Distribution and polarity of actin in the sensory hair cells of the chinchilla cochlea

Summary The distribution and polarity of actin in sensory hair cells of the chinchilla cochlea has been determined by decoration of actin filaments with myosin sub fragment S₁. Decorated actin filaments of the same polarity were present within the stereocilia above the cuticular plate. However the filaments in the rootlets and the thin filaments projecting laterally from the rootlets into the cuticular plate did not decorate with S₁. Decorated actin filaments were present within the cuticular plate, and near the plasma-membrane filaments of opposite polarity were observed. In the cross-striated region at the base of the cuticular plate of inner hair cells, decorated filaments were present in the dense bands of the cross-striations but the thin filaments perpendicular to the dense bands were not decorated. These results are discussed with respect to the two mechanisms that have been suggested for actin-myosin mediated movement of the stereocilia of inner-ear sensory cells.     

Cytoplasmic actin and cochlear outer hair cell motility

Summary Isolated outer hair cells of the guinea pig lacking a cuticular plate and its associated infracuticular network retain the ability to shorten longitudinally and become thinner. Membrane ghosts lacking cytoplasm retain the cylindrical shape of the hair-cell, and although they do not shorten, they retain the ability to constrict and become thinner. These data suggest that cytoplasmic components are associated with outer hair-cell longitudinal shortening and that the lateral wall is responsible for maintaing cell shape and for constriction. Actin, a protein associated with the cytoskeleton and cell motility, is thought to be involved in outer hair-cell motility. To study its role, actin was localized in isolated outer hair cells by use of phalloidin labeled with fluorescein and antibodies against actin coupled to colloidal gold. In permeabilized guinea-pig hair cells stained with phalloidin, actin filaments are found along the lateral wall. In frozen-fixed hair cells actin filaments are distributed uniformly throughout the cytoplasm. Electron-microscopic studies show that antibodies label actin throughout the outer hair-cell body. Thus cytoplasmic actin filaments may provide the structural basis for the contraction-like events.     

The role of Hsp27 and actin in the regulation of movement in human cancer cells responding to heat shock

Human heat shock 27-kDa protein 1 (HSPB1)/heat shock protein (Hsp) 27 is a small heat shock protein which is thought to have several roles within the cell. One of these roles includes regulating actin filament dynamics in cell movement, since Hsp27 has previously been found to inhibit actin polymerization in vitro. In this study, the role of Hsp27 in regulating actin filament dynamics is further investigated. Hsp27 protein levels were reduced using siRNA in SW480 cells, a human colon cancer cell line. An in vitro wound closure assay showed that cells with knocked down Hsp27 levels were unable to close wounds, indicating that this protein is involved in regulating cell motility. Immunoprecipitation pull down assays were done, to observe if and when Hsp27 and actin are in the same complex within the cell, before and after heat shock. At all time points tested, Hsp27 and actin were present in the same cell lysate fraction. Lastly, indirect immunostaining was done before and after heat shock to evaluate Hsp27 and actin interaction in cells. Hsp27 and actin showed colocalization before heat shock, little association 3 h after heat shock, and increased association 24 h after heat shock. Cytoprotection was observed as early as 3 h after heat shock, yet cells were still able to move. These results show that Hsp27 and actin are in the same complex in cells and that Hsp27 is important for cell motility. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Visualisation of microtubules and actin filaments in fixed BY-2 suspension cells using an optimised whole mount immunolabelling protocol

Excellent visualisation of microtubules and actin filaments was obtained in fixed tobacco BY-2 suspension cells after optimising a protocol for whole mount immunolabelling. The procedure is based on modification of fixation, cell wall digestion, dimethyl sulfoxide (DMSO) treatment, post fixation, and blocking. The most critical aspects of successful preservation and visualization of cytoskeletal elements appeared to be: a two-step fixation with paraformaldehyde and glutaraldehyde before enzymatic cell wall digestion and a post fixation with aldehydes thereafter. The method allows the improved visualization of the organisation of the microtubular and actin filament arrays during the successive stages of cell division and at interphase. Although we present the application of our protocols for cytoskeleton labelling, the excellent results show the potential of using this method for the analysis of various proteins and molecules in plant cells.Electronic Supplementary Material Supplementary material is available for this article at     

Relative actin nucleation promotion efficiency by WASP and WAVE proteins in endothelial cells

The mammalian genome encodes multiple Wiskott-Aldrich syndrome protein (WASP)/WASP-family Verprolin homologous (WAVE) proteins. Members of this family interact with the actin related protein (Arp) 2/3 complex to promote growth of a branched actin network near the plasma membrane or the surface of moving cargos. Arp2/3 mediated branching can further lead to formation of comet tails (actin rockets). Despite their similar domain structure, different WASP/WAVE family members fulfill unique functions that depend on their subcellular location and activity levels. We measured the relative efficiency of actin nucleation promotion of full-length WASP/WAVE proteins in a cytoplasmic extract from primary human umbilical vein endothelial cells (HUVEC). In this assay WAVE2 and WAVE3 complexes showed higher nucleation efficiency than WAVE1 and N-WASP, indicating distinct cellular controls for different family members. Previously, WASP and N-WASP were the only members that were known to stimulate comet formation. We observed that in addition to N-WASP, WAVE3 also induced short actin tails, and the other WAVEs induced formation of asymmetric actin shells. Differences in shape and structure of actin-based growth may reflect varying ability of WASP/WAVE proteins to break symmetry of the actin shell, possibly by differential recruitment of actin bundling or severing (pruning or debranching) factors.     

Desmosome dynamics in migrating epithelial cells requires the actin cytoskeleton

Re-modeling of epithelial tissues requires that the cells in the tissue rearrange their adhesive contacts in order to allow cells to migrate relative to neighboring cells. Desmosomes are prominent adhesive structures found in a variety of epithelial tissues that are believed to inhibit cell migration and invasion. Mechanisms regulating desmosome assembly and stability in migrating cells are largely unknown. In this study we established a cell culture model to examine the fate of desmosomal components during scratch wound migration. Desmosomes are rapidly assembled between epithelial cells at the lateral edges of migrating cells and structures are transported in a retrograde fashion while the structures become larger and mature. Desmosome assembly and dynamics in this system are dependent on the actin cytoskeleton prior to being associated with the keratin intermediate filament cytoskeleton. These studies extend our understanding of desmosome assembly and provide a system to examine desmosome assembly and dynamics during epithelial cell migration.     

Immunocytochemical demonstration of caldesmon and actin in thyroid glands of rats

Summary The distribution of caldesmon (a calmodulin-binding, F-actin interacting protein; Sobue et al. 1982) and actin was studied in the rat thyroid gland by means of light-microscopic immunocytochemistry, and the fine-structural distribution of actin filaments was examined by use of heavy meromyosin (HMM). Caldesmon and actin were demonstrated in the apical cytoplasm of almost all the follicle epithelial cells in normal as well as TSH-treated animals. Immunoreactivities for both caldesmon and actin showed almost the same pattern in localization. The smooth muscle cells of the blood vessels were also positive for caldesmon and actin. By electron microscopy, numerous actin filaments decorated by HMM and running perpendicularly or randomly to the apical surface were recognized in the apical cytoplasm of the follicle epithelial cell. These results suggest that caldesmon and actin, in conjugation with calmodulin, play a role in the regulation of cellular activity such as exocytosis and endocytosis in the apical portion of the follicle epithelial cell.This study was supported by grants from the Ministry of Education, Science and Culture, Japan     

In vivo visualization of actin dynamics and actin interactions by BiFC

The method of bimolecular fluorescence complementation (BiFC) enables selective visualization of protein interactions. While BiFC complex formation under in vitro conditions is considered to be essentially irreversible, there are hints that under in vivo conditions BiFC complex formation can be reversible. In the present study we used the BiFC method to visualize in vivo actin cytoskeleton dynamics. We demonstrate that in living cells formation of actin/actin BiFC complexes is reversible. Furthermore, we show heterologous binding between actin and protein kinase C delta (PKCdelta). Treatment with phorbol esters caused translocation of actin/PKCdelta complexes from the cytosol to the plasma membrane independent of an intact actin cytoskeleton. Our experiments demonstrate that the BiFC method might be a useful tool to investigate participation of the actin cytoskeleton in regulation of cell function.     

Correlative immuno-electron-microscopic and immunofluorescent localization of actin in sensory and supporting cells of the inner ear by use of a low-temperature embedding resin

Summary The cochleas from chinchilla inner ears were processed in the cold through Lowicryl K4M, and cured by UV light. Thick (2 m) sections were reacted with primary antibodies raised against actin, and anti-actin antibodies localized by FITC epifluorescence. On thin sections from the same blocks anti-actin antibodies were localized ultrastructurally with secondary antibodies coupled to colloidal gold.In the hair cells, actin was present in the stereocilia and cuticular plate, regions where thin filaments were observed by electron microscopy. Colloidal gold was uniformly distributed over these regions and over the stereocilia rootlets demonstrating that actin was present in this region although previously in permeabilized cells, the rootlet was not decorated with myosin subfragment S-1. Actin was present in the pillar and Deiters supporting cells at the reticular lamina and at the basilar membrane, where a meshwork of thin filaments was seen by electron microscopy. Colloidal gold particles were also localized over the thin processes of the pillar and Deiters cells, and over the region of the Deiters cell which envelops the base of the outer hair cell. In these regions actin co-localized with microtubules along the entire length of the supporting cells.     

Distribution of actin and myosin in muscle and non-muscle cells

Summary Specific anti-actin and anti-myosin antibodies were shown to react in single and double immunofluorescence sandwich tests with identical sites in non-muscle cells in frozen sections of tissues and in cultured cells. In tissues, both antibodies reacted with liver cell membranes, parts of renal glomeruli, brush borders and peritubular fibrils of renal tubules, brain synaptic junctions, and membranes of lymphoid cells in thymic medulla, lymph nodes and spleen. Both antibodies reacted strongly with long parallel cytoplasmic fibrils in cultured fibroblasts, and with disrupted fibrils in cytochalasin-B treated cells. In neuroblastoma cells both antibodies gave prominent staining of growth cones and microspikes. The observation that the distribution of myosin parallels that of actin in non-muscle cells argues strongly in favour of a functional interaction between the two molecules in the generation of contractile activity in nonmuscle cells.The authors thank Dr. M. Owen, National Institute of Medical Research, Mill Hill, for the gift of rabbit anti-actin antibodyOn sabbatical leave from Monash University, and supported by a Commonwealth Medical FellowshipThe Brompton Hospital, London     

Closing the loops: new insights into the role and regulation of actin during cell polarization

In this review, we summarize recent results on the understanding of actin organization and cell polarization with an emphasis on the critical role of actin during this process. We first report on the advances made in understanding the function and mechanism of formin family proteins in the nucleation of actin filaments. We also discuss how formins and other regulators of actin dynamics are thought to be involved in the generation of cell polarity. In the second part we discuss new findings indicating that, rather than using a linear pathway from signal transduction to cytoskeleton re-organization, cell polarity is established through bidirectional interplay between these processes. We describe the various types of feedback loops identified and point out common schemes. Finally we briefly summarize the emerging role of actinlike proteins in the generation of polarity in prokaryotes that implies an early origin of actin's role in cell polarity.     

Actin filament branching and protrusion velocity in a simple 1D model of a motile cell

We formulate and analyse a 1D model for the spatial distribution of actin density at the leading edge of a motile cell. The model incorporates nucleation, capping, growth and decay of actin filaments, as well as retrograde flow of the actin meshwork and known parameter values based on the literature. Using a simplified geometry, and reasonable assumptions about the biochemical processes, we derive PDEs for the density of actin filaments and their tips. Analytic travelling wave solutions are used to predict how the speed of the cell depends on rates of nucleation, capping, polymerization and membrane resistance. Analysis and simulations agree with experimental profiles for measured actin distributions. Extended versions of the model are studied numerically. We find that our model produces stable travelling wave solutions with reasonable cell speeds. Increasing the rate of nucleation of filaments (by the actin related protein Arp2/3) or the rate of actin polymerization leads to faster cell speed, whereas increasing the rate of capping or the membrane resistance reduces cell speed. We consider several variants of nucleation (spontaneous, tip, and side branching) and find best agreement with experimentally measured spatial profiles of filament and tip density in the side branching case.     

Control of tubulin and actin gene expression in Tetrahymena pyriformis during the cell cycle

Poly(A)-containing mRNA was isolated from division synchronized populations of the ciliated protozoan, Tetrahymena pyriformis. The level of tubulin and actin mRNA at specific cell cycle stages was analyzed by hybridization to tubulin and actin cDNA probes and by gel analysis of their in vitro translation products. The pattern of fluctuation of tubulin mRNA levels was similar to that observed for the in vivo tubulin synthesis previously reported [1]. This suggests that as the cells progress through the cell cycle, tubulin synthesis is controlled at the mRNA level. There was little fluctuation of actin synthesis or actin mRNA levels during the cell cycle, which may be indicative of a different regulatory mechanism for actin than for tubulin.     

Ultrastructural immunolocalization of actin in a fungus

Summary We have successfully localized fungal actin for the first time using immuno-electron microscopy and hyphal tips of the rice blast pathogenMagnaporthe grisea. Following ultrarapid freezing, samples were processed in a novel substitution fluid of 10% acrolein in anhydrous ethanol and embedded in LR White resin. A monoclonal anti-actin antibody, previously shown to recognizeM. grisea actin, bound specifically to filasomes concentrated in the peripheral cytoplasm of subapical regions, and to the core-region of the Spitzenkörper.Abbreviations IEM immuno-electron microscopy - TEM transmission electron microscopy     

Unphosphorylated calponin enhances the binding force of unphosphorylated myosin to actin

Background

Smooth muscle has the distinctive ability to maintain force for long periods of time and at low energy costs. While it is generally agreed that this property, called the latch-state, is due to the dephosphorylation of myosin while attached to actin, dephosphorylated-detached myosin can also attach to actin and may contribute to force maintenance. Thus, we investigated the role of calponin in regulating and enhancing the binding force of unphosphorylated tonic muscle myosin to actin.

Methods

To measure the effect of calponin on the binding of unphosphorylated myosin to actin, we used the laser trap assay to quantify the average force of unbinding (F_unb) in the absence and presence of calponin or phosphorylated calponin.

Results

F_unb from F-actin alone (0.12 ± 0.01 pN; mean ± SE) was significantly increased in the presence of calponin (0.20 ± 0.02 pN). This enhancement was lost when calponin was phosphorylated (0.12 ± 0.01 pN). To further verify that this enhancement of F_unb was due to the cross-linking of actin to myosin by calponin, we repeated the measurements at high ionic strength. Indeed, the F_unb obtained at a [KCl] of 25 mM (0.21 ± 0.02 pN; mean ± SE) was significantly decreased at a [KCl] of 150 mM, (0.13 ± 0.01 pN).

Conclusions

This study provides direct molecular level-evidence that calponin enhances the binding force of unphosphorylated myosin to actin by cross-linking them and that this is reversed upon calponin phosphorylation. Thus, calponin might play an important role in the latch-state.

General significance

This study suggests a new mechanism that likely contributes to the latch-state, a fundamental and important property of smooth muscle that remains unresolved.     

Mutants in the Dictyostelium Arp2/3 complex and chemoattractant-induced actin polymerization

We have investigated the role of the Arp2/3 complex in Dictyostelium cell chemotaxis towards cyclic-AMP and in the actin polymerization that is triggered by this chemoattractant. We confirm that the Arp2/3 complex is recruited to the cell perimeter, or into a pseudopod, after cyclic-AMP stimulation and that this is coincident with actin polymerization. This recruitment is inhibited when actin polymerization is blocked using latrunculin suggesting that the complex binds to pre-existing actin filaments, rather than to a membrane associated signaling complex. We show genetically that an intact Arp2/3 complex is essential in Dictyostelium and have produced partially active mutants in two of its subunits. In these mutants both phases of actin polymerization in response to cyclic-AMP are greatly reduced. One mutant projects pseudopodia more slowly than wild type and has impaired chemotaxis, together with slower movement. The second mutant chemotaxes poorly due to an adhesion defect, suggesting that the Arp2/3 complex plays a crucial part in adhering cells to the substratum as they move. We conclude that the Arp2/3 complex largely mediates the actin polymerization response to chemotactic stimulation and contributes to cell motility, pseudopod extension and adhesion in Dictyostelium chemotaxis.     

Initiation of cytoskeletal asymmetry for cell polarization and movement

A variety of mathematical models for the actin driven motility of eucaryotic cells have been discussed over the last decades. However, most of them do model polarized cells which are already in motion or at least have established lamellipods. Here we investigate the stimulus induced transition from a symmetric resting state of a cell to a polarized one. Our goal is to find a minimal scenario for this rearrangement of the cytoskeleton and to figure out which of the manifold proteins and processes associated with actin dynamics are essential for the initiation of movement.