia and flagella results from sliding between the microtubular outer doublets, driven by dynein motor enzymes. This review reminds us that many questions remain to be answered before we can understand how dynein-driven sliding causes the oscillatory bending of cilia and flagella. Does oscillation require switching between two distinct, persistent modes of dynein activity? Only one mode, an active forward mode, has been characterized, but an alternative mode, either inactive or reverse, appears to be required. Does switching between modes use information from curvature, sliding direction, or both? Is there a mechanism for reciprocal inhibition? Can a localized capability for oscillatory sliding become self-organized to produce the metachronal phase differences required for bend...

Correlated waves of actin filaments and PIP3 in Dictyostelium cells

Cell Motility and the Cytoskeleton — Thu, 25 Sep 2008 04:00:00 +0100

Chemotaxis-deficient amiB-null mutant Dictyostelium cells show two distinct movements: (1) they extend protrusions randomly without net displacements; (2) they migrate persistently and unidirectionally in a keratocyte-like manner. Here, we monitored the intracellular distribution of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to gain insight into roles PIP3 plays in those spontaneous motilities. In keratocyte-like cells, PIP3 showed convex distribution over the basal membrane, with no anterior enrichment. In stalled cells, as well as in wild type cells, PIP3 repeated wave-like changes, including emergence, expansion and disappearance, on the basal membrane. The waves induced lamellipodia when they approached the cell edge, and the advancing speed of the waves was comparable to the mi...

Cooperativity between the [beta]-tubulin carboxy tail and the body of the molecule is required for microtubule function

Cell Motility and the Cytoskeleton — Fri, 19 Sep 2008 04:00:00 +0100

Using Drosophila spermatogenesis as a model, we show that function of the [beta]-tubulin C-terminal tail (CTT) is not independent of the body of the molecule. For optimal microtubule function, the [beta]-tubulin CTT and body must match. [beta]2 is the only [beta]-tubulin used in meiosis and spermatid differentiation. [beta]1-tubulin is used in basal bodies, but [beta]1 cannot replace [beta]2. However, when [beta]1 is co-expressed with [beta]2, both [beta]-tubulins are equally incorporated into all microtubules, and males exhibit near wild type fertility. In contrast, co-expression of [beta]2[beta]1C and [beta]1[beta]2C, two reciprocal chimeric molecules with bodies and tails swapped, results in defects in meiosis, cytoskeletal microtubules, and axonemes; males produce few functional sperm ...

Expression and alternative splicing of N-RAP during mouse skeletal muscle development

Cell Motility and the Cytoskeleton — Wed, 17 Sep 2008 04:00:00 +0100

N-RAP alternative splicing and protein localization were studied in developing skeletal muscle tissue from pre- and postnatal mice and in fusing primary myotubes in culture. Messages encoding N-RAP-s and N-RAP-c, the predominant isoforms of N-RAP detected in adult skeletal muscle and heart, respectively, were present in a 5:1 ratio in skeletal muscle isolated from E16.5 embryos. N-RAP-s mRNA levels increased three-fold over the first 3 weeks of postnatal development, while N-RAP-c mRNA levels remained low. N-RAP alternative splicing during myotube differentiation in culture was similar to the pattern observed in embryonic and neonatal muscle, with N-RAP-s expression increasing and N-RAP-c mRNA levels remaining low. In both developing skeletal muscle and cultured myotubes, N-RAP protein was...

The ability to survive mitosis in the presence of microtubule poisons differs significantly between human nontransformed (RPE-1) and cancer (U2OS, HeLa) cells

Cell Motility and the Cytoskeleton — Sat, 13 Sep 2008 04:00:00 +0100

We used live cell imaging to compare the fate of human nontransformed (RPE-1) and cancer (HeLa, U2OS) cells as they entered mitosis in nocodazole or taxol. In the same field, and in either drug, a cell in all lines could die in mitosis, exit mitosis and die within 10 h, or exit mitosis and survive [ge]10 h. Relative to RPE-1 cells, significantly fewer HeLa or U2OS cells survived mitosis or remained viable after mitosis: in nocodazole concentrations that inhibit spindle microtubule assembly, or in 500 nM taxol, 30% and 27% of RPE-1 cells, respectively, died in or within 10 h of exiting mitosis while 90% and 49% of U2OS and 78% and 81% of HeLa died. This was even true for clinically relevant taxol concentrations (5 nM) which killed 93% and 46%, respectively, of HeLa and U2OS cells in mitosis...

Inflammatory cytokines augments TGF-[beta]1-induced epithelial-mesenchymal transition in A549 cells by up-regulating T[beta]R-I

Cell Motility and the Cytoskeleton — Fri, 12 Sep 2008 04:00:00 +0100

In this study, we report that cytomix (a mixture of IL-1[beta], TNF-[alpha] and IFN-[gamma]) significantly enhances TGF-[beta]1-induced EMT in A549 cells as evidenced by acquisition of fibroblast-like cell shape, loss of E-cadherin, and reorganization of F-actin. IL-1[beta] or TNF-[alpha] alone can also augment TGF-[beta]1-induced EMT. However, a combination of IL-1[beta] and TNF-[alpha] or the cytomix is more potent to induce EMT. Cytomix, but not individual cytokine of IL-1[beta], TNF-[alpha] or IFN-[gamma], significantly up-regulates expression of TGF-[beta] receptor type I (T[beta]R-I). Suppression of T[beta]R-I, Smad2 or Smad3 by siRNA partially blocks EMT induction by cytomix plus TGF-[beta]1, indicating cytomix augments TGF-[beta]1-induced EMT through enhancing T[beta]R-I and Smad s...

The involvement of phospholipases C and D in the asymmetric division of subsidiary cell mother cells of Zea mays

Cell Motility and the Cytoskeleton — Fri, 12 Sep 2008 04:00:00 +0100

In the present study, the involvement of phospholipase C and D (PLC and PLD) pathways in the asymmetric divisions that produce the stomatal complexes of Zea mays was investigated. In particular, the polar organization of microtubules (MTs) and actin filaments (AFs) and the process of asymmetric division were studied in subsidiary cell mother cells (SMCs) treated with PLC and PLD modulators. In SMCs treated with butanol-1 (but-1), which blocks phosphatidic acid (PA) production via PLDs, AF-patch formation laterally to the inducing guard cell mother cell (GMC) and the subsequent asymmetric division were inhibited. In these SMCs, cell division plane determination, as expressed by MT preprophase band (MT-PPB) formation, was not disturbed. Exogenously applied PA partially relieved the but-1 eff...

Arp2 depletion inhibits sheet-like protrusions but not linear protrusions of fibroblasts and lymphocytes

Cell Motility and the Cytoskeleton — Fri, 22 Aug 2008 04:00:00 +0100

The Arp2/3 complex-mediated assembly and protrusion of a branched actin network at the leading edge occurs during cell migration, although some studies suggest it is not essential. In order to test the role of Arp2/3 complex in leading edge protrusion, Swiss 3T3 fibroblasts and Jurkat T cells were depleted of Arp2 and evaluated for defects in cell morphology and spreading efficiency. Arp2-depleted fibroblasts exhibit severe defects in formation of sheet-like protrusions at early time points of cell spreading, with sheet-like protrusions limited to regions along the length of linear protrusions. However, Arp2-depleted cells are able to spread fully after extended times. Similarly, Arp2-depleted Jurkat T lymphocytes exhibit defects in spreading on anti-CD3. Interphase Jurkats in suspension a...

Localization of spindle checkpoint proteins in cells undergoing mitosis with unreplicated genomes

Cell Motility and the Cytoskeleton — Wed, 20 Aug 2008 04:00:00 +0100

CHO cells can be arrested with hydoxyurea at the beginning of the DNA synthesis phase of the cell cycle. Subsequent treatment with the xanthine, caffeine, induces cells to bypass the S-phase checkpoint and enter unscheduled mitosis [Schlegel and Pardee,, Science 232:1264-1266]. These treated cells build a normal spindle and distribute kinetochores, unattached to chromosomes, to their daughter cells [Brinkley et al.,, Nature 336:251-254; Zinkowski et al.,, J Cell Biol 113:1091-1110; Wise and Brinkley,, Cell Motil Cytoskeleton 36:291-302; Balczon et al.,, Chromosoma 112:96-102]. To investigate how these cells distribute kinetochores to daughter cells, we analyzed the spindle checkpoint components, Mad2, CENP-E, and the 3F3 phosphoepitope, using immunofluorescence and digital microscopy. Even...

Kinesin-5 is not essential for mitotic spindle elongation in Dictyostelium

Cell Motility and the Cytoskeleton — Wed, 20 Aug 2008 04:00:00 +0100

The proper assembly and operation of the mitotic spindle is essential to ensure the accurate segregation of chromosomes and to position the cytokinetic furrow during cell division in eukaryotes. Not only are dynamic microtubules required but also the concerted actions of multiple motor proteins are necessary to effect spindle pole separation, chromosome alignment, chromatid segregation, and spindle elongation. Although a number of motor proteins are known to play a role in mitosis, there remains a limited understanding of their full range of functions and the details by which they interact with other spindle components. The kinesin-5 (BimC/Eg5) family of motors is largely considered essential to drive spindle pole separation during the initial and latter stages of mitosis. We have deleted ...

Chemotaxis-mediated scission contributes to efficient cytokinesis in Dictyostelium

Cell Motility and the Cytoskeleton — Sat, 09 Aug 2008 04:00:00 +0100

Interphase amoeba of Entamoeba invadens are attracted to the furrowing region of a neighboring dividing cell to assist with the division. A seemingly similar behavior has been observed in Dictyostelium discoideum, but in this case, it has not been shown whether the movements were truly directed toward the furrowing region or whether they have any relevance. We thus used myosin II-null cells, which spend more time than wild type cells in cytokinesis, and successfully demonstrated that nearly half of the division events involve the attraction of a neighbor cell to the furrowing region. Cells lacking the [beta] subunit of the trimeric G protein (G[beta]), which are incapable of chemotaxis, did not show such midwifery. Culturing wild type cells flattened under agarose sheets also slowed the cy...

Jasplakinolide, an actin stabilizing agent, alters anaphase chromosome movements in crane-fly spermatocytes

Cell Motility and the Cytoskeleton — Thu, 07 Aug 2008 04:00:00 +0100

We added jasplakinolide to anaphase crane-fly spermatocytes and determined its effects on chromosome movement. Previous work showed that the actin depolymerizing agents cytochalasin D or latrunculin B blocked or slowed chromosome movements. We studied the effects of jasplakinolide, a compound that stabilizes actin filaments. Jasplakinolide had the same effect on movements of each half- bivalent in a separating pair of half-bivalents, but different half-bivalent pairs in the same cell often responded differently, even when the concentrations of jasplakinolide varied by a factor of two. Jasplakinolide had no effect on about 20% of the pairs, but otherwise caused movements to slow, or to stop, or, rarely, to accelerate. When cells were kept in jasplakinolide, stopped pairs eventually resumed ...

Cell adhesion in zebrafish myogenesis: Distribution of intermediate filaments, microfilaments, intracellular adhesion structures and extracellular matrix

Cell Motility and the Cytoskeleton — Tue, 05 Aug 2008 04:00:00 +0100

To overcome the limitations of in vitro studies, we have been studying myogenesis in situ in zebrafish embryos, at a sub-cellular level. While in previous works we focused on myofibrillogenesis and some aspects of adhesion structures, here we describe in more detail cell adhesion structures and interactions among cytoskeletal components, membrane and extracellular matrix during zebrafish muscle development. We studied the intermediate filaments, and we describe the full range of desmin distribution in zebrafish development, from perinuclear to striated, until its deposition around the intersomite septa of older somites. This adhesion structure, positive for desmin and actin, has not been previously observed in myogenesis in vitro. We also show that actin is initially located in the interso...

Subcellular curvature at the perimeter of micropatterned cells influences lamellipodial distribution and cell polarity

Cell Motility and the Cytoskeleton — Mon, 04 Aug 2008 04:00:00 +0100

This study demonstrates that the cytoskeleton modulates cell polarity in response to multiple geometric cues in the extracellular environment. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. (Source: Cell Motility and the Cytoskeleton)

Ectopic expression of LIM-nebulette (LASP2) reveals roles in cell migration and spreading

Cell Motility and the Cytoskeleton — Fri, 01 Aug 2008 04:00:00 +0100

LIM-nebulette (LASP2) is a small focal adhesion protein and a member of the nebulin family of actin binding proteins. This recently identified splice variant of the nebulette locus is widely expressed and highly enriched in neuronal tissue. Other than that LIM-nebulette is a focal adhesion protein and interacts with zyxin, nothing is known about its function. Given that LIM-nebulette has an identical modular organization and overlapping tissue distributions to that of LASP1, we have analyzed the role of LIM-nebulette in comparison with that of LASP1. We find that LIM-nebulette is a dynamic focal adhesion protein that increases the rate of attachment and spreading of fibroblasts on fibronectin coated surfaces. Additionally, LIM-nebulette is recruited from the cortical cytoskeleton in non-mo...

Direction and speed of microtubule movements driven by kinesin motors arranged on catchin thick filaments

Cell Motility and the Cytoskeleton — Sun, 20 Jul 2008 04:00:00 +0100

Conventional kinesin (Kinesin-1) is a microtubule-based molecular motor that supports intracellular vesicle/organelle transport in various eukaryotic cells. To arrange kinesin motors similarly to myosin motors on thick filaments in muscles, the motor domain of rat conventional kinesin (amino acid residues 1-430) fused to the C-terminal 829 amino acid residues of catchin (KHC430Cat) was bacterially expressed and attached to catchin filaments that can attach to and arrange myosin molecules in a bipolar manner on their surface. Unlike the case of myosin where actin filaments move toward the center much faster than in the opposite direction along the catchin filaments, microtubules moved at the same speed in both directions. In addition, many microtubules moved across the filaments at the same...

The translocation of signaling molecules in dark adapting mammalian rod photoreceptor cells is dependent on the cytoskeleton

Cell Motility and the Cytoskeleton — Sun, 13 Jul 2008 04:00:00 +0100

In vertebrate rod photoreceptor cells, arrestin and the visual G-protein transducin move between the inner segment and outer segment in response to changes in light. This stimulus dependent translocation of signalling molecules is assumed to participate in long term light adaptation of photoreceptors. So far the cellular basis for the transport mechanisms underlying these intracellular movements remains largely elusive. Here we investigated the dependency of these movements on actin filaments and the microtubule cytoskeleton of photoreceptor cells. Co-cultures of mouse retina and retinal pigment epithelium were incubated with drugs stabilizing and destabilizing the cytoskeleton. The actin and microtubule cytoskeleton and the light dependent distribution of signaling molecules were subseque...

Living microlens arrays

Cell Motility and the Cytoskeleton — Thu, 10 Jul 2008 04:00:00 +0100

We report that a monolayer of epithelial cells exerts more stress on the substrate than fibroblasts and attribute this to the collective behavior of the epithelium. By subsequently adding different chemical triggers to the system, the contractile nature of the cells changes, thus modifying the focal length of the microlenses. Together, these findings demonstrate the importance of studying the mechanics of cell sheets and also introduce a new design paradigm for advanced materials, offering great promise for a range of applications. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. (Source: Cell Motility and the Cytoskeleton)

Regulation of polarity in cells devoid of actin bundle system after treatment with inhibitors of myosin II activity

Cell Motility and the Cytoskeleton — Wed, 09 Jul 2008 04:00:00 +0100

Interplay of two cytoskeletal systems - microfilaments and microtubules is essential for directional cell movement. To better understand the role of those cytoskeletal systems in polarization of cells, rat fibroblasts were incubated with drugs inhibiting activity of myosin II: blebbistatin and Y-27632. Both drugs led to disappearance of actin-myosin bundles and mature focal cell-matrix adhesions but did not affect polarization and directional motility. The rate of motility even increased after inhibitor treatment. The characteristic feature of inhibitor-treated fibroblasts was collapse of the cytoplasm accompanied by bundling of microtubules that led to transformation of lamellae into long immobile tails. The only exception was the leading anterior lamella which was not transformed into th...

Cortactin branches out: Roles in regulating protrusive actin dynamics

Cell Motility and the Cytoskeleton — Wed, 09 Jul 2008 04:00:00 +0100

Since its discovery in the early 1990's, cortactin has emerged as a key signaling protein in many cellular processes, including cell adhesion, migration, endocytosis, and tumor invasion. While the list of cellular functions influenced by cortactin grows, the ability of cortactin to interact with and alter the cortical actin network is central to its role in regulating these processes. Recently, several advances have been made in our understanding of the interaction between actin and cortactin, providing insight into how these two proteins work together to provide a framework for normal and altered cellular function. This review examines how regulation of cortactin through post-translational modifications and interactions with multiple binding partners elicits changes in cortical actin cyto...

Role of nonmuscle myosin IIB and N-RAP in cell spreading and myofibril assembly in primary mouse cardiomyocytes

Cell Motility and the Cytoskeleton — Wed, 09 Jul 2008 04:00:00 +0100

We investigated the role of nonmuscle myosin heavy chain (NMHC) IIB in cultured embryonic mouse cardiomyocytes by specific knockdown using RNA interference. NMHC IIB protein levels decreased 90% compared with mock-transfected cells by 3 days post transfection. NMHC IIB knockdown resulted in a slow decrease in N-RAP protein levels over 6 days with no change in N-RAP transcript levels. N-RAP is a scaffold for [alpha]-actinin and actin assembly during myofibrillogenesis, and we quantitated myofibril accumulation by morphometric analysis of [alpha]-actinin organization. Between 3 and 6 days, NMHC IIB knockdown was accompanied by the abolishment of cardiomyocyte spreading. During this period the rate of myofibril accumulation steadily decreased, correlating with the slowly decreasing levels of ...

[beta]-tubulin isotype classes II and V expression patterns in nonsmall cell lung carcinomas

Cell Motility and the Cytoskeleton — Tue, 08 Jul 2008 04:00:00 +0100

Previous studies suggest that [beta]-tubulin isotype protein levels could be useful as indicators of nonsmall cell lung cancer (NSCLC) aggressiveness. However, measurement of protein amounts in tissue samples by staining techniques is semiquantitative at best. Since technologies for measuring mRNA levels have become more efficient and quantitative, we wanted to determine whether [beta]-tubulin message levels may be useful as biomarkers. Quantitative real-time RT-PCR was used to measure the seven classes of [beta]-tubulin isotypes, stathmin and MAP4 mRNA levels in 64 NSCLC and 12 normal lung tissue samples. We found significantly higher fractions of [beta]-tubulin classes II and V mRNA compared to the other isotypes in all lung tumor samples (P < 0.05). In addition, the ratio of [beta]-tubu...

The knock-out of ARP3a gene affects F-actin cytoskeleton organization altering cellular tip growth, morphology and development in moss Physcomitrella patens

Cell Motility and the Cytoskeleton — Tue, 08 Jul 2008 04:00:00 +0100

The seven subunit Arp2/3 complex is a highly conserved nucleation factor of actin microfilaments. We have isolated the genomic sequence encoding a putative Arp3a protein of the moss Physcomitrella patens. The disruption of this ARP3A gene by allele replacement has generated loss-of-function mutants displaying a complex developmental phenotype. The loss-of function of ARP3A gene results in shortened, almost cubic chloronemal cells displaying affected tip growth and lacking differentiation to caulonemal cells. In moss arp3a mutants, buds differentiate directly from chloronemata to form stunted leafy shoots having differentiated leaves similar to wild type. Yet, rhizoids never differentiate from stem epidermal cells. To characterize the F-actin organization in the arp3a-mutated cells, we disr...

CaMK-II promotes focal adhesion turnover and cell motility by inducing tyrosine dephosphorylation of FAK and paxillin

Cell Motility and the Cytoskeleton — Tue, 08 Jul 2008 04:00:00 +0100

In this study, evidence is provided to show that CaMK-II, a Ca2+/calmodulin dependent protein kinase, influences fibroblast adhesion and motility. TIRF microscopy reveals a dynamic population of CaMK-II at the cell surface in migrating cells. Inhibition of CaMK-II with two mechanistically distinct, membrane permeant inhibitors (KN-93 and myr-AIP) freezes lamellipodial dynamics, accelerates spreading on fibronectin, enlarges paxillin-containing focal adhesions and blocks cell motility. In contrast, constitutively active CaMK-II is not found at the cell surface, reduces cell attachment, eliminates paxillin from focal adhesions and decreases the phospho-tyrosine levels of both FAK and paxillin; all of these events can be reversed with myr-AIP. Thus, both CaMK-II inhibition and constitutive ac...

Sarcomere alignment is regulated by myocyte shape

Cell Motility and the Cytoskeleton — Mon, 16 Jun 2008 04:00:00 +0100

Cardiac organogenesis and pathogenesis are both characterized by changes in myocyte shape, cytoskeletal architecture, and the extracellular matrix (ECM). However, the mechanisms by which the ECM influences myocyte shape and myofibrillar patterning are unknown. We hypothesized that geometric cues in the ECM align sarcomeres by directing the actin network orientation. To test our hypothesis, we cultured neonatal rat ventricular myocytes on islands of micro-patterned ECM to measure how they remodeled their cytoskeleton in response to extracellular cues. Myocytes spread and assumed the shape of circular and rectangular islands and reorganized their cytoskeletons and myofibrillar arrays with respect to the ECM boundary conditions. Circular myocytes did not assemble predictable actin networks no...

Heterogeneous modes of uptake for latex beads revealed through live cell imaging of phagocytes expressing a probe for phosphatidylinositol-(3,4,5)-trisphosphate and phosphatidylinositol-(3,4)-bisphosphate

Cell Motility and the Cytoskeleton — Mon, 16 Jun 2008 04:00:00 +0100

Latex beads are the preferred phagocytic substrate in biochemical studies of phagosome composition and maturation. Using living Dictyostelium cells and fluorescent probes, we compared the properties of phagosomes formed to ingest latex beads or digestible prey. Significant differences were found during the initial steps of phagocytosis. During uptake of bacteria or yeast, PHcrac-GFP, a probe that binds to membranes enriched in PI(3,4,5)P3 and PI(3,4)P2, always labeled the nascent phagosome and faded shortly after it sealed. However, labeling of bead-containing phagosomes was highly variable. Beads were engulfed by phagosomes either lacking or displaying the PHcrac-GFP label, and that label, if present, often persisted for many minutes, revealing that early trafficking steps for bead-contai...

A minor [beta]-tubulin essential for mammalian cell proliferation

Cell Motility and the Cytoskeleton — Sat, 14 Jun 2008 04:00:00 +0100

Mammals use tubulin from multiple genes to construct microtubules. Some genes are expressed in a tissue specific manner, while others are expressed in almost all cell types. [beta]5-Tubulin is a minor, ubiquitous isoform whose overexpression was recently shown to disrupt microtubules. Using inhibitory RNA, we now report that suppression of [beta]5 production in both human and hamster cells blocks cell proliferation. Cells depleted of [beta]5 either trigger the mitotic checkpoint and undergo apoptosis; or they experience a transient mitotic block, a high incidence of lagging chromosomes, and progression into G1 without cytokinesis to become large, flat cells with elevated DNA content. Microtubules appear to be normally organized in cells depleted of [beta]5, but they are rich in acetylated ...

Plectin deposition at podosome rings requires myosin contractility

Cell Motility and the Cytoskeleton — Fri, 13 Jun 2008 04:00:00 +0100

Metalloproteinase-dependent tissue invasion requires the formation of podosomes and invadopodia for localized matrix degradation. Actin cytoskeleton remodeling via Arp2/3-mediated actin polymerization is essential for podosome formation, and dynamic microtubules have an important role in maintaining podosome turnover in macrophages and osteoclasts. Little is known, however, about the involvement of the intermediate filament cytoskeleton in formation, stabilization, and turnover of podosomes. Here we show that vimentin intermediate filaments colocalize with the early sites of podosome formation at the stress fiber - focal adhesion interface in cultured vascular smooth muscle cells, but do not directly contribute to podosome formation, or stabilization. In unstimulated A7r5 cells the cytolin...

Novel interactions of fission yeast kinesin 8 revealed through in vivo expression of truncation alleles

Cell Motility and the Cytoskeleton — Fri, 13 Jun 2008 04:00:00 +0100

Fission yeast expresses two kinesin 8s, klp5+ and klp6+, which are important for diverse cellular functions: mitosis, meiosis, and the maintenance of normal cell morphology. During vegetative growth these motors display complex localization patterns, moving from the cytoplasm during interphase to the kinetochores in early mitosis, the interpolar spindle in anaphase B, and then back into the cytoplasm. We have expressed GFP-tagged alleles of domains from these motors, seeking the signals required for their localizations. The tail of Klp5p localized to the interphase nucleus, more specifically to telomeres. Addition of the neck re-directed this fragment to microtubules in the cytoplasm. Klp6-tail and the neck-tail domains of both motors localized at microtubule ends. Klp6-neck-tail localized...

Expression of the fast twitch troponin complex, fTnT, fTnI and fTnC, in vascular smooth muscle

Cell Motility and the Cytoskeleton — Thu, 12 Jun 2008 04:00:00 +0100

It is generally believed that proteins of the troponin complex are not expressed in smooth muscle. We have directly assayed for expression of troponin transcripts in mouse vascular smooth muscle and found that troponin sequences normally associated with fast twitch skeletal muscle (fTnT, fTnI, fTnC) were present at significant levels in the thoracic aorta. In situ hybridization experiments demonstrated that fTnT, fTnI and fTnC transcripts were expressed in the smooth muscle layer of mouse blood vessels of all sizes. Protein blot analysis using rat tissue showed that at least two members of the troponin complex, Troponin T and Troponin I, were translated in vascular smooth muscle of the aorta. Finally, immuno-fluorescence microscopy of rat aortic smooth muscle revealed that TnT and TnI are ...

The spindle function of CDCA4

Cell Motility and the Cytoskeleton — Fri, 23 May 2008 04:00:00 +0100

In an attempt to discover novel proteins functioning in both interphase nucleus and mitotic spindle as NuMA does, we carried out cDNA library screening with pooled autoimmune antibodies. Among positive clones we found a recently identified transcription regulatory protein (CDCA4) with the distinctive nuclear-mitotic apparatus distribution. CDCA4 localizes at metaphase spindle poles and the midzone in later stages. Additionally, an intensive CDCA4 accumulation parallel to spindle was observed in half of metaphase cells but not in later stages, implying a transient form of CDCA4 binding to midzone from anaphase. Mitotic arrest dissolved CDCA4 from centrosomes but during the spindle recovery, CDCA4 invariably colocalized with the microtubule nucleation foci as a component of microtubule organ...

Live-cell analysis of mitotic spindle formation in taxol-treated cells

Cell Motility and the Cytoskeleton — Wed, 14 May 2008 04:00:00 +0100

Taxol functions to suppress the dynamic behavior of individual microtubules, and induces multipolar mitotic spindles. However, little is known about the mechanisms by which taxol disrupts normal bipolar spindle assembly in vivo. Using live imaging of GFP-[alpha] tubulin expressing cells, we examined spindle assembly after taxol treatment. We find that as taxol-treated cells enter mitosis, there is a dramatic re-distribution of the microtubule network from the centrosomes to the cell cortex. As they align there, the cortical microtubules recruit NuMA to their embedded ends, followed by the kinesin motor HSET. These cortical microtubules then bud off to form cytasters, which fuse into multipolar spindles. Cytoplasmic dynein and dynactin do not re-localize to cortical microtubules, and disrup...

Coordination of outer arm dynein activity along axonemal doublet microtubules

Cell Motility and the Cytoskeleton — Tue, 13 May 2008 04:00:00 +0100

This study considers the mechanism by which ODA based sliding is produced and the relationship of that mechanism to the determination of beat frequency. Two models of activity have been examined: a stochastic model, where ODA activity is random and a metachronal model, where activity is sequentially triggered along a doublet. Inactivation of a few ODAs would have virtually no effect on stochastic activity, but would completely block metachronal activity. We (Seetharam and Satir []: Cell Motil Cytoskeleton 60:96-103) previously demonstrated that ODAs produce high speed sliding of about 200 [mu]m/s, followed by a pause. IDAs produce slow, 5 [mu]m/s, continuous sliding. We have examined the effects of nM concentrations of vanadate on sliding, measuring velocity and extent of high speed slidin...

HMWMAP2: New perspectives on a pathway to dendritic identity

Cell Motility and the Cytoskeleton — Mon, 12 May 2008 04:00:00 +0100

Neuronal polarity is established by the differentiation of two types of cytoplasmic processes: dendrites and the axon. These processes can be distinguished by their composition in microtubule-associated proteins, the high molecular weight MAP2 proteins (HMWMAP2) being found in the dendrites and tau proteins in the axon. It is believed that the main contribution of HMWMAP2 to the acquisition and maintenance of dendrites is to promote microtubule assembly and stability. However, recent studies force us to enlarge our view on how HMWMAP2 might contribute to defining the role of the dendritic microtubules. The purpose of this article is to convey our view that HMWMAP2 are important players in defining the contribution of microtubules to dendritic identity by anchoring membranous organelles and...

Treponema denticola Msp-deduced peptide conjugate, P34BSA, promotes RhoA-dependent actin stress fiber formation independent of its internalization by fibroblasts

Cell Motility and the Cytoskeleton — Mon, 12 May 2008 04:00:00 +0100

No abstract. (Source: Cell Motility and the Cytoskeleton)

Unsolved motility looking for answer

Cell Motility and the Cytoskeleton — Tue, 29 Apr 2008 04:00:00 +0100

A rod-like axostyle complex turns the anterior end of a termite flagellate, including the plasma membrane, continually in the same direction relative to the rest of the cell at speeds up to [sim]1 Hz. This motility provides direct visual evidence for the fluid nature of cell membranes. Torque is generated along the length of the axostyle complex by an unknown mechanism. Here I describe findings not published before and promising experiments that may help to solve this remarkable motility. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. (Source: Cell Motility and the Cytoskeleton)

The roles of cys124 and ser239 in the functional properties of human [beta]III tubulin

Cell Motility and the Cytoskeleton — Fri, 25 Apr 2008 04:00:00 +0100

Tubulin is the target for some very powerful anti-mitotic and anti-tumor drugs. The [beta]III tubulin isotype is found in very few normal tissues, but is often found in tumors, where it has been implicated in resistance to anti-tumor drugs. The [beta]III isotype occurs in fish, amphibians, birds and mammals and its unique features are highly conserved in evolution. One of these features is the replacement of cys239 by ser239. Cys239 is unusual in being highly sensitive to oxidation; in fact, oxidation of this residue inhibits microtubule assembly. The [beta]III isotype also has a very unusual cys124, where other [beta] isotypes have ser/ala124. The striking conservation in [beta]III of vertebrates strongly suggests that cys124 and ser239 play functional roles. We have prepared the C124S an...

Luminal fluid tonicity regulates airway ciliary beating by altering membrane stretch and intracellular calcium

Cell Motility and the Cytoskeleton — Thu, 24 Apr 2008 04:00:00 +0100

The coordinated, directional beating of airway cilia drives airway mucociliary clearance. Here we explore the hypothesis that airway surface liquid osmolarity is a key regulator of ciliary beating. Cilia in freshly isolated human and murine airways visualized with streaming video-microscopy exhibited a reciprocal dependence on a physiological range of luminal fluid osmolarities, across the entire range of ciliary activity (0-20 beats per sec). Increasing osmolarity slowed or completely abrogated, while lower osmolarity dramatically stimulated ciliary beating. In parallel, epithelial cell height and importantly, intracellular calcium levels (as judged by fluorescence imaging) also changed. Moreover, ciliary beating was stimulated by isosmotic solutions containing membrane permeant osmolytes...

Theory of diffraction from eukaryotic flagellar axonemes

Cell Motility and the Cytoskeleton — Wed, 23 Apr 2008 04:00:00 +0100

Existing theories for diffraction from cylindrically symmetrical and helical objects are applied to the axonemes of eukaryotic flagella and cilia, motile organelles responsible for locomotion of cells, transportation of food and mucus, etc. Each axoneme has a scaffold of microtubules arranged in the "9 + 2" scheme. Motor proteins dynein and other proteins are associated with the scaffold with apparent axial periodicities, and their exact arrangement would be best explored by X-ray diffraction. Here, various symmetries are assumed (helical and nonhelical) in the arrangement of these proteins, and the reflections that will emerge are predicted, hereby providing a practical means to analyze diffraction patterns currently being recorded in the latest synchrotron radiation facilities. Cell Moti...

The motor activity of mammalian axonemal dynein studied in situ on doublet microtubules

Cell Motility and the Cytoskeleton — Thu, 17 Apr 2008 04:00:00 +0100

Flagellar dynein generates forces that produce relative shearing between doublet microtubules in the axoneme; this drives propagated bending of flagella and cilia. To better understand dynein's role in coordinated flagellar and ciliary motion, we have developed an in situ assay in which polymerized single microtubules glide along doublet microtubules extruded from disintegrated bovine sperm flagella at a pH of 7.8. The exposed, active dynein remain attached to their respective doublet microtubules, allowing gliding of individual microtubules to be observed in an environment that allows direct control of chemical conditions. In the presence of ATP, translocation of microtubules by dynein exhibits Michaelis-Menten type kinetics, with Vmax = 4.7 ± 0.2 [mu]m/s and Km = 124 ± 11 [mu]M. The ch...

Protein kinase A RII-like (R2D2) proteins exhibit differential localization and AKAP interaction

Cell Motility and the Cytoskeleton — Thu, 17 Apr 2008 04:00:00 +0100

In this report, we demonstrate that all four R2D2 proteins are expressed in a wide variety of tissues and three of the proteins SP17, CABYR, and ASP are located in motile cilia of human bronchus and fallopian tubes. In addition, we detect SP17 in primary cilia. We also provide evidence that ROPN1 and ASP bind to a variety of AKAPs and this interaction can be disrupted with anchoring inhibitor peptides. The interaction of SP17 and CABYR with AKAPs appears to be much more limited. None of the R2D2 proteins appears to bind cAMP, a fundamental characteristic of the regulatory subunits of PKA. These observations suggest that R2D2 proteins utilize docking interactions with AKAPs to accomplish their function of regulating cilia and flagella. Based on location, affinity for AKAPs and lack of affin...

Dynamic rearrangement of F-actin organization triggered by host-specific plant signal is linked to morphogenesis of Aphanomyces cochlioides zoospores

Cell Motility and the Cytoskeleton — Wed, 16 Apr 2008 04:00:00 +0100

Cochliophilin A (5-hydroxy-6,7-methylenedioxyflavone), a root releasing host-specific plant signal triggers chemotaxis and subsequent morphological changes in pathogenic Aphanomyces cochlioides zoospores before host penetration. The present study illustrates time-course changing patterns of cytoskeletal filamentous actin (F-actin) organization in the zoospores of A. cochlioides during rapid morphological changes (encystment and germination) after exposure to cochliophilin A. Confocal laser scanning microscopic analysis revealed that F-actin microfilaments remained concentrated at ventral groove and diffusely distributed in peripheral cytoplasm of the zoospore. These microfilaments dramatically rearranged and changed into granular F-actin plaques interconnected with fine arrays during encys...

Dynamic compartmentalization of protein tyrosine phosphatase receptor Q at the proximal end of stereocilia: Implication of myosin VI-based transport

Cell Motility and the Cytoskeleton — Tue, 15 Apr 2008 04:00:00 +0100

Hair cell stereocilia are apical membrane protrusions filled with uniformly polarized actin filament bundles. Protein tyrosine phosphatase receptor Q (PTPRQ), a membrane protein with extracellular fibronectin repeats has been shown to localize at the stereocilia base and the apical hair cell surface, and to be essential for stereocilia integrity. We analyzed the distribution of PTPRQ and a possible mechanism for its compartmentalization. Using immunofluorescence we demonstrate that PTPRQ is compartmentalized at the stereocilia base with a decaying gradient from base to apex. This distribution can be explained by a model of transport directed toward the stereocilia base, which counteracts diffusion of the molecules. By mathematical analysis, we show that this counter transport is consistent...

Phosphorylation of neurofilament subunit NF-M is regulated by activation of NMDA receptors and modulates cytoskeleton stability and neuronal shape

Cell Motility and the Cytoskeleton — Tue, 15 Apr 2008 04:00:00 +0100

The cytoskeleton is essential for the structural organization of neurons and is influenced during development by excitatory stimuli such as activation of glutamate receptors. In particular, NMDA receptors are known to modulate the function of several cytoskeletal proteins and to influence cell morphology, but the underlying molecular and cellular mechanisms remain unclear. Here, we characterized the neurofilament subunit NF-M in cultures of developing mouse cortical neurons chronically exposed to NMDA receptor antagonists. Western blots analysis showed that treatment of cortical neurons with MK801 or AP5 shifted the size of NF-M towards higher molecular weights. Dephosphorylation assay revealed that this increased size of NF-M observed after chronic exposure to NMDA receptor antagonists wa...

Localization of [beta]v tubulin in the cochlea and cultured cells with a novel monoclonal antibody

Cell Motility and the Cytoskeleton — Tue, 15 Apr 2008 04:00:00 +0100

Tubulin, the dimeric structural protein of microtubules, is a heterodimer of [alpha] and [beta] subunits; both [alpha] and [beta] exist as numerous isotypes encoded by different genes. In vertebrates the sequence differences among the [beta]I, [beta]II, [beta]III, [beta]IV and [beta]V isotypes are highly conserved in evolution, implying that the isotypes may have functional significance. Isotype-specific monoclonal antibodies have been useful in determining the cellular and sub-cellular distributions and possible functions of the [beta]I, [beta]II, [beta]III, and [beta]IV isotypes; however, little is known about the [beta]V isotype. We here report the creation and purification of a monoclonal antibody (SHM.12G11) specific for [beta]V. The antibody was designed to be specific for the C-term...

The Arabidopsis class VIII myosin ATM2 is involved in endocytosis

Cell Motility and the Cytoskeleton — Tue, 08 Apr 2008 04:00:00 +0100

In this study the class VIII myosin ATM2 from the model plant Arabidopsis thaliana was selected for the examination of cargo specificity in vivo. Fluorescent protein-fusion plasmid constructs with fragments of the ATM2 cDNA were generated and used for Agrobacterium tumefaciens-based transient transformation of Nicotiana benthamiana leaves. The resulting subcellular localization patterns were recorded by live imaging with confocal laser scanning microscopy (CLSM) in epidermal leaf cells. Expression of a nearly full-length construct displayed labeling of filaments and vesicles, a head + neck fragment led to decoration of filaments only. However, expression of fluorescent protein-tagged C-terminal tail domain constructs labeled vesicular structures of different appearance. Most importantly, c...

Myosin-Va mediates RNA distribution in primary fibroblasts from multiple organs

Cell Motility and the Cytoskeleton — Fri, 21 Mar 2008 04:00:00 +0100

Myosin-Va has been shown to have multiple functions in a variety of cell types, including a role in RNA transport in neurons. Using primary cultures of cells from organs of young dilute-lethal (Myo5ad-l/Myo5ad-l) null mutant mice and wild-type controls, we show that in some, but not all, tissues, RNA distribution is dramatically different in the homozygous null mutant cells. The dependence of RNA localization on myosin-Va correlates with the relative abundance of the brain-specific splicing pattern of the myosin-Va tail. We also show that myosin-Va is involved in RNA localization soon after synthesis, because the effects of its absence are diminished for RNAs that are more than 30 min old. Finally, we show that localization of [beta]-actin mRNA is significantly changed by the absence of my...

Myosin Va phosphorylated on Ser1650 is found in nuclear speckles and redistributes to nucleoli upon inhibition of transcription

Cell Motility and the Cytoskeleton — Tue, 11 Mar 2008 04:00:00 +0100

In this study, myosin-Va, phosphorylated on a conserved serine in the tail domain (phospho-ser1650 MVa), was localized to subnuclear compartments. A monoclonal antibody, 9E6, raised against a peptide corresponding to phosphoserine1650 and flanking regions of the murine myosin Va sequence, was immunoreactive to myosin Va heavy chain in cellular and nuclear extracts of HeLa cells, PC12 cells and B16-F10 melanocytes. Immunofluorescence microscopy with this antibody revealed discrete irregular spots within the nucleoplasm that colocalized with SC35, a splicing factor that earmarks nuclear speckles. Phospho-ser1650 MVa was not detected in other nuclear compartments, such as condensed chromatin, Cajal bodies, gems and perinucleolar caps. Although nucleoli also were not labeled by 9E6 under norma...

Tracking changes in Z-band organization during myofibrillogenesis with FRET imaging

Cell Motility and the Cytoskeleton — Mon, 10 Mar 2008 04:00:00 +0100

There are a large number of proteins associated with Z-bands in myofibrils, but the precise arrangements of most of these proteins in Z-bands are largely unknown. Even less is known about how these arrangements change during myofibrillogenesis. We have begun to address this issue using Sensitized Emission Fluorescence Resonance Energy Transfer (SE-FRET) microscopy. Cultured skeletal muscle cells from quail embryos were transfected to express fusions of alpha-actinin, FATZ, myotilin, or telethonin with cyan and yellow fluorescent proteins in various pair wise combinations. FATZ and myotilin were selected because previous biochemical studies have suggested that they bind to alpha-actinin, the major protein of the Z-band. Telethonin was selected for its reported ability to bind FATZ. Statisti...

Anisotropic contraction in forisomes: Simple models won't fit

Cell Motility and the Cytoskeleton — Mon, 10 Mar 2008 04:00:00 +0100

Forisomes are ATP-independent, Ca2+-driven contractile protein bodies acting as reversible valves in the phloem of plants of the legume family. Forisome contraction is anisotropic, as shrinkage in length is associated with radial expansion and vice versa. To test the hypothesis that changes in length and width are causally related, we monitored Ca2+- and pH-dependent deformations in the exceptionally large forisomes of Canavalia gladiata by high-speed photography, and computed time-courses of derived geometric parameters (including volume and surface area). Soybean forisomes, which in the resting state resemble those of Canavalia geometrically but have less than 2% of the volume, were also studied to identify size effects. Calcium induced sixfold volume increases in forisomes of both speci...