CLP-36 PDZ-LIM protein associates with nonmuscle alpha-actinin-1 and alpha-actinin-4

The PDZ-LIM family of proteins (Enigma/LMP-1, ENH, ZASP/Cypher, RIL, ALP, and CLP-36) has been suggested to act as adapters that direct LIM-binding proteins to the cytoskeleton. Most interactions of PDZ-LIM proteins with the cytoskeleton have been identified in striated muscle, where several PDZ-LIM proteins are predominantly expressed. By contrast, CLP-36 mRNA is expressed in several nonmuscle tissues, and here we demonstrate high expression of CLP-36 in epithelial cells by in situ hybridization analysis. Our subcellular localization studies indicate that in nonmuscle cells, CLP-36 protein localizes to actin stress fibers. This localization is mediated via the PDZ domain of CLP-36 that associates with the spectrin-like repeats of alpha-actinin. Interestingly, immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis indicate that both nonmuscle alpha-actinin-1 and alpha-actinin-4 form complexes with CLP-36. The high expression of alpha-actinin-4 in the colon, together with these results, suggests a specific function for the alpha-actinin-4-CLP-36 complex in the colonic epithelium. More generally, results presented here demonstrate that the association of PDZ-LIM proteins with the cytoskeleton extends to the actin stress fibers of nonmuscle cells.     

Molecular identification and localization of cellular titin, a novel titin isoform in the fibroblast stress fiber

We previously discovered a large titin-like protein-c-titin-in chicken epithelial brush border and human blood platelet extracts that binds alpha-actinin and organizes arrays of myosin II bipolar filaments in vitro. RT-PCR analysis of total RNA from human megakaryoblastic (CHRF-288-11) and mouse fibroblast (3T3) nonmuscle cells reveal sequences identical to known titin gene exon sequences that encode parts of the Z-line, I-band, PEVK domain, A-band, and M-line regions of striated muscle titins. In the nonmuscle cells, these sequences are differentially spliced in patterns not reported for any striated muscle titin isoform. Rabbit polyclonal antibodies raised against expressed protein fragments encoded by the Z-repeat and kinase domain regions react with the c-titin band in Western blot analysis of platelet extracts and immunoprecipitate c-titin in whole platelet extracts. Immunofluorescent localization demonstrates that the majority of the c-titin colocalizes with alpha-actinin and actin in 3T3 and Indian Muntjac deer skin fibroblast stress fibers. Our results suggest that differential expression of titin gene exons in nonmuscle cells yields multiple novel isoforms of the protein c-titin that are associated with the actin stress fiber structures.     

The ZASP-like motif in actinin-associated LIM protein is required for interaction with the alpha-actinin rod and for targeting to the muscle Z-line

The Z-line is a specialized structure connecting adjacent sarcomeres in muscle cells. alpha-Actinin cross-links actin filaments in the Z-line. Several PDZ-LIM domain proteins localize to the Z-line and interact with alpha-actinin. Actinin-associated LIM protein (ALP), C-terminal LIM domain protein (CLP36), and Z band alternatively spliced PDZ-containing protein (ZASP) have a conserved region named the ZASP-like motif (ZM) between PDZ and LIM domains. To study the interactions and function of ALP we used purified recombinant proteins in surface plasmon resonance measurements. We show that ALP and alpha-actinin 2 have two interaction sites. The ZM motif was required for the interaction of ALP internal region with the alpha-actinin rod and for targeting of ALP to the Z-line. The PDZ domain of ALP bound to the C terminus of alpha-actinin. This is the first indication that the ZM motif would have a direct role in a protein-protein interaction. These results suggest that the two interaction sites of ALP would stabilize certain conformations of alpha-actinin 2 that would strengthen the Z-line integrity.     

Zasp/Cypher internal ZM-motif containing fragments are sufficient to co-localize with alpha-actinin--analysis of patient mutations

Z-band alternatively spliced PDZ-containing protein (ZASP/Cypher) has an important role in maintaining Z-disc stability in striated and cardiac muscle. ZASP/Cypher interacts through its PDZ domain with the major Z-disc actin cross-linker, alpha-actinin. ZASP/Cypher also has a conserved sequence called the ZM-motif, and it is found in two alternatively spliced exons 4 and 6. We have shown earlier that the ZM-motif containing internal regions of two related proteins ALP and CLP36 interact with alpha-actinin rod region, and that the ZM-motif is important in targeting ALP to the alpha-actinin containing structures in cell. Here, we show that the ZASP/Cypher internal fragments containing either ZM exon 4 or 6 co-localized with alpha-actinin in cultured myoblasts and nonmuscle cells. Fragments of 130 residues around the ZM-consensus were sufficient for localization, which is similar to our previous results of ALP. Moreover, ZASP/Cypher protein interacted directly with the alpha-actinin rod and competed with ALP in binding to the rod. During the inhibition of stress fiber assembly ZASP/Cypher and alpha-actinin co-localization could be partially disturbed, suggesting that ZASP/Cypher is bound to alpha-actinin mainly when alpha-actinin is localizing in stress fibers. Many point mutations found in cardiomyopathy patients are located in the internal region of ZASP/Cypher. However, we found no evidence that human patient mutations in the internal domain would affect the ZASP/Cypher co-localization with alpha-actinin, or that the mutations would destabilize the ZASP/Cypher protein.     

CLP36 and RIL recruit α-actinin-1 to stress fibers and differentially regulate stress fiber dynamics in F2408 fibroblasts

CLP36 is a member of the ALP/Enigma protein family and has been shown to be localized to stress fibers in various cells. We previously reported that depletion of CLP36 caused loss of stress fibers in BeWo choriocarcinoma cells, but it remains unclear how CLP36 contributes to stress fiber formation. In this study, we generated CLP36-depleted F2408 fibroblasts and found that stress fibers showed abnormal non-oriented organization in these cells. In addition to CLP36, F2408 cells contained RIL, another ALP/Enigma protein, and we demonstrated that RIL could compensate for the role of CLP36 in stress fiber formation. CLP36 and RIL form a complex with α-actinin-1 and palladin. We found a strong correlation between loss of CLP36/RIL and failure of α-actinin-1 or palladin to localize on stress fibers. In addition, time lapse observation revealed that incorporation of RIL stabilizes stress fibers and that CLP36 influences the dynamic architecture of these fibers. Our findings indicate that CLP36 and RIL have a redundant role in the formation of stress fibers, but have different effects on stress fiber dynamics in F2408 cells.     

Stress fibers in the splenic sinus endothelium in situ: molecular structure, relationship to the extracellular matrix, and contractility

In the present study, we investigated structural and functional aspects of stress fibers in a cell type in situ, i.e., the sinus endothelium of the human spleen. In this cell type, stress fibers extend underneath the basal plasma membrane and are arranged parallel to the cellular long axis. Ultrastructurally, the stress fibers were found to be composed of thin actin-like filaments (5-8 nm) and thick myosin-like filaments (10-15 nm X 300 nm). Actin filaments displayed changes in polarity (determined by S-1-myosin subfragment decoration), which may allow a sliding filament mechanism. At their plasmalemmal attachment sites, actin filaments exhibited uniform polarity with the S-1-arrowhead complexes pointing away from the plasma membrane. Fluorescence microscopy showed that the stress fibers have a high affinity for phalloidin and antibodies to actin, myosin, tropomyosin, and alpha-actinin. Vinculin was confined to the cytoplasmic aspect of the plasmalemmal termination sites of stress fibers, while laminin, fibronectin, and collagens were located at the extracellular aspect of these stress fiber-membrane associations. Western blot analysis revealed polypeptide bands that contained actin, myosin, and alpha-actinin to be major components of isolated cells. Exposure of permeabilized cells to MgATP results in prominent changes in cellular shape caused by stress fiber contraction. It is concluded that the stress fibers in situ anchored to cell-to-extracellular matrix contacts can create tension that might allow the endothelium to resist the fluid shear forces of blood flow.     

The PDZ-LIM protein CLP36 is required for actin stress fiber formation and focal adhesion assembly in BeWo cells

CLP36 belongs to the ALP subfamily of PDZ-LIM proteins and has a PDZ domain at its N-terminal and a LIM domain at its C-terminal. It has been shown that CLP36 is localized to stress fibers through interaction with α-actinin, but its function is still unclear. To investigate the role of CLP36 in stress fibers, we suppressed CLP36 expression in BeWo cells by RNAi and examined the phenotypic changes. CLP36-knockdown resulted in cell spreading and the loss of stress fibers and focal adhesions. These changes were reversed by addition of exogenous CLP36, but not by addition of mutant forms of CLP36 that lacked the PDZ or LIM domain. These findings indicate that CLP36 plays a critical role in stress fiber formation and the assembly of focal adhesions in BeWo cells. In addition, the PDZ and LIM domains are both essential for CLP36 to function.     

Perturbations of Drosophila alpha-actinin cause muscle paralysis, weakness, and atrophy but do not confer obvious nonmuscle phenotypes

We have investigated accumulation of alpha-actinin, the principal cross-linker of actin filaments, in four Drosophila fliA mutants. A single gene is variably spliced to generate one nonmuscle and two muscle isoforms whose primary sequence differences are confined to a peptide spanning the actin binding domain and first central repeat. In fliA3 the synthesis of an adult muscle-specific isoform is blocked in flight and leg muscles, while in fliA4 the synthesis of nonmuscle and both muscle-specific isoforms is severely reduced. Affected muscles are weak or paralyzed, and, in the case of fliA3, atrophic. Their myofibrils, while structurally irregular, are remarkably normal considering that they are nearly devoid of a major contractile protein. Also surprising is that no obvious nonmuscle cell abnormalities can be discerned despite the fact that both the fliA1- and fliA4-associated mutations perturb the nonmuscle isoform. Our observations suggest that alpha-actinin stabilizes and anchors thin filament arrays, rather than orchestrating their assembly, and further imply that alpha-actinin function is redundant in both muscle and nonmuscle cells.     

Actin filament organization regulates the induction of lens cell differentiation and survival

The actin cytoskeleton has the unique capability of integrating signaling and structural elements to regulate cell function. We have examined the ability of actin stress fiber disassembly to induce lens cell differentiation and the role of actin filaments in promoting lens cell survival. Three-dimensional mapping of basal actin filaments in the intact lens revealed that stress fibers were disassembled just as lens epithelial cells initiated their differentiation in vivo. Experimental disassembly of actin stress fibers in cultured lens epithelial cells with either the ROCK inhibitor Y-27632, which destabilizes stress fibers, or the actin depolymerizing drug cytochalasin D induced expression of lens cell differentiation markers. Significantly, short-term disassembly of actin stress fibers in lens epithelial cells by cytochalasin D was sufficient to signal lens cell differentiation. As differentiation proceeds, lens fiber cells assemble actin into cortical filaments. Both the actin stress fibers in lens epithelial cells and the cortical actin filaments in lens fiber cells were found to be necessary for cell survival. Sustained cytochalasin D treatment of undifferentiated lens epithelial cells suppressed Bcl-2 expression and the cells ultimately succumbed to apoptotic cell death. Inhibition of Rac-dependent cortical actin organization induced apoptosis of differentiating lens fiber cells. Our results demonstrate that disassembly of actin stress fibers induced lens cell differentiation, and that actin filaments provide an essential survival signal to both lens epithelial cells and differentiating lens fiber cells.     

Phosphoinositide binding regulates alpha-actinin dynamics: mechanism for modulating cytoskeletal remodeling

The active association-dissociation of dynamic protein-protein interactions is critical for the ability of the actin cytoskeleton to remodel. To determine the influence of phosphoinositide binding on the dynamic interaction of alpha-actinin with actin filaments and integrin adhesion receptors, fluorescence recovery after photobleaching (FRAP) microscopy was carried out comparing wild-type green fluorescent protein (GFP)-alpha-actinin and a GFP-alpha-actinin mutant with a decreased affinity for phosphoinositides (Fraley, T. S., Tran, T. C., Corgan, A. M., Nash, C. A., Hao, J., Critchley, D. R., and Greenwood, J. A. (2003) J. Biol. Chem. 278, 24039-24045). In fibroblasts, recovery of the mutant alpha-actinin protein was 2.2 times slower than the wild type along actin stress fibers and 1.5 times slower within focal adhesions. FRAP was also measured in U87MG glioblastoma cells, which have higher levels of 3-phosphorylated phosphoinositides. As expected, alpha-actinin turnover for both the stress fiber and focal adhesion populations was faster in U87MG cells compared with fibroblasts with recovery of the mutant protein slower than the wild type along actin stress fibers. To understand the influence of alpha-actinin turnover on the modulation of the actin cytoskeleton, wild-type or mutant alpha-actinin was co-expressed with constitutively active phosphoinositide (PI) 3-kinase. Co-expression with the alpha-actinin mutant inhibited actin reorganization with the appearance of enlarged alpha-actinin containing focal adhesions. These results demonstrate that the binding of phosphoinositides regulates the association-dissociation rate of alpha-actinin with actin filaments and integrin adhesion receptors and that the dynamics of alpha-actinin is important for PI 3-kinase-induced reorganization of the actin cytoskeleton. In conclusion, phosphoinositide regulation of alpha-actinin dynamics modulates the plasticity of the actin cytoskeleton influencing remodeling.     

Heterogeneous distribution of isoactins in cultured vascular smooth muscle cells does not reflect segregation of contractile and cytoskeletal domains.

We have previously demonstrated that alpha-smooth muscle (alpha-SM) actin is predominantly distributed in the central region and beta-non-muscle (beta-NM) actin in the periphery of cultured rabbit aortic smooth muscle cells (SMCs). To determine whether this reflects a special form of segregation of contractile and cytoskeletal components in SMCs, this study systematically investigated the distribution relationship of structural proteins using high-resolution confocal laser scanning fluorescent microscopy. Not only isoactins but also smooth muscle myosin heavy chain, alpha-actinin, vinculin, and vimentin were heterogeneously distributed in the cultured SMCs. The predominant distribution of beta-NM actin in the cell periphery was associated with densely distributed vinculin plaques and disrupted or striated myosin and alpha-actinin aggregates, which may reflect a process of stress fiber assembly during cell spreading and focal adhesion formation. The high-level labeling of alpha-SM actin in the central portion of stress fibers was related to continuous myosin and punctate alpha-actinin distribution, which may represent the maturation of the fibrillar structures. The findings also suggest that the stress fibers, in which actin and myosin filaments organize into sarcomere-like units with alpha-actinin-rich dense bodies analogous to Z-lines, are the contractile structures of cultured SMCs that link to the network of vimentin-containing intermediate filaments through the dense bodies and dense plaques.     

Conditional expression of a truncated fragment of nonmuscle myosin II-A alters cell shape but not cytokinesis in HeLa cells

A truncated fragment of the nonmuscle myosin II-A heavy chain (NMHC II-A) lacking amino acids 1-591, delta N592, was used to examine the cellular functions of this protein. Green fluorescent protein (GFP) was fused to the amino terminus of full-length human NMHC II-A, NMHC II-B, and delta N592 and the fusion proteins were stably expressed in HeLa cells by using a conditional expression system requiring absence of doxycycline. The HeLa cell line studied normally expressed only NMHC II-A and not NMHC II-B protein. Confocal microscopy indicated that the GFP fusion proteins of full-length NMHC II-A, II-B, and delta N592 were localized to stress fibers. However, in vitro assays showed that baculovirus-expressed delta N592 did not bind to actin, suggesting that delta N592 was localized to actin stress fibers through incorporation into endogenous myosin filaments. There was no evidence for the formation of heterodimers between the full-length endogenous nonmuscle myosin and truncated nonmuscle MHCs. Expression of delta N592, but not full-length NMHC II-A or NMHC II-B, induced cell rounding with rearrangement of actin filaments and disappearance of focal adhesions. These cells returned to their normal morphology when expression of delta N592 was repressed by addition of doxycycline. We also show that GFP-tagged full-length NMHC II-A or II-B, but not delta N592, were localized to the cytokinetic ring during mitosis, indicating that, in vertebrates, the amino-terminus part of mammalian nonmuscle myosin II may be necessary for localization to the cytokinetic ring.     

Stress fiber and cleavage furrow formation in living cells microinjected with fluorescently labeled alpha-actinin

alpha-Actinins, isolated from muscle and nonmuscle sources and labeled with various fluorescent dyes, were microinjected into living PtK2 cells during interphase to observe the reformation of stress fibers following cell division. Fluorescently labeled ovalbumin and bovine serum albumin were also injected as control proteins. alpha-Actinin was incorporated into stress fibers within 5 minutes after injection and remained present in the fibers for up to 11 days. The pattern of incorporation was the same regardless of whether the alpha-actinin was isolated from muscle or nonmuscle tissues or whether it was labeled with fluorescein, Lucifer Yellow, or rhodamine dyes. In contrast, neither labeled ovalbumin nor bovine serum albumin were incorporated into stress fibers. When the injected cells entered prophase, all stress fibers disassembled, resulting in a distribution of the fluorescent alpha-actinin throughout the cytoplasm. During cytokinesis, the fluorescent alpha-actinin was concentrated in the broad area between the separated chromosomes and along the edge of the cell in the cleavage area. Within 10 minutes after the completion of cleavage, the first fluorescent stress fibers reformed parallel to the spreading edges of the daughter cells and in close association with the midbody with a concomitant loss of alpha-actinin in the former cleavage furrow. Additional fibers formed adjacent to these first stress fibers. In some cases, new stress fibers formed between two existing stress fibers and some stress fibers moved up to 4 micron apart from one another in the course of 2 hours. Thus, fluorescent alpha-actinin, injected into living cells, undergoes the same cyclical changes in distribution as endogenous alpha-actinin during the cell cycle: from stress fibers to cleavage furrow and back to stress fibers.     

Phospholipase D activity is required for actin stress fiber formation in fibroblasts

Phospholipase D (PLD) is a ubiquitously expressed enzyme of ill-defined function. In order to explore its cellular actions, we inactivated the rat PLD1 (rPLD1) isozyme by tagging its C terminus with a V5 epitope (rPLD1-V5). This was stably expressed in Rat-2 fibroblasts to see if it acted as a dominant-negative mutant for PLD activity. Three clones that expressed rPLD1-V5 were selected (Rat2V16, Rat2V25, and Rat2V29). Another clone (Rat2V20) that lost expression of rPLD1-V5 was also obtained. In the three clones expressing rPLD1-V5, PLD activity stimulated by phorbol myristate acetate (PMA) or lysophosphatidic acid (LPA) was reduced by ~50%, while the PLD activity of Rat2V20 cells was normal. Changes in the actin cytoskeleton in response to LPA or PMA were examined in these clones. All three clones expressing rPLD1-V5 failed to form actin stress fibers after treatment with LPA. However, Rat2V20 cells formed stress fibers in response to LPA to the same extent as wild-type Rat-2 cells. In contrast, there was no significant change in membrane ruffling induced by PMA in the cells expressing rPLD1-V5. Since Rho is an activator both of rPLD1 and stress fiber formation, the activation of Rho was monitored in wild-type Rat-2 cells and Rat2V25 cells, but no significant difference was detected. The phosphorylation of vimentin mediated by Rho-kinase was also intact in Rat2V25 cells. Rat2V25 cells also showed normal vinculin-containing focal adhesions. However, the translocation of alpha-actinin to the cytoplasm and to the detergent-insoluble fraction in Rat2V25 cells was reduced. These results indicate that PLD activity is required for LPA-induced rearrangement of the actin cytoskeleton to form stress fibers and that PLD might be involved in the cross-linking of actin filaments mediated by alpha-actinin.     

Caldesmon affects actin organization at the leading edge and inhibits cell migration

The effect of the suppression of expression of the actin-binding protein caldesmon on the motility of nonmuscle cells has been studied. A more than a fivefold decrease in the content of this protein in cells by RNA interference led to the disturbance of the formation of actin stress fibers and acceleration of cell migration to the zone of injury of the monolayer. A stimulation of stationary cells by serum induced more than 1,5-fold accumulation of stress fibers only in control cells, but not in caldesmon-deficient cells. Similarly, the accumulation of actin filaments was observed in actively migrating cells of only wild type, but not in the cells with low caldesmon content. These changes occurred mainly at the leading edge of the migrating cell where the distinct structure of actin filaments was not seen in the absence of caldesmon. It was assumed that caldesmon inhibits cell migration due to the stabilization of actin in filaments and a decrease in the dynamics of monomeric actin at the leading edge of the migrating cell.     

Stress fiber dynamics as probed by antibodies against myosin

The dynamics of microfilament bundles (stress fibers) in tissue culture cells were studied by microinjecting an affinity-purified polyclonal antibody against chicken gizzard myosin. This antibody cross-reacted exclusively with the light chains of nonmuscle myosin and should therefore bind to the head portion of myosin molecules. When injected in high concentrations (13-26 mg/ml), it disrupted stress fibers in a high proportion (60-80%) of rat and chicken embryo fibroblasts, as well as in PtK2 cells. Myosin was found collected in large aggregates probably comprising protein: antibody precipitates, while actin and alpha-actinin were not localized in any defined structures in stress fiber depleted cells. Fibroblasts rounded up, probably because of lack of tension-generating microfilament bundles. After several hours, stress fibers were seen to regrow again in the afflicted cells, even when myosin precipitates and excess antibody were still present. The extent of stress fiber disruption and the time point of their reappearance were dependent on the concentration of the injected antibody.     

Phosphatidylinositol 4,5-bisphosphate phosphatase regulates the rearrangement of actin filaments.

Phosphatidylinositol 4,5-bisphosphate (PIP2) reorganizes actin filaments by modulating the functions of a variety of actin-regulatory proteins. Until now, it was thought that bound PIP2 is hydrolyzed only by tyrosine-phosphorylated phospholipase Cgamma (PLCgamma) after the activation of tyrosine kinases. Here, we show a new mechanism for the hydrolysis of bound PIP2 and the regulation of actin filaments by PIP2 phosphatase (synaptojanin). We isolated a 150-kDa protein (p150) from brains that binds the SH3 domains of Ash/Grb2. The sequence of this protein was found to be homologous to that of synaptojanin. The expression of p150 in COS 7 cells produces a decrease in the number of actin stress fibers in the center of the cells and causes the cells to become multinuclear. On the other hand, the expression of a PIP2 phosphatase-negative mutant does not disrupt actin stress fibers or produce the multinuclear phenotype. We have also shown that p150 forms the complexes with Ash/Grb2 and epidermal growth factor (EGF) receptors only when the cells are treated with EGF and that it reorganizes actin filaments in an EGF-dependent manner. Moreover, the PIP2 phosphatase activity of native p150 purified from bovine brains is not inhibited by profilin, cofilin, or alpha-actinin, although PLCdelta1 activity is markedly inhibited by these proteins. Furthermore, p150 suppresses actin gelation, which is induced by smooth muscle alpha-actinin. All these data suggest that p150 (synaptojanin) hydrolyzes PIP2 bound to actin regulatory proteins, resulting in the rearrangement of actin filaments downstream of tyrosine kinase and Ash/Grb2.     

Probing the mechanism of incorporation of fluorescently labeled actin into stress fibers

The mechanism of actin incorporation into and association with stress fibers of 3T3 and WI38 fibroblasts was examined by fluorescent analog cytochemistry, fluorescence recovery after photobleaching (FRAP), image analysis, and immunoelectron microscopy. Microinjected, fluorescein-labeled actin (AF-actin) became associated with stress fibers as early as 5 min post-injection. There was no detectable cellular polarity in the association of AF-actin with pre-existing stress fibers relative to perinuclear or peripheral regions. The rate of incorporation was quantified by image analysis of images generated with a two-dimensional photon counting microchannel plate camera. After equilibration of up to 2 h post-injection, FRAP demonstrated that actin subunits exchanged rapidly between filaments in stress fibers and the surrounding cytoplasm. When co-injected with rhodamine-labeled bovine serum albumin as a control, only actin was detected in the phase-dense stress fibers. The control protein was excluded from fibers and any linear fluorescence of the control was demonstrated as a pathlength artifact. The incorporation of AF-actin into stress fibers was studied by immunoelectron microscopy using anti-fluorescein as the primary antibody and goat anti-rabbit IgG coupled to peroxidase as the secondary antibody. At 5 min post-injection, reaction product was localized periodically in some fibers with a periodicity of approximately 0.75 microns. In large diameter fibers at 5 min post-injection, the analog was seen first on the surface of fibers, with individual filaments resolvable within the core. In the same cell, thinner diameter fibers were labeled uniformly throughout the diameter. By 20 min post-injection, most fibers were uniformly labeled. We conclude that the rate of actin subunit exchange in vivo is extremely rapid with molecular incorporation into actin filaments of stress fibers occurring as early as a few minutes post-injection. Exchange appears to first occur in filaments along the surface of stress fibers and then into more central regions in a periodic manner. We suggest that the periodic localization of actin at very early time points is due to a local microheterogeneity in which microdomains of fast vs. slower incorporation result from the periodic localization of actin-binding protein, such as alpha-actinin, along the length of the fiber.     

Stress fiber reformation after ATP depletion

Fluorescently labeled heavy meromyosin, alpha-actinin, and vinculin were used to localize actin, alpha-actinin, and vinculin, respectively, in permeabilized and living cells during the process of stress fiber reassembly, which occurred when cells were removed from ATP-depleting medium (20 mM sodium azide and 10 mM 2-deoxyglucose). In 80% of the cells recovering from ATP depletion, small, scattered plaques containing actin, alpha-actinin, and vinculin were replaced by long, thin, periodic fibers within 5 minutes of removal of the inhibitors. These nascent stress fibers grew broader as recovery progressed, until they attained the thickness of stress fibers in control cells. In the other 20% of the cells, the scattered plaques aggregated within 5 minutes of reversal, and almost all the actin, alpha-actinin, and vinculin in the cells became localized in one perinuclear aggregate, with a diameter of approximately 15-25 micron. As recovery progressed, all aggregates resembled rings, with diameters that increased at about 0.5 micron/minute and grew to as large as 70 micron in some giant cells. As the size of the rings increased, fibers radiated outward from them and sometimes spanned the diameter of the rings. The shape of the cells did not change during this time. By 1 hour after reversal, the rings were no longer present and all cells had networks of stress fibers. Indirect immunofluorescence techniques used to localize tubulin and vimentin indicated that microtubules and intermediate filaments were not constituents of the rings, and the rings were not closely apposed to the substrate, judging from reflection contrast optics. The rapid rearrangement of attachment plaques into a perinuclear aggregate that spreads radially in the cytoplasm occurs at the same speed as fibroblast and chromosomal movement, but is unlike other types of intracytoplasmic motility.     

Rho-kinase--mediated contraction of isolated stress fibers

It is widely accepted that actin filaments and the conventional double-headed myosin interact to generate force for many types of nonmuscle cell motility, and that this interaction occurs when the myosin regulatory light chain (MLC) is phosphorylated by MLC kinase (MLCK) together with calmodulin and Ca(2+). However, recent studies indicate that Rho-kinase is also involved in regulating the smooth muscle and nonmuscle cell contractility. We have recently isolated reactivatable stress fibers from cultured cells and established them as a model system for actomyosin-based contraction in nonmuscle cells. Here, using isolated stress fibers, we show that Rho-kinase mediates MLC phosphorylation and their contraction in the absence of Ca(2+). More rapid and extensive stress fiber contraction was induced by MLCK than was by Rho-kinase. When the activity of Rho-kinase but not MLCK was inhibited, cells not only lost their stress fibers and focal adhesions but also appeared to lose cytoplasmic tension. Our study suggests that actomyosin-based nonmuscle contractility is regulated by two kinase systems: the Ca(2+)-dependent MLCK and the Rho-kinase systems. We propose that Ca(2+) is used to generate rapid contraction, whereas Rho-kinase plays a major role in maintaining sustained contraction in cells.