Shroom4 (Kiaa1202) is an actin-associated protein implicated in cytoskeletal organization

All animal cells utilize a specialized set of cytoskeletal proteins to determine their overall shape and the organization of their intracellular compartments and organelles. During embryonic development, the dynamic nature of the actin cytoskeleton is critical for virtually all morphogenic events requiring changes in cell shape, migration, adhesion, and division. The behavior of the actin cytoskeleton is modulated by a myriad of accessory proteins. Shroom3 is an actin binding protein that regulates neural tube morphogenesis by eliciting changes in cell shape through a myosin II-dependent pathway. The Shroom-related gene SHROOM4 (formerly called KIAA1202) has also been implicated in neural development, as mutations in this gene are associated with human X-linked mental retardation. To better understand the function of Shrm4 in embryonic development, we have cloned mouse Shroom4 and characterized its protein product in vivo and in vitro. Shroom4 is expressed in a wide range of cell types during mouse development, including vascular endothelium and the polarized epithelium of the neural tube and kidney. In endothelial cells and embryo fibroblasts, endogenous Shroom4 co-distributes with myosin II to a distinct cytoplasmic population of F-actin and ectopic expression of Shroom4 in multiple cell types enhances or induces the formation of this actin-based structure. This localization is mediated, at least in part, by the direct interaction of Shroom4 and F-actin. Our results suggest that Shroom4 is a regulator of cytoskeletal architecture that may play an important role in vertebrate development.     

Structure of Shroom domain 2 reveals a three-segmented coiled-coil required for dimerization, Rock binding, and apical constriction

Shroom (Shrm) proteins are essential regulators of cell shape and tissue morpho-logy during animal development that function by interacting directly with the coiled-coil region of Rho kinase (Rock). The Shrm-Rock interaction is sufficient to direct Rock subcellular localization and the subsequent assembly of contractile actomyosin networks in defined subcellular locales. However, it is unclear how the Shrm-Rock interaction is regulated at the molecular level. To begin investigating this issue, we present the structure of Shrm domain 2 (SD2), which mediates the interaction with Rock and is required for Shrm function. SD2 is a unique three-segmented dimer with internal symmetry, and we identify conserved residues on the surface and within the dimerization interface that are required for the Rock-Shrm interaction and Shrm activity in vivo. We further show that these residues are critical in both vertebrate and invertebrate Shroom proteins, indicating that the Shrm-Rock signaling module has been functionally and molecularly conserved. The structure and biochemical analysis of Shrm SD2 indicate that it is distinct from other Rock activators such as RhoA and establishes a new paradigm for the Rock-mediated assembly of contractile actomyosin networks.     

Shroom2 regulates contractility to control endothelial morphogenesis

The intrinsic contractile, migratory, and adhesive properties of endothelial cells are central determinants in the formation of vascular networks seen in vertebrate organisms. Because Shroom2 (Shrm2) is expressed within the endothelium, is localized to cortical actin and cell-cell adhesions, and contains a conserved Rho kinase (Rock) binding domain, we hypothesized that Shrm2 may participate in the regulation of endothelial cell behavior during vascular morphogenesis. Consistent with this hypothesis, depletion of Shrm2 results in elevated branching and sprouting angiogenic behavior of endothelial cells. This is recapitulated in human umbilical vein endothelial cells and in a vasculogenesis assay in which differentiated embryonic stem cells depleted for Shrm2 form a more highly branched endothelial network. Further analyses indicate that the altered behavior observed following Shrm2 depletion is due to aberrant cell contractility, as evidenced by decreased stress fiber organization and collagen contraction with an increase in cellular migration. Because Shrm2 directly interacts with Rock, and Shrm2 knockdown results in the loss of Rock and activated myosin II from sites of cell-cell adhesion, we conclude that Shrm2 facilitates the formation of a contractile network within endothelial cells, the loss of which leads to an increase in endothelial sprouting, migration, and angiogenesis.     

Hypoxia induces the activation of hepatic stellate cells through the PVT1-miR-152-ATG14 signaling pathway

Molecular and Cellular Biochemistry - Shrm4 is a protein that is exclusively expressed in polarized tissues. The physiological function of Shrm4 in the brain was required to be elucidated. Thus, we...     

Regulation of neural differentiation by normal and mutant (G654A, amyloidogenic) gelsolin.

Gelsolin belongs to a family of proteins that modulate the structural dynamics of cytoskeletal actin. Gelsolin activity is required for the redistribution of actin occurring during membrane ruffling, cell crawling, and platelet activation. A point mutation (G654A) in the gelsolin gene causes a dominantly inherited systemic amyloidosis called familial amyloidosis of the Finnish type (FAF). This disease is characterized by a cranial neuropathy that cannot be explained solely by amyloid deposits. To address the question of whether gelsolin has a specific role in neural cell development, we transfected cDNA for wild type and G654A point-mutated gelsolin into a neural cell line, Paju, which can be induced to differentiate by treatment with phorbol 12-myristate 13-acetate. Overexpressed wild type gelsolin inhibited neural differentiation whereas mutated gelsolin did not, indicating that appropriate gelsolin activity is essential for neural sprouting. The G654A mutant gelsolin induced stabilization of F-actin and reduced the plasticity of neural development. This provides a novel etiopathogenetic mechanism for the neuronal dysfunction in FAF.     

The effect of the 540-kilodalton actin cross-linking protein, actin-binding protein, on the mechanical properties of F-actin

This study describes the effect of actin-binding protein derived from rabbit lung macrophages on the mechanical properties of F-actin. The dynamic storage modulus, G'(omega), and loss modulus, G"(omega) of F-actin, at concentrations from 1 to 4 mg/ml, in the absence or presence of actin-binding protein at molar ratios to actin of 1:1000 to 1:125, were measured at frequencies ranging from 3 X 10(-3) to 0.5 Hz. Actin-binding protein increased the dynamic moduli of F-actin, but this increase was much greater as either the actin-binding protein/actin ratio or the total protein concentration increased. Moreover, there was a convergence of the values of G' and G" at high frequencies for F-actin which became more prominent upon the addition of actin-binding protein. The value of the modulus obtained by an extrapolation of these data to actin concentrations similar to that found in the cell cortex was close to values which have been obtained by direct measurements. The addition of actin-binding protein to an F-actin solution enabled it to reach an equilibrium strain following the application of a stress, in contrast to pure F-actin. These data allow a more rigorous definition of the "sol" to "gel" transition and suggest that the cross-linking of actin filaments by actin-binding protein leads to the formation of a network structure whose underlying mechanism of mechanical behavior is short range intrafilament bending in contrast to the classical rubber network.     

Inhibition of "self" engulfment through deactivation of myosin-II at the phagocytic synapse between human cells

Phagocytosis of foreign cells or particles by macrophages is a rapid process that is inefficient when faced with "self" cells that display CD47-although signaling mechanisms in self-recognition have remained largely unknown. With human macrophages, we show the phagocytic synapse at cell contacts involves a basal level of actin-driven phagocytosis that, in the absence of species-specific CD47 signaling, is made more efficient by phospho-activated myosin. We use "foreign" sheep red blood cells (RBCs) together with CD47-blocked, antibody-opsonized human RBCs in order to visualize synaptic accumulation of phosphotyrosine, paxillin, F-actin, and the major motor isoform, nonmuscle myosin-IIA. When CD47 is functional, the macrophage counter-receptor and phosphatase-activator SIRPalpha localizes to the synapse, suppressing accumulation of phosphotyrosine and myosin without affecting F-actin. On both RBCs and microbeads, human CD47 potently inhibits phagocytosis as does direct inhibition of myosin. CD47-SIRPalpha interaction initiates a dephosphorylation cascade directed in part at phosphotyrosine in myosin. A point mutation turns off this motor's contribution to phagocytosis, suggesting that self-recognition inhibits contractile engulfment.     

Effects of two familial hypertrophic cardiomyopathy mutations in alpha-tropomyosin, Asp175Asn and Glu180Gly, on the thermal unfolding of actin-bound tropomyosin

Differential scanning calorimetry was used to investigate the thermal unfolding of native alpha-tropomyosin (Tm), wild-type alpha-Tm expressed in Escherichia coli and the wild-type alpha-Tm carrying either of two missense mutations associated with familial hypertrophic cardiomyopathy, D175N or E180G. Recombinant alpha-Tm was expressed with an N-terminal Ala-Ser extension to substitute for the essential N-terminal acetylation of the native Tm. Native and Ala-Ser-Tm were indistinguishable in our assays. In the absence of F-actin, the thermal unfolding of Tm was reversible and the heat sorption curve of Tm with Cys-190 reduced was decomposed into two separate calorimetric domains with maxima at approximately 42 and 51 degrees C. In the presence of phalloidin-stabilized F-actin, a new cooperative transition appears at 46-47 degrees C and completely disappears after the irreversible denaturation of F-actin. A good correlation was found to exist between the maximum of this peak and the temperature of half-maximal dissociation of the F-actin/Tm complex as determined by light scattering experiments. We conclude that Tm thermal denaturation only occurs upon its dissociation from F-actin. In the presence of F-actin, D175N alpha-Tm shows a melting profile and temperature dependence of dissociation from F-actin similar to those for wild-type alpha-Tm. The actin-induced stabilization of E180G alpha-Tm is significantly less than for wild-type alpha-Tm and D175N alpha-Tm, and this property could contribute to the more severe myopathy phenotype reported for this mutation.     

Conformational change in actin filament induced by the interaction with heavy meromyosin: effects of pH, tropomyosin and deoxy-ATP.

Conformational changes in pure and tropomyosin-containing F-actin during interaction with heavy meromyosin in the absence and presence of deoxy-ATP, were studied by measurements of the changes in fluorescence intensity of e-ADP² incorporated into the F-actin instead of ADP. The actin filaments were found to be stabilized by tropomyosin and were more stable at pH 7 than at pH 8. The rigor binding of HMM to F-actin caused an increase in the fluorescence intensity. The increase with F-actin containing TM was higher than that with pure F-actin at each HMM concentration. A linear relation between the fluoresence change and moles of HMM per actin was found regardless of the presence of TM, with a maximum value of 0.5 moles of HMM per actin. In the presence of deoxy-ATP, (which is a substrate for acto-HMM but cannot bind to actin) no changes in fluorescence intensity of e-ADP bound to pure F-actin were observed. In the case of F-actin containing TM, the fluorescence intensity increased with increasing HMM concentration, although the light scattering intensity of the acto-HMM solutions indicated that almost all the HMM was dissociated from the F-actin. This suggests that the conformational change in F-actin-TM induced by the interaction with HMM in the presence of deoxy-ATP has a long lifetime which continues for some time even after the detachment of the HMM.     

Myosin-induced movement of alphaalpha, alphabeta, and betabeta smooth muscle tropomyosin on actin observed by multisite FRET

The interaction of the alphaalpha, betabeta, and alphabeta smooth muscle tropomyosin (Tm) isoforms with F-actin was systematically studied in the absence and in the presence of myosin subfragment 1 (S1) using multifrequency phase/modulation F?rster resonance energy transfer (FRET). A Gaussian double distance distribution model was adopted to fit FRET data between a 5-(2-iodoacetyl-amino-ethyl-amino)naphthalene-1-sulfonic acid donor at either Cys-36 of the beta-chain or Cys-190 of the alpha-chain and a 4-dimethylaminophenylazophenyl 4'-maleimide acceptor at Cys-374 of F-actin. Experimental data were obtained for singly and doubly labeled alphabeta Tm (donor only at alpha, only at beta, or both) and for doubly labeled alphaalpha or betabeta Tm. Data for singly labeled alphabetaTm were combined in a global analysis with doubly labeled alphabetaTm. In all doubly labeled isoforms, upon S1 binding, one donor-acceptor "apparent" distance increased slightly by 0.5-2 A, whereas the other decreased by 6-9 A. These changes are consistent with a uniform "rolling" motion of Tm over the F-actin surface. The analysis indicates that Tm occupies relatively well-defined positions, with some flexibility, in both the predominantly closed (-S1) and open (+S1) thin-filament states. The results for the alphabetaTm heterodimer indicate that the local twofold symmetry of alphaalpha or betabeta Tm is effectively broken in alphabetaTm bound to F-actin, which implies a difference between the alpha- and beta-chains in terms of their interaction with F-actin.     

Replacement of threonine 558, a critical site of phosphorylation of moesin in vivo, with aspartate activates F-actin binding of moesin. Regulation by conformational change

Point and deletion mutants of moesin were examined for F-actin binding by blot overlay and co-sedimentation, and for intra- and intermolecular interactions with N- and C-terminal domains with yeast two-hybrid and in vitro binding assays. Wild-type moesin molecules interact poorly with F-actin and each other, and bind neither C- nor N-terminal fragments. Interaction with F-actin is strongly enhanced by replacement of Thr558 with aspartate (T558D), by deletion of 11 N-terminal residues (DelN11), by deletion of the entire N-terminal membrane-binding domain of both wild type and T558D mutant molecules, and by exposure to phosphatidylinositol 4, 5-diphosphate. Activation of F-actin binding is accompanied by changes in inter- and intramolecular domain interactions. The T558D mutation renders moesin capable of binding wild type but not mutated (T558D) C-terminal or wild type N-terminal fragments. The interaction between the latter two is prevented. DelN11 truncation enables binding of wild type N and C domain fragments. These changes suggest that the T558D mutation, mimicking phosphorylation of Thr558, promotes F-actin binding by disruption of interdomain interactions between N and C domains and exposure of the high affinity F-actin binding site in the C-terminal domain. Oscillation between activated and resting state could thus provide the structural basis for transient interactions between moesin and the actin cytoskeleton in protruding and retracting microextensions.     

Functional synergy of actin filament cross-linking proteins

The organization of filamentous actin (F-actin) in resilient networks is coordinated by various F-actin cross-linking proteins. The relative tolerance of cells to null mutations of genes that code for a single actin cross-linking protein suggests that the functions of those proteins are highly redundant. This apparent functional redundancy may, however, reflect the limited resolution of available assays in assessing the mechanical role of F-actin cross-linking/bundling proteins. Using reconstituted F-actin networks and rheological methods, we demonstrate how alpha-actinin and fascin, two F-actin cross-linking/bundling proteins that co-localize along stress fibers and in lamellipodia, could synergistically enhance the resilience of F-actin networks in vitro. These two proteins can generate microfilament arrays that "yield" at a strain amplitude that is much larger than each one of the proteins separately. F-actin/alpha-actinin/fascin networks display strain-induced hardening, whereby the network "stiffens" under shear deformations, a phenomenon that is non-existent in F-actin/fascin networks and much weaker in F-actin/alpha-actinin networks. Strain-hardening is further enhanced at high rates of deformation and high concentrations of actin cross-linking proteins. A simplified model suggests that the optimum results of the competition between the increased stiffness of bundles and their decreased density of cross-links. Our studies support a re-evaluation of the notion of functional redundancy among cytoskeletal regulatory proteins.     

beta-actinin, a regulatory protein of muscle. Purification, characterization and function.

beta-Actinin, a minor regulatory protein of muscle, was purified from skeletal muscles of rabbit and chicken by DEAE-Sephadex chromatography. beta-Actinin consisted of two subunits, beta I and betaII, with chain weights of 37,000 and 34,000 daltons, respectively. The amino acid compositions were similar, though not identical. It appears that each of the two subunits is associated in solution. beta-Actinin had the following effects on actin: (1) inhibition of reassociation of F-actin fragments; (2) inhibition of network formation of F-actin; (3) inhibition of growth of F-actin fragments; (4) retardation of depolymerization of F-actin and (5) acceleration of polymerization of G-actin. All these actions of beta-actinin can be explained in terms of action as an "ending factor". Experimental evidence favored the view that beta-actinin is bound to one end of the F-actin filament, namely to the end opposite to the direction of polymerization. Fluorescence-labeled anti-beta-actinin stained the middle portion of the A band of myofibrils. Based on the finding that the stain was unchanged on removal of myosin, it is suggested that beta-actinin is located at the free ends of the I filaments of myofibrils. Thus is seems likely that beta-actinin functions as an ending factor for actin filaments.     

Myosin Va bound to phagosomes binds to F-actin and delays microtubule-dependent motility

We established a light microscopy-based assay that reconstitutes the binding of phagosomes purified from mouse macrophages to preassembled F-actin in vitro. Both endogenous myosin Va from mouse macrophages and exogenous myosin Va from chicken brain stimulated the phagosome-F-actin interaction. Myosin Va association with phagosomes correlated with their ability to bind F-actin in an ATP-regulated manner and antibodies to myosin Va specifically blocked the ATP-sensitive phagosome binding to F-actin. The uptake and retrograde transport of phagosomes from the periphery to the center of cells in bone marrow macrophages was observed in both normal mice and mice homozygous for the dilute-lethal spontaneous mutation (myosin Va null). However, in dilute-lethal macrophages the accumulation of phagosomes in the perinuclear region occurred twofold faster than in normal macrophages. Motion analysis revealed saltatory phagosome movement with temporarily reversed direction in normal macrophages, whereas almost no reversals in direction were observed in dilute-lethal macrophages. These observations demonstrate that myosin Va mediates phagosome binding to F-actin, resulting in a delay in microtubule-dependent retrograde phagosome movement toward the cell center. We propose an "antagonistic/cooperative mechanism" to explain the saltatory phagosome movement toward the cell center in normal macrophages.     

Fission yeast Mor2/Cps12, a protein similar to Drosophila Furry,is essential for cell morphogenesis and its mutation induces Wee1-dependent G(2) delay

Fission yeast cells identify growing regions at the opposite ends of the cell, producing the rod-like shape. The positioning of the growth zone(s) and the polarized growth require CLIP170-like protein Tip1 and the Ndr kinase Orb6, respectively. Here, we show that the mor2/cps12 mutation disrupts the localization of F-actin at the cell ends, producing spherical cells and concomitantly inducing a G(2) delay at 36 degrees C. Mor2 is important for the localization of F-actin at the cell end(s) but not at the medial region, and is essential for the restriction of the growth zone(s) where Tip1 targets. Mor2 is homologous to the Drosophila Furry protein, which is required to maintain the integrity of cellular extensions, and is localized at both cell ends and the medial region of the cell in an actin-dependent fashion. Cellular localization of Mor2 and Orb6 was interdependent. The tyrosine kinase Wee1 is necessary for the G(2) delay and maintenance of viability of the mor2 mutant. These results indicate that Mor2 plays an essential role in cell morphogenesis in concert with Orb6, and the mutation activates the mechanism coordinating morphogenesis with cell cycle progression.     

Moesin, ezrin, and p205 are actin-binding proteins associated with neutrophil plasma membranes.

Actin-binding proteins in bovine neutrophil plasma membranes were identified using blot overlays with 125I-labeled F-actin. Along with surface-biotinylated proteins, membranes were enriched in major actin-binding polypeptides of 78, 81, and 205 kDa. Binding was specific for F-actin because G-actin did not bind. Further, unlabeled F-actin blocked the binding of 125I-labeled F-actin whereas other acidic biopolymers were relatively ineffective. Binding also was specifically inhibited by myosin subfragment 1, but not by CapZ or plasma gelsolin, suggesting that the membrane proteins, like myosin, bind along the sides of the actin filaments. The 78- and 81-kDa polypeptides were identified as moesin and ezrin, respectively, by co-migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoprecipitation with antibodies specific for moesin and ezrin. Although not present in detectable amounts in bovine neutrophils, radixin (a third and closely related member of this gene family) also bound 125I-labeled F-actin on blot overlays. Experiments with full-length and truncated bacterial fusion proteins localized the actin-binding site in moesin to the extreme carboxy terminus, a highly conserved sequence. Immunofluorescence micrographs of permeabilized cells and cell "footprints" showed moesin co-localization with actin at the cytoplasmic surface of the plasma membrane, consistent with a role as a membrane-actin-linking protein.     

The kinetics of chemotactic peptide-induced change in F-actin content, F-actin distribution, and the shape of neutrophils

Formyl-met-leu-phe (fMLP) induces actin assembly in neutrophils; the resultant increase in F-actin content correlates with an increase in the rate of cellular locomotion at fMLP concentrations less than or equal to 10(-8) M (Howard, T.H., and W.H. Meyer, 1984, J. Cell Biol., 98:1265-1271). We studied the time course of change in F-actin content, F-actin distribution, and cell shape after fMLP stimulation. F-actin content was quantified by fluorescence activated cell sorter analysis of nitrobenzoxadiazole-phallacidin-stained cells (Howard, T.H., 1982, J. Cell Biol., 95(2, Pt. 2:327a). F-actin distribution and cell shape were determined by analysis of fluorescence photomicrographs of nitrobenzoxadiazole-phallacidin-stained cells. After fMLP stimulation at 25 degrees C, there is a rapid actin polymerization that is maximal (up to 2.0 times the control level) at 45 s; subsequently, the F-actin depolymerizes to an intermediate F-actin content 5-10 min after stimulation. The depolymerization of F-actin reflects a true decrease in F-actin content since the quantity of probe extractable from cells also decreases between 45 s and 10 min. The rate of actin polymerization (3.8 +/- 0.3-4.4 +/- 0.6% increase in F-actin/s) is the same for 10(-10) - 10(-6) M fMLP and the polymerization is inhibited by cytochalasin D. The initial rate of F-actin depolymerization (6.0 +/- 1.0-30 +/- 5% decrease in F-actin/min) is inversely proportional to fMLP dose. The F-actin content of stimulated cells at 45 s and 10 min is greater than control levels and varies directly with fMLP dose. F-actin distribution and cell shape also vary as a function of time after stimulation. 45 s after stimulation the cells are rounded and F-actin is diffusely distributed; 10 min after stimulation the cell is polarized and F-actin is focally distributed. These results indicate that actin polymerization and depolymerization follow fMLP stimulation in sequence, the rate of depolymerization and the maximum and steady state F-actin content but not the rate of polymerization are fMLP dose dependent, and concurrent with F-actin depolymerization, F-actin is redistributed and the cell changes shape.     

UNC-60B, an ADF/cofilin family protein, is required for proper assembly of actin into myofibrils in Caenorhabditis elegans body wall muscle

The Caenorhabditis elegans unc-60 gene encodes two functionally distinct isoforms of ADF/cofilin that are implicated in myofibril assembly. Here, we show that one of the gene products, UNC-60B, is specifically required for proper assembly of actin into myofibrils. We found that all homozygous viable unc-60 mutations resided in the unc-60B coding region, indicating that UNC-60B is responsible for the Unc-60 phenotype. Wild-type UNC-60B had F-actin binding, partial actin depolymerizing, and weak F-actin severing activities in vitro. However, mutations in UNC-60B caused various alterations in these activities. Three missense mutations resulted in weaker F-actin binding and actin depolymerizing activities and complete loss of severing activity. The r398 mutation truncated three residues from the COOH terminus and resulted in the loss of severing activity and greater actin depolymerizing activity. The s1307 mutation in a putative actin-binding helix caused greater activity in actin-depolymerizing and severing. Using a specific antibody for UNC-60B, we found varying protein levels of UNC-60B in mutant animals, and that UNC-60B was expressed in embryonic muscles. Regardless of these various molecular phenotypes, actin was not properly assembled into embryonic myofibrils in all unc-60 mutants to similar extents. We conclude that precise control of actin filament dynamics by UNC-60B is required for proper integration of actin into myofibrils.     

Arabidopsis RIC1 Severs Actin Filaments at the Apex to Regulate Pollen Tube Growth

Pollen tubes deliver sperms to the ovule for fertilization via tip growth. The rapid turnover of F-actin in pollen tube tips plays an important role in this process. In this study, we demonstrate that Arabidopsis thaliana RIC1, a member of the ROP-interactive CRIB motif-containing protein family, regulates pollen tube growth via its F-actin severing activity. Knockout of RIC1 enhanced pollen tube elongation, while overexpression of RIC1 dramatically reduced tube growth. Pharmacological analysis indicated that RIC1 affected F-actin dynamics in pollen tubes. In vitro biochemical assays revealed that RIC1 directly bound and severed F-actin in the presence of Ca²⁺ in addition to interfering with F-actin turnover by capping F-actin at the barbed ends. In vivo, RIC1 localized primarily to the apical plasma membrane (PM) of pollen tubes. The level of RIC1 at the apical PM oscillated during pollen tube growth. The frequency of F-actin severing at the apex was notably decreased in ric1-1 pollen tubes but was increased in pollen tubes overexpressing RIC1. We propose that RIC1 regulates F-actin dynamics at the apical PM as well as the cytosol by severing F-actin and capping the barbed ends in the cytoplasm, establishing a novel mechanism that underlies the regulation of pollen tube growth.     

Effect of denervation, reinnervation and hypertrophy on the state of actin filaments in skeletal muscle fibres

The conformational state of actin filaments was studied in the rat soleus muscle atrophying after denervation, recovering following reinnervation, hypertrophying following tenotomy of synergists and in intact muscle. Intrinsic (tryptophan residues of F-actin) and extrinsic (rhodamine-phalloidin or 1,5-IAEDANS attached to F-actin) polarized fluorescence was measured. In parallel, the influence of ATP or NEM on the state of F-actin was studied. The results show that the conformational state of F-actin is changed in all experimental muscles. These changes of the denervated muscle differ from those of the reinnervated and hypertrophying muscles. In the reinnervated muscle, beginning with the first days of recovery, the structure of F-actin seems to "recover" to the state in intact muscle. In the later stage of muscle recovery, the state of F-actin is similar to that in hypertrophying muscle. Differences between the mentioned muscles in the conformational state of actin monomers, in the orientation of monomers and in the flexibility of thin filaments are discussed.