A new twist on tropomyosin binding to actin filaments: perspectives on thin filament function,assembly and biomechanics

Tropomyosin, best known for its role in the steric regulation of muscle contraction, polymerizes head-to-tail to form cables localized along the length of both muscle and non-muscle actin-based thin filaments. In skeletal and cardiac muscles, tropomyosin, under the control of troponin and myosin, moves in a cooperative manner between blocked, closed and open positions on filaments, thereby masking and exposing actin-binding sites necessary for myosin crossbridge head interactions. While the coiled-coil signature of tropomyosin appears to be simple, closer inspection reveals surprising structural complexity required to perform its role in steric regulation. For example, component α-helices of coiled coils are typically zippered together along a continuous core hydrophobic stripe. Tropomyosin, however, contains a number of anomalous, functionally controversial, core amino acid residues. We argue that the atypical residues at this interface, including clusters of alanines and a charged aspartate, are required for preshaping tropomyosin to readily fit to the surface of the actin filament, but do so without compromising tropomyosin rigidity once the filament is assembled. Indeed, persistence length measurements of tropomyosin are characteristic of a semi-rigid cable, in this case conducive to cooperative movement on thin filaments. In addition, we also maintain that tropomyosin displays largely unrecognized and residue-specific torsional variance, which is involved in optimizing contacts between actin and tropomyosin on the assembled thin filament. Corresponding twist-induced stiffness may also enhance cooperative translocation of tropomyosin across actin filaments. We conclude that anomalous core residues of tropomyosin facilitate thin filament regulatory behavior in a multifaceted way.

     

The thin filaments of smooth muscles

Summary Contraction in vertebrate smooth and striated muscles results from the interaction of the actin filaments with crossbridges arising from the myosin filaments. The functions of the actin based thin filaments are (1) interaction with myosin to produce force; (2) regulation of force generation in response to Ca²⁺ concentration; and (3) transmission of the force to the ends of the cell. The major protein components of smooth muscle thin filaments are actin, tropomyosin and caldesmon, present in molar ratios of 28:4:1 respectively. Other smooth muscle proteins which may be associated with the thin filaments in the cell are filamin, vinculin, -actinin, myosin light chain kinase and calmodulin. We have reviewed the structural and functional properties of these proteins and where possible we have suggested what their function and mechanism of action may be. We propose that actin and tropomyosin are involved in the force producing interaction with myosin, and that this interaction is controlled by a Ca²⁺-dependent mechanism involving caldesmon, tropomyosin and calmodulin. Vinculin, -actinin and filamin appear to be involved in the attachment of the thin filaments to the cell membrane and their spatial organization within the cell. We conclude that the filaments of smooth muscles share many common properties with those from skeletal muscle, but that they are also quite distinct in terms of both their caldesmon based regulatory mechanism and their mode of organization into a contractile apparatus.     

Filamin and gelsolin influence Ca2+-sensitivity of smooth muscle thin filaments

Summary Sheep aorta thin filaments were prepared by ultracentrifugation of an ATP-containing extract in the presence of different concentrations of ethanediol. Thin filaments prepared without ethanediol contained small quantities of tropomyosin (0.027 Tm:actin) and caldesmon (0.017 CD:actin) and activated the MgATPase of skeletal myosin independently of Ca²⁺. Ultracentrifugation in the presence of 10–20% ethanediol resulted in preparation of thin filaments with increased content of tropomyosin (0.17 Tm:actin) and caldesmon (0.04 CD:actin). These thin filaments possessed high Ca²⁺-sensitivity in activation of skeletal muscle myosin ATPase. Besides actin, tropomyosin and caldesmon, thin filaments contained gelsolin and filamin. Gelsolin content (0.007 gelsolin:actin) was independent of the presence of ethanediol. The filamin content decreased from 0.015 to 0.007 mol:mol actin when the ethanediol concentration was increased from 0 to 20%, and was negatively correlated with the Ca²⁺ sensitivity of thin filaments. In a reconstituted system, pure filamin or gelsolin affected caldesmon regulation of actomyosin ATPase. Gelsolin (0.01:actin) reduced the inhibition of actomyosin ATPase caused by caldesmon and increased the potency of Ca²⁺-calmodulin in reversing this inhibition. Filamin (0.007:actin) also decreased the inhibitory action of caldesmon on actin-activated myosin ATPase and also potentiated the reversal of this inhibition by calmodulin. We conclude that minor components of smooth muscle thin filaments (gelsolin and filamin) significantly modify caldesmon mediated regulation of actomyosin ATPase. We suggest a tropomyosin-mediated mechanism by which filamin or gelsolin could exert similar effects.     

Structural interactions between actin,tropomyosin, caldesmon and calcium binding protein and the regulation of smooth muscle thin filaments

The basic structure and functional properties of smooth muscle thin filaments were established about 10 years ago. Since then we and others have been working on the details of how tropomyosin, caldesmon and the Ca²⁺-binding protein regulate actin interaction with myosin. Our work has tended to emphasize the similarities between caldesmon and troponin function whilst others have been more concerned with the differences. The need to resolve the resulting differences has stimulated us to find new and more direct ways of investigating the mechanism of thin filament regulation. In recent years an apparent divergence has opened up between functional measurements, which indicate an allosteric-cooperative regulatory mechanism in which caldesmon and Ca²⁺-binding protein control actin—tropomyosin state in the same way as troponin, and structural measurements which show thin filament structures unlike striated muscle thin filaments. The challenge is to interpret function in terms of structure. We have combined functional studies with expression and mutagenesis of caldesmon and with structural methods including X-ray crystalography of tropomyosin—caldesmon crystals, electron microscopy and helical reconstruction of actin—tropomyosincaldesmon complexes and high resolution nuclear magnetic resonance spectroscopy of the C-terminus of caldesmon in interaction with actin and calmodulin. We have used this information to propose a structural mechanism for caldesmon regulation of the smooth muscle thin filament.     

Use of thin filament reconstituted muscle fibres to probe the mechanism of force generation

The technique of selective removal of the thin filament by gelsolin in bovine cardiac muscle fibres, and reconstitution of the thin filament from isolated proteins is reviewed, and papers that used reconstituted preparations are discussed. By comparing the results obtained in the absence/presence of regulatory proteins tropomyosin (Tm) and troponin (Tn), it is concluded that the role of Tm and Tn in force generation is not only to expose the binding site of actin to myosin, but also to modify actin for better stereospecific and hydrophobic interaction with myosin. This conclusion is further supported by experiments that used a truncated Tm mutant and the temperature study of reconstituted fibres. The conclusion is consistent with the hypothesis that there are three states in the thin filament: blocked state, closed state, and open state. Tm is the major player to produce these effects, with Tn playing the role of Ca²⁺ sensing and signal transmission mechanism. Experiments that changed the number of negative charges at the N-terminal finger of actin demonstrates that this part of actin is essential to promote the strong interaction between actin and myosin molecules, in addition to the well-known weak interaction that positions the myosin head at the active site of actin prior to force generation.     

Purification and properties of Ca2+-regulated thin filaments and F-actin from sheep aorta smooth muscle

Summary We have investigated the conditions for isolation of Ca²⁺-regulated thin filaments from sheep aorta. Inhibition of proteolysis by 2 µg ml^–1 leupeptin and chymostatin and of oxidation with 5mm dithiothreitol were essential. Washed homogenates were extracted in 10mm ATP of low ionic strength at pH 6.1 to minimize coextraction of myosin with thin filaments. Thin filaments were separated from myosin by high speed sedimentation; 20% glycol was added to prevent loss of regulatory factors and tropomyosin. The resulting thin filaments (yield 2.5 mg protein g^–1 artery wet weight) were made up of actin, tropomyosin and a 120 000M _r protein (molar ratio 1:1/5:1/29) and were up to 4 µm long. They activated skeletal muscle myosin at least 50 times in presence of Ca²⁺. Up to 80% inhibition was observed in the absence of Ca²⁺. We also prepared pure arterial F-actin, which activated skeletal myosin more than the thin filaments, but was similar to skeletal F-actin. We conclude that Ca²⁺ regulation is negative, involves cooperative interactions between actin, myosin and tropomyosin and suggest that it is mediated by the 120 000M _r protein.     

Effect of unphosphorylated smooth muscle myosin on caldesmon-mediated regulation of actin filament velocity

Summary The effect of smooth muscle myosin at different levels of light chain phosphorylation on caldesmon-mediated movement of actin filaments was investigated using an in vitro motility assay. Myosin at different levels of phosphorylation was obtained by mixing different proportions of fully phosphorylated and unphosphorylated myosin in monomeric form, while keeping the total myosin concentration constant. The average velocity of actin filaments containing tropomyosin was 1.20±0.046 m s^–1 at 30°C with fully phosphorylated myosin. This velocity was not altered when the percentage of unphosphorylated myosin coated on the nitrocellulose surface was increased to 80%; further increases lowered the velocity. When the actin filaments with caldesmon bound at stoichiometric levels were used, filament velocity was unaffected until 50% of the myosin was unphosphorylated, but further increases in the percentage of unphosphorylated myosin induced a decrease in the velocity, and at 95% unphosphorylated myosin, filament movement had ceased. The decreased filament velocity in the presence of caldesmon was also observed when phosphorylated myosin was mixed with myosin rod instead of unphosphorylated myosin, but was not observed when the 38 kDa caldesmon C-terminal fragment, which lacks the myosin-binding domain, was used instead of intact caldesmon. These data indicate that the decreased filament velocity in the presence of caldesmon reflects the mechanical load produced by the tethering of actin to myosin through the interaction of the caldesmon N-terminal domain and the myosin S-2 region. The tethering effect mediated by caldesmon may play a role in smooth muscle contraction when a large number of myosin heads are dephosphorylated, as in force maintenance.     

Properties of tropomyosin from the dual-regulated obliquely striated body wall muscle of the earthworm (Lumbricus terrestris L.)

Summary The obliquely straited body wall muscle of the earthwormLumbricus terrestris L. possesses a dual actin-linked and myosin-linked regulatory system. Tropomyosin from this muscle has now been purified and its functional properties compared to tropomyosin from vertebrate skeletal muscle.Earthworm tropomyosin has a molecular weight of about 70 000 and is composed of two polypeptide chains of molecular weight of 34 000 and 37 000. Structural and functional similarities to skeletal muscle tropomyosin were demonstrated with respect to the formation and periodicity of paracrystals and nets and the potentiation of skeletal muscle acto-SF1 ATPase activity at low ATP concentration. Likewise, earthworm tropomyosin inhibited skeletal muscle acto-HMM ATPase activity at normal ATP concentrations but to a much greater extent than skeletal muscle tropomyosin; this inhibition was removed by skeletal muscle troponin, in the presence of Ca²⁺. In a system containing earthworm myosin and skeletal muscle actin, earthworm tropomyosin had no detectable influence on the actin-activated ATPase activity. It is concluded that earthworm tropomyosin plays an active role in the actin-linked troponin-dependent regulatory system and has no measurable effect on the regulation via myosin.     

Structure of myosin decorated actin filaments and natural thin filaments

Summary Negatively stained paracrystals of reconstituted thin filaments decorated with myosin subfragment 1 (S1), at high calcium concentrations (10^–5 m), exhibitpgg plane group symmetry with component filaments having 28 subunits in 13 turns of the actin genetic helix. Isolated S1 decorated F-actin filaments trapped in a stain film were also observed to form spontaneously paracrystals withpgg plane group symmetry. We conclude that a favourable S1-S1 interaction must exist in order to stabilize these structures. Three-dimensional helical reconstructions, calculated from these paracrystals show S1 to be curved, 12 to 14 nm long and tilted with respect to the helical axis, in broad agreement with previous reconstructions calculated from isolated particles. Reconstructions of S1 and HMM decorated filaments that resolve actin show a principal myosin binding site located on the side of the actin subunit reported by Taylor & Amos [J. molec. Biol. 147, 297–324 (1981)] and a possible small interaction on the opposite side.The appearance, symmetry and helical reconstructions of iolated F-actin filaments decorated with heavy meromyosin (HMM) were similar to those of S1 decorated filaments, except at high radii where extra mass was observed. This probably arose from the connection between the two heads of HMM bound to the same long-pitch strand of actin.In contrast to most studies on thin filaments, which use reconstituted filaments, we present data on natural I-segments of muscle homogenates. Individual filaments exhibited actin helical symmetry which on reconstruction gave a two-domain motif oriented constitently with its long axis approximately perpendicular to the helical axis, but inclined towards the 5.9 nm genetic helix. Our original interpretation of these maps [Seymour & O'Brien,Nature, Lond. 283, 680–2 (1980)] depended upon reconstructions from F-actin paracrystals, which suggested actin was rather symmetrical in shape. New data from tw- and three-dimensional crystal studies and reconstructions of actin-tropomyosin filaments show that actin is rather elongated and consists of two domains. These results indicate that actin contributes towards both domains of our I-segment motif and are consistent with the monomer long axis lying approximately perpendicular to the helical axis. Although tropomyosin is not resolved, comparison of the actin-tropomyosin and I-segment reconstructions suggests that tropomyosin is strongly merged with the actin domain at a lower radius from the helical axis and that the domain at higher radius arises solely from actin.     

Complex stiffness of smooth muscle cytoplasm in the presence of Ca-activated brevin

Summary Brevin, an F-actin severing protein, regulates actin gel-sol transformation in a Ca²⁺-dependent way. Here, we tested its effect on the stiffness of the cytoplasm of skinned smooth muscle, in the absence of actin-myosin interaction (inhibited myosin ATPase). Complex stiffness was measured by imposing sinusoidal stretches and releases at different frequencies (1–50 Hz). In the presence of Ca-activated brevin, the stiffness decreased by about 30%, at all frequencies, from its initial values in Ca-free, relaxing solution. This decrease reflected a fall in both elasticity and viscosity of the cytoplasm. We propose that brevin specifically operates on an actin network in parallel with the contractile apparatus, e.g. on the actin-filamin gel.This paper is dedicated to the memory of the late Dr Ichiro Matsubara.     

A novel Ca2+ binding protein associated with caldesmon in Ca2+-regulated smooth muscle thin filaments: evidence for a structurally altered form of calmodulin

Smooth muscle thin filaments are made up of actin, tropomyosin, the inhibitory protein caldesmon and a Ca²⁺-binding protein. Thin filament activation of myosin MgATPase is Ca²⁺-regulated but thin filaments assembled from smooth muscle actin, tropomyosin and caldesmon plus brain or aorta calmodulin are not Ca²⁺-regulated at 25°C/50 mM KCl. We isolated the Ca²⁺-binding protein (CaBP) from smooth muscle thin filaments by DEAE fast-flow chromatography in 6 M urea and phenyl sepharose chromatography using sheep aorta as our starting material. CaBP combines with smooth muscle actin, tropomyosin and caldesmon to reconstitute a normally regulated thin filament at 25°C/50 mM KCl. It reverses caldesmon inhibition at pCa5 under conditions where CaM is largely inactive, it binds to caldesmon when complexed with actin and tropomyosin rather than displacing it and it binds to caldesmon independently of [Ca²⁺]. Amino acid sequencing, and electrospray mass spectrometry show the CaBP is identical to CaM. Structural probes indicate it is different: calmodulin increases caldesmon tryptophan fluorescence but CaBP does not. The distribution of charged species in electrospray mass spectrometry and nozzle skimmer fragmentation patterns are different indicating a less stable N-terminal lobe for CaBP. Brief heating abolishes these special properties of the CaBP. Mass spectrometry in aqueous buffer showed no evidence for the presence of any covalent or non-covalently bound adduct. The only remaining conclusion is that CaBP is calmodulin locked in a metastable altered state.     

On the mechanics of the actin filament: the linear relationship between stiffness and yield strength allows estimation of the yield strength of thin filament in vivo

Comparison of the behaviour of actin filaments either modified with tetramethylrhodamine iodoacetamide or decorated with tetramethylrhodamine-phalloidin or with tropomyosin or with myosin subfragment 1 shows that, in all the cases, yield strength is linearly related to stiffness.     

Disease causing mutations of troponin alter regulated actin state distributions

Striated muscle contraction is regulated primarily through the action of tropomyosin and troponin that are bound to actin. Activation requires Ca²⁺ binding to troponin and/or binding of high affinity myosin complexes to actin. Mutations within components of the regulatory complex may lead to familial cardiomyopathies and myopathies. In several cases examined, either physiological or pathological changes in troponin alter the distribution among states of actin?Ctropomyosin?Ctroponin that differ in their abilities to stimulate myosin ATPase activity. These observations open possibilities for managing disorders of the troponin complex. Furthermore, analyses of mutant forms of troponin give insights into the regulation of striated muscle contraction.     

Fesselin binds to actin and myosin and inhibits actin-activated ATPase activity

Fesselin is an actin binding protein that bundles actin filaments and accelerates nucleation of actin polymerization. The effect of fesselin on actin polymerization is regulated by Ca⁺⁺-calmodulin. Because actin filaments serve both structural and contractile functions we also examined the effect of fesselin on activation of myosin S1 ATPase activity. Fesselin inhibited the activation of S1-catalyzed ATP hydrolysis in a similar manner in both the presence and absence of tropomyosin. This inhibition was unaffected by Ca⁺⁺-calmodulin. Fesselin inhibited the binding of myosin-S1 to actin during steady-state ATP hydrolysis. Fesselin also displaced caldesmon from actin. S1 displaced fesselin from actin in the absence of nucleotide when the affinity of S1 for actin was much greater than the affinity of fesselin for actin. It is likely that fesselin and S1 share common binding sites on F-actin. We also observed that fesselin could bind to smooth muscle myosin with μM affinity. Fesselin shares some similarities to caldesmon in binding to several other proteins and having multiple potential functions. Parts of this work were presented in preliminary form at the 45th Annual Biophysical Society Meeting, Baltimore, MD, February 2004 and the 46th Annual Meeting, Long Beach, CA, February 2005.     

Myosin light chain kinase: functional domains and structural motifs

Conventional myosin light chain kinase found in differentiated smooth and non-muscle cells is a dedicated Ca²⁺/calmodulin-dependent protein kinase which phosphorylates the regulatory light chain of myosin II. This phosphorylation increases the actin-activated myosin ATPase activity and is thought to play major roles in a number of biological processes, including smooth muscle contraction. The catalytic domain contains residues on its surface that bind a regulatory segment resulting in autoinhibition through an intrasteric mechanism. When Ca²⁺/calmodulin binds, there is a marked displacement of the regulatory segment from the catalytic cleft allowing phosphorylation of myosin regulatory light chain. Kinase activity depends upon Ca²⁺/calmodulin binding not only to the canonical calmodulin-binding sequence but also to additional interactions between Ca²⁺/calmodulin and the catalytic core. Previous biochemical evidence shows myosin light chain kinase binds tightly to actomyosin containing filaments. The kinase has low-affinity myosin and actin binding sites in Ig-like motifs at the N- and C-terminus, respectively. Recent results show the N-terminus of myosin light chain kinase is responsible for filament binding in vivo. However, the apparent binding affinity is greater for smooth muscle myofilaments, purified thin filaments, or actin-containing filaments in permeable cells than for purified smooth muscle F-actin or actomyosin filaments from skeletal muscle. These results suggest a protein on actin thin filaments that may facilitate kinase binding. Myosin light chain kinase does not dissociate from filaments in the presence of Ca²⁺/calmodulin raising the interesting question as to how the kinase phosphorylates myosin in thick filaments if it is bound to actin-containing thin filaments.     

Nebulin: big protein with big responsibilities

Nebulin, encoded by NEB, is a giant skeletal muscle protein of about 6669 amino acids which forms an integral part of the sarcomeric thin filament. In recent years, the nebula around this protein has been largely lifted resulting in the discovery that nebulin is critical for a number of tasks in skeletal muscle. In this review, we firstly discussed nebulin’s role as a structural component of the thin filament and the Z-disk, regulating the length and the mechanical properties of the thin filament as well as providing stability to myofibrils by interacting with structural proteins within the Z-disk. Secondly, we reviewed nebulin’s involvement in the regulation of muscle contraction, cross-bridge cycling kinetics, Ca²⁺-homeostasis and excitation contraction (EC) coupling. While its role in Ca²⁺-homeostasis and EC coupling is still poorly understood, a large number of studies have helped to improve our knowledge on how nebulin affects skeletal muscle contractile mechanics. These studies suggest that nebulin affects the number of force generating actin-myosin cross-bridges and may also affect the force that each cross-bridge produces. It may exert this effect by interacting directly with actin and myosin and/or indirectly by potentially changing the localisation and function of the regulatory complex (troponin and tropomyosin). Besides unravelling the biology of nebulin, these studies are particularly helpful in understanding the patho-mechanism of myopathies caused by NEB mutations, providing knowledge which constitutes the critical first step towards the development of therapeutic interventions. Currently, effective treatments are not available, although a number of therapeutic strategies are being investigated.

     

Tropomyosin dynamics

Tropomyosin is a two chained α-helical coiled coil protein that binds actin filaments and interacts with various actin binding proteins. Tropomyosin function depends on its ability to move to distinct locations on the surface of actin in response to the binding of different thin filament effectors. Tropomyosin dynamics plays an important role in these fluctuating interactions with actin and is thought to be fundamental to many of its biological activities. For example tropomyosin concerted movement on the surface of actin triggered by Ca²⁺ binding to troponin or myosin head binding to actin has been argued to be key to the cooperative allosteric regulation of muscle contraction. These large-scale motions are affected by tropomyosin internal dynamics and mechanical properties. Tropomyosin internal dynamics corresponding to smaller and more localised structural fluctuations are increasingly recognised to play an important role in its function. A thorough understanding of the coupling between local and global structural fluctuations in tropomyosin is required to understand how time dependent structural fluctuations in tropomyosin contribute to the overall thin filament dynamics and dictate their various biological activities.     

Regulation of Structure and Function of Sarcomeric Actin Filaments in Striated Muscle of the Nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans has been used as a valuable system to study structure and function of striated muscle. The body wall muscle of C. elegans is obliquely striated muscle with highly organized sarcomeric assembly of actin, myosin, and other accessory proteins. Genetic and molecular biological studies in C. elegans have identified a number of genes encoding structural and regulatory components for the muscle contractile apparatuses, and many of them have counterparts in mammalian cardiac and skeletal muscles or striated muscles in other invertebrates. Applicability of genetics, cell biology, and biochemistry has made C. elegans an excellent system to study mechanisms of muscle contractility and assembly and maintenance of myofibrils. This review focuses on the regulatory mechanisms of structure and function of actin filaments in the C. elegans body wall muscle. Sarcomeric actin filaments in C. elegans muscle are associated with the troponin–tropomyosin system that regulates the actin–myosin interaction. Proteins that bind to the side and ends of actin filaments support ordered assembly of thin filaments. Furthermore, regulators of actin dynamics play important roles in initial assembly, growth, and maintenance of sarcomeres. The knowledge acquired in C. elegans can serve as bases to understand the basic mechanisms of muscle structure and function. Anat Rec, 297:1548–1559, 2014. © 2014 Wiley Periodicals, Inc.     

Alpha-tropomyosin mutations in inherited cardiomyopathies

The inherited cardiac diseases hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) can both be caused by missense mutations in the TPM1 gene which encodes the thin filament regulatory protein α-tropomyosin. Different mutations are responsible for either HCM or DCM, suggesting that distinct changes in tropomyosin structure and function can lead to the different diseases. Various biophysical and physiological approaches have been used to investigate the structure–function effects of the mutations, and animal models developed. The reported effects of the mutations include changes to the secondary structure of tropomyosin, its binding to actin and its position on the thin filament, and alterations to actin–myosin interactions and myofilament Ca²⁺ sensitivity. The latter changes have been found to be particularly consistent, with HCM mutations increasing Ca²⁺ sensitivity and DCM mutations in general decreasing this parameter and uncoupling the effect of troponin phosphorylation upon Ca²⁺ responsiveness. As well as impacting on contractility, these changes are likely to alter intracellular Ca²⁺ handling and signaling, and a combination of these alterations may provide the trigger for disease remodeling.     

Contractile units in stress fibers of fetal human astroglia in tissue culture

The interaction between myosin and F-actin requires the enzyme, myosin light chain kinase (MLCK), as well as Ca2⁺-calmodulin and the calmodulin binding protein, caldesmon, which also binds to F-actin. Using immunofluorescence staining, we have demonstrated that in human fetal astroglia as in mouse astroglia (Abd-El-Basset et al., 1991) the stress fibers contain these contractile elements: F-actin, myosin, tropomyosin and caldesmon. F-actin extends continuously along the stress fibers, whereas myosin, tropomyosin and caldesmon are localized discontinuously in a periodic pattern. In addition, we have demonstrated that fetal human astroglia have the enzyme MLCK and calmodulin. The association of the contractile elements listed above together with calmodulin and MLCK constitutes what may be termed ‘contractile units’, suggesting that the stress fibers in astroglia may be contractile. Contractile stress fibers would enable astroglia to exert tension on the matrix surrounding them, thus facilitating rapid changes in cell shape.