Search

The molecular mechanism of processive movement of single myosin molecules from classes V and VI along their actin tracks has recently attracted extraordinary attention. Another member of the myosin superfamily, myosin VII, plays vital roles in the sensory function of Drosophila and mammals. We studied the molecular mechanism of Drosophila myosin VIla, using transient kinetics and single-molecule motility assays. Myosin Vlla moves along actin filaments as a processive, double-headed single molecule when dimerized by the inclusion of a leucine zipper at the C terminus of the coiled-coil domain. Its motility is [approximately equal to] 8-10 times slower than that of myosin V, and its step size is 30 nm, which is consistent with the presence of five IQ motifs in its neck region. The kinetic basis for the processive motility of myosin Vlla is the relative magnitude of the release rate constants of phosphate (fast) and ADP (slow) as in myosins V and VI. The ATPase pathway is rate- limited by a reversible interconversion between two distinct ADP-bound actomyosin states, which results in high steady-state occupancy of a strongly actin-bound myosin species. The distinctive features of myosin Vlla (long run lengths, slow motility) will be very useful in video-based single-molecule applications. In cells, this kinetic behavior would allow myosin Vlla to exert and hold tension on actin filaments and, if dimerized, to function as a processive cargo transporter. fluorescence imaging with one-nanometer accuracy | kinetics | processivity

Like all mucosal surfaces, the intestine forms a barrier that separates the external environment, i.e., the gut lumen, from the protected internal milieu. The intestinal barrier is formed by the epithelial cells that line the luminal surface. Plasma membranes of these cells prevent free passage of hydrophilic molecules across this barrier but do not seal the space between cells. This function is provided by the tight junction. Each cell is encircled at the apicolateral boundary by the tight junction, which seals the paracellular space. The tight junction does not form a completely imperlneant seal, however, because that would prevent paracellular absorption of essential nutrients and ions; intestinal tight junctions are 'leaky' and allow solutes to be transported paracellularly according to size and charge. Abundant data are available to demonstrate that barrier properties of tight junctions can be modulated in response to physiological, pharmacological, and pathophysiological stimuli, but the structural modifications responsible for these responses are poorly defined. Recent advances in understanding the role of tight junction dynamics in response to such stimuli are the focus of this review. barrier, cytoskeleton; cytokine; nutrient transport; myosin

The classic type of transient receptor potential channel (TRPC) is a molecular candidate for [Ca.sup.2+]-permeable cation channels in mammalian cells. Because TRPC channels have calmodulin (CAM) binding sites at their COOH termini, we investigated the effect of CaM on mTRPCS. TRPC5 was initially activated by muscarinic stimulation with 50 [micro]M carbachol and then decayed rapidly even in the presence of carbachol. Intracellular CaM (150 [micro]g/ml) increased the amplitude of mTRPC5 current activated by muscarinic stimulation. CaM antagonists (W-7 and calmidazolium) inhibited mTRPC5 currents when they were applied during the activation of mTRPC5. Pretreatment of W-7 and calmidazolium also inhibited the activation of mTRPC5 current. Inhibitors of myosin light chain kinase (MLCK) inhibited the activation of mTRPC5 currents, whereas inhibitors of CaM-dependent protein kinase II did not. Small interfering RNA against cardiac type MLCK also inhibited the activation of mTRPC5 currents. However, inhibitors of CaM or MLCK did not show any effect on GTP[gamma]S-induced currents. Application of both Rho kinase inhibitor and MLCK inhibitor inhibited GTP[gamma]S-induced currents. We conclude that CaM and MLCK modulates the activation process of mTRPC5. transient receptor potential channel; nonselective cation channel

Myosins are eukaryotic actin-dependent molecular motors important for a broad range of functions like muscle contraction, vision, hearing, cell motility, and host cell invasion of apicomplexan parasites. Myosin heavy chains consist of distinct head, neck, and tail domains and have previously been categorized into 18 different classes based on phylogenetic analysis of their conserved heads. Here we describe a comprehensive phylogenetic examination of many previously unclassified myosins, with particular emphasis on sequences from apicomplexan and other chromalveolate protists including the model organism Toxoplasma, the malaria parasite Plasmodium, and the ciliate Tetrahymena. Using different phylogenetic inference methods and taking protein domain architectures, specific amino acid polymorphisms, and organismal distribution into account, we demonstrate a hitherto unrecognized common origin for ciliate and apicomplexan class XIV myosins. Our data also suggest common origins for some apicomplexan myosins and class VI, for classes II and XVIII, for classes XII and XV, and for some microsporidian myosins and class V, thereby reconciling evolutionary history and myosin structure in several cases and corroborating the common coevolution of myosin head, neck, and tail domains. Six novel myosin classes are established to accommodate sequences from chordate metazoans (class XIX), insects (class XX), kinetoplastids (class XXI), and apicomplexans and diatom algae (classes XXII, XXIII, and XXIV). These myosin (sub)classes include sequences with protein domains (FYVE, WW, UBA, ATS1-like, and WD40) previously unknown to be associated with myosin motors. Regarding the apicomplexan ''myosome,'' we significantly update class XIV classification, propose a systematic naming convention, and discuss possible functions in these parasites. Apicomplexa | Chromalveolata

Urinary bladder dysfunction caused by the alteration of detrusor smooth muscle (DSM) is one of the complications of diabetes. It is well established that smooth muscle contractility is regulated by an elevation of cytosolic [Ca.sup.2+] via myosin light chain (MLC) phosphorylation. However. recent studies have shown the modulation of MLC phosphorylation without a rise in [Ca.sup.2+] in smooth muscle and that two key molecules (Rho-kinase and CPI-17) are involved in the regulation of calcium sensitization. This study investigates the effect of diabetes on DSM calcium sensitization. Diabetes was induced by alloxan in New Zealand White rabbits, and age-matched rabbits given 5% sucrose in the drinking water served as control for diuresis. Two-dimensional gel electrophoresis showed that basal MLC phosphorylation level was significantly higher in diabetic animals than normal or diuretic controls, and Rho-kinase-specific inhibitor, Y-27632, decreased MLC phosphorylation level. Adding Y-27632 to bethanechol-precontracted DSM strips can induce muscle relaxation, but it occulted much more slowly in diabetic samples compared with controls. RT-PCR, Western blot analysis, and immunohistochemistry revealed the over-expression of Rho-kinase [beta] and CP1-17 at both mRNA and protein levels in response to diabetes. In conclusion, our results demonstrate that Rho-kinase contributes to DSM MLC phosphorylation and there is a higher basal MLC phosphorylation level in diabetic DSM. Our results also suggest that this high basal MLC phosphorylation may be due to the upregulation of Rho-kinase and CPI-17. Thus Rho-kinase- and CPI-17-mediated [Ca.sup.2+] sensitization might play a role in diabetes-induced alteration of the detrusor contractility and bladder dysfunction. urinary bladder dysfunction; calcium sensitization: hyperglycemia

We and others have shown that the fetal pulmonary arterial smooth muscle potential for contraction and relaxation is significantly reduced compared with the adult. Whether these developmental changes relate to age differences in the expression and/or activity of key enzymes regulating the smooth muscle mechanical properties has not been previously evaluated. Therefore, we studied the catalytic activities and expression of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) catalytic (PP1c[delta]) and regulatory (MYPT) subunits in late fetal, early newborn, and adult rat intrapulmonary arterial tissues. In keeping with the greater force development and relaxation of adult pulmonary artery, Western blot analysis showed that the MLCK, MYPT, and PP1c[delta] contents increased significantly with age and were highest in the adult rat. In contrast, their specific activities (activity/enzyme content) were significantly higher in the fetal compared with the adult tissue. The fetal and newborn pulmonary arterial muscle relaxant response to the Rho-kinase inhibitor Y-27632 was greater than the adult tissue. In addition to the 130-kDa isoform of MLCK, we documented the presence of minor higher-molecular-weight embryonic isoforms in the fetus and newborn. During fetal life, the lung pulmonary arterial MLCK- and MLCP-specific activities are highest and appear to be related to Rho-kinase activation during lung morphogenesis. lung; transitional circulation; pulmonary vascular resistance

The mechanoenzymatic function of human myosin Vb (HuM5B) if reported for the first time. The results suggest that ADP release is the rate-limiting step for the actin-activated ATPase cycle and the actin filament assay reveals that a single HuM5B molecule is enough to move the actin filament continuously, indicating that HuM5b is a processive motor.

Myosin-1 is the single-headed member of the myosin superfamily that associates with acidic phospholipids through its basic tail domain. Membrane association is essential for proper myosin-I localization and function. However, little is known about the physiological relevance of the direct association of myosin-I with phospholipids or about phospholipid headgroup-binding specificity. To better understand the mechanism of myosin-l-membrane association, we measured effective dissociation constants for the binding of a recombinant my01c tail construct (which includes three IQ domains and bound calmodulins) to large unilamellar vesicles (LUVs) composed of phosphatidylcholine and various concentrations of phosphatidylserine (PS) or phosphatidylinositol 4,5-bisphosphate ([PIP.sub.2]). We found that the myo1c-tail binds tightly to LUVs containing >60% PS but very weakly to LUVs containing physiological PS concentrations ( cell motility | unconventional myosin | lipid signaling

During ischemia intracellular concentrations of [P.sub.i] and [H.sub.+] increase. Also, changes in myosin heavy chain (MHC) isoform toward [beta]-MHC have been reported after ischemia and infarction associated with coronary artery disease. The purpose of this study was to investigate the effects of myoplasmic changes of [P.sub.i] and [H.sup.+] on the loaded shortening velocity and power output of cardiac myocytes expressing either [alpha]- or [beta]-MHC. Skinned cardiac myocyte preparations were obtained from adult male Sprague-Dawley rats (control or treated with 5-n-propyl-2-thiouracil to induce [beta]-MHC) and mounted between a force transducer and servomotor system. Myocyte preparations were subjected to a series of isotonic force clamps to determine shortening velocity and power output during [Ca.sup.2+] activations in each of the following solutions: 1) pCa 4.5 and pH 7.0; 2) pCa 4.5, pH 7.0, and 5 mM [P.sub.i]; 3) pCa 4.5 and pH 6.6; and 4) pCa 4.5, pH 6.6, and 5 mM [P.sub.i]. Added [P.sub.i] and lowered pH each caused isometric force to decline to the same extent in [alpha]-MHC and [beta]-MHC myocytes; however, [beta]-MHC myocytes were more resistant to changes in absolute power output. For example, peak absolute power output fell 53% in [alpha]-MHC myocytes, whereas power fell only 38% in [beta]-MHC myocytes in response to elevated [P.sub.i] and lowered pH (i.e., solution 4). The reduced effect on power output was the result of a greater increase in loaded shortening velocity induced by [P.sub.i] in [beta]-MHC myocytes and an increase in loaded shortening velocity at pH 6.6 that occurred only in [beta]-MHC myocytes. We conclude that the functional response to elevated [P.sub.i] and lowered pH during ischemia is MHC isoform-dependent with [beta]-MHC myocytes being more resistant to declines in power output. ischemia; inorganic phosphate; loaded shortening velocity; power output

We studied the distribution of the giant sarcomeric protein obscurin during de novo myofibrillogenesis in [C.sub.2][C.sub.12] myotubes to learn when it is integrated into developing sarcomeres. Obscurin becomes organized first at the developing M band and later at the mature Z disk. Primordial M bands consisting of obscurin, myomesin, and M band epitopes of titin assemble before adult fast-twitch sarcomeric myosin is organized periodically and nearly concurrently with primitive Z disks, which are composed of [alpha]-actinin and Z disk epitopes of titin. Z disks and M bands can assemble independently at spatially distant sites. As sarcomerogenesis proceeds, these structures interdigitate to produce a more mature organization. Fast-twitch muscle myosin accumulates in the myoplasm and assembles into A bands only after Z disks and M bands assume their typical interdigitated striations. The periodicities of M bands remain constant at ~1.8 [micro]m throughout sarcomerogenesis, whereas distances between Z disks increase from ~1.1 [micro]m in early sarcomeres to ~1.8 [micro]m in more mature structures. Our findings indicate for the first time that primitive M bands self-assemble independently of Z disks, that obscurin is a component of these primitive M bands in skeletal muscle cells, and that A bands assemble only after M bands and Z disks integrate into maturing sarcomeres. obscurin; titin; myosin; myomesin; [alpha]-actinin; A band

The internal dynamics of triosephosphate isomerase is investigated with elastic networks, with and without a substrate bound. It is shown that elastic network computations on protein system combine atoms for functional parts of the structure with coarse-grained representations of the remainder of the structure in several different ways.

Myosin generates force by a rotation of its lever arm. Crystal structures of myosin II indicate an unloaded working stroke of 10-12 nm, a range confirmed by recent x-ray interference experiments. However, when an actin filament, held between two weakly, optically trapped beads is made to interact with a single head of skeletal myosin, the bead displacements have often been reported as having a mean value of 5-6 nm, a value that is commonly interpreted as the working stroke. In general, the observed displacement is not expected to be equal to the working stroke because the kinetics of the stroke is necessarily strain-dependent: this effect biases the frequency of binding events to different actin sites so that displacements smaller than the working stroke are preferentially selected. Our analysis is tailored to current trap experiments, in which the time resolution is insufficient to detect prerigor states. If the preceding transitions are in equilibrium, the mean displacement is zero, contrary to observations in the presence of ATP. However, under ATP-cycling conditions, we find that the mean displacement is deflated to 0.3-0.7 of the true working stroke, depending on the equilibrium constant of the stroke and the rate at which the first myosin product state can detach from actin. The primary working stroke of processive myosin motors as measured by optical trapping is similarly uncertain. optical | tweezers | molecular motor

During skeletal muscle contraction, regular arrays of actin and myosin filaments slide past each other driven by the cyclic ATP-dependent interaction of the motor protein myosin II (the crossbridge) with actin. The rate of the cross-bridge cycle and its load-dependence, defining shortening velocity and energy consumption at the molecular level, vary widely among different isoforms of myosin II. However, the underlying mechanisms remain poorly understood. We have addressed this question by applying a single-molecule approach to rapidly ([approximately equal to] 300 [micro]s) and precisely ([approximately equal to] 0.1 nm) detect acto-myosin interactions of two myosin isoforms having large differences in shortening velocity. We show that skeletal myosin propels actin filaments, performing its conformational change (working stroke) in two steps. The first step ([approximately equal to] 3.4-5.2 nm) occurs immediately after myosin binding and is followed by a smaller step ([approximately equal to] 1.0-1.3 nm), which occurs much faster in the fast myosin isoform than in the slow one, independently of ATP concentration. On the other hand, the rate of the second phase of the working stroke, from development of the latter step to dissociation of the acto-myosin complex, is very similar in the two isoforms and depends linearly on ATP concentration. The finding of a second mechanical event in the working stroke of skeletal muscle myosin provides the molecular basis for a simple model of actomyosin interaction. This model can account for the variation, in different fiber types, of the rate of the cross-bridge cycle and provides a common scheme for the chemo-mechanical transduction within the myosin family. isoforms | optical tweezers | single molecule

Agonist-induced activation of the RhoA/Rho kinase (ROCK) pathway results in inhibition of myosin phosphatase and maintenance of myosin light chain ([MLC.sub.20]) phosphorylation. We have shown that RhoA/ROCKII translocates and associates with heat shock protein (HSP)27 in the particulate fraction. We hypothesize that inhibition of the 130-kDa regulatory myosin-binding subunit (MYPT) requires its association with HSP27 in the particulate fraction. Furthermore, it is not certain whether regulation of MYPT by CPI-17 or by ROCKII is due to cross talk between RhoA and PKC-[alpha]. Presently, we examined the cross talk between RhoA and PKC-[alpha] in the regulation of MYPT phosphorylation in rabbit colon smooth muscle cells. Acetylcholine induced 1) sustained phosphorylation of PKC-[alpha], CPI-17, and MYPT; 2) an increase in the association of phospho-MYPT with HSP27 in the particulate fraction; 3) a decrease in myosin phosphatase activity (66.21 [+ or -] 3.52 and 42.19 [+ or -] 3.85%nM/ml lysate at 30 s and 4 min); and 4) an increase in PKC activity (298.12 [+ or -] 46.60% and 290.59 [+ or -] 22.07% at 30 s and 4 min). Inhibition of RhoA/ROCKII by Y-27632 inhibited phosphorylation of MYPT and its association with HSP27. Both Y27632 and a negative dominant construct of RhoA inhibited phosphorylation of MYPT and CPI-17. Inhibition of PKCs or calphostin C or selective inhibition of PKC-[alpha] by negative dominant constructs inhibited phosphorylation of MYPT and CPI-17. The results suggest that 1) acetylcholine induces activation of both RhoA and/or PKC-[alpha] pathways, suggesting cross talk between RhoA and PKC-[alpha] resulting in phosphorylation of MYPT, inhibition of myosin phosphatase activity, and maintenance of MLC phosphorylation; and 2) phosphorylated MYPT is associated with HSP27 and translocated to the particulate fraction, suggesting a scaffolding role for HSP27 in mediating the association of the complex MYPT/RhoA-ROCKII. Thus both pathways (PKC and RhoA) converge on the regulation of myosin phosphatase activities and modulate sustained phosphorylation of [MLC.sub.20]. myosin light chain; contraction; CPI-17; acetylcholine; Rho kinase; colon; phosphatase; protein kinase C; heat shock protein

Tropomyosin is a two-chain [alpha]-helical coiled coil whose periodic interactions with the F-actin helix are critical for thin filament stabilization and the regulation of muscle contraction. Here we deduce the mechanical and chemical basis of these interactions from the 2.3-[Angstrom]-resolution crystal structure of the middle three of tropomyosin's seven periods. Geometrically specific bends of the coiled coil, produced by clusters of core alanines, and variable bends about gaps in the core, produced by isolated alanines, occur along the molecule. The crystal packing is notable in signifying that the functionally important fifth period includes an especially favorable protein-binding site, comprising an unusual apolar patch on the surface together with surrounding charged residues. Based on these and other results, we have constructed a specific model of the thin filament, with the N-terminal halves of each period (i.e., the so-called '[alpha] zones') of tropomyosin axially aligned with subdomain 3 of each monomer in F-actin. alanine | [alpha]-helix | cardiomyopathy | coiled coil | packing

Repetitive stimulation potentiates contractile tension of fast-twitch skeletal muscle. We examined the role of myosin regulatory light chain (RLC) phosphorylation in this physiological response by ablating [Ca.sup.2+]/calmodulin-dependent skeletal muscle myosin light chain kinase (MLCK) gene expression. Western blot and quantitative-PCR showed that MLCK is expressed predominantly in fasttwitch skeletal muscle fibers with insignificant amounts in heart and smooth muscle. In contrast, smooth muscle MLCK had a more ubiquitous tissue distribution, with the greatest expression observed in smooth muscle tissue. Ablation of the MYLK2 gene in mice resulted in loss of skeletal muscle MLCK expression, with no change in smooth muscle MLCK expression. In isolated fast-twitch skeletal muscles from these knockout mice, there was no significant increase in RLC phosphorylation in response to repetitive electrical stimulation. Furthermore, isometric twitch-tension potentiation after a brief tetanus (posttetanic twitch potentiation) or low-frequency twitch potentiation (staircase) was attenuated relative to responses in muscles from wild-type mice. Interestingly, the site of phosphorylation of the small amount of monophosphorylated RLC in the knockout mice was the same site phosphorylated by MLCK, indicating a potential alternative signaling pathway affecting contractile potentiation. Loss of skeletal muscle MLCK expression had no effect on cardiac RLC phosphorylation. These results identify myosin light chain phosphorylation by the dedicated skeletal muscle [Ca.sup.2+]/calmodulin-dependent MLCK as a primary biochemical mechanism for tension potentiation due to repetitive stimulation in fast-twitch skeletal muscle. calcium | calmodulin | twitch

Two smooth muscle myosin heavy chain isoforms differ in their amino terminus by the presence [(+)insert] or absence [(-)insert] of a seven-amino acid insert. Animal studies show that the (+)insert isoform is predominantly expressed in rapidly contracting phasic muscle and the (-)insert isoform is mostly found in slowly contracting tonic muscle. The expression of the (+)insert isoform has never been demonstrated in human smooth muscle. We hypothesized that the (+)insert isoform is present in humans and that its expression is commensurate with the organ's functional requirements. We report, for the first time, the sequence of the human (+)insert isoform and quantification of its expression by real-time PCR and Western blot analysis in a panel of human organs. The (+)insert isoform mRNA and protein expression levels are significantly greater in small intestine compared with all organs studied except for trachea and are significantly greater in trachea compared with uterus and aorta. To assess the functional significance of this differential myosin isoform expression between organs, we measured the rate of actin filament movement ([v.sub.max]) when propelled by myosin purified from rat organs, because the rat and human inserts are identical and their remaining sequences show 93% identity. [v.sub.max] exhibits a rank correlation from the most tonic to the most phasic organ. The selective expression of the (+)insert isoform observed among human organs suggests that it is an important determinant of tissue shortening velocity. A differential expression of the (+)insert isoform could also account for altered contractile properties observed in human pathology. phasic and tonic smooth muscle; real-time polymerase chain reaction; in vitro motility assay

Vascular smooth muscle cell (VSMC) phenotypic modulation is a key factor in vascular pathology. We have investigated the role of Notch receptor signaling in controlling human vascular smooth muscle cell (hVSMC) differentiation in vitro and established a role for cyclic strain-induced changes in Notch signaling in promoting this phenotypic response. The expression of [actin]-actin, calponin, myosin, and smoothelin was examined by performing immunocytochemistry, Western blot analysis, and quantitative real-time PCR in hVSMCs cultured under static conditions after forced overexpression of constitutively active Notch 1 and 3 receptors, inhibition of endogenous Cp-binding factor 1 (CBF-1)/recombination signal sequence-binding protein-J[kappa] (RBP-J[kappa]) signaling, and exposure to cyclic strain using a Flexercell Tension Plus unit. Overexpression of constitutively active Notch intracellular (IC) receptors (Notch 1 IC and Notch 3 IC) resulted in a significant downregulation of [alpha]-actin, calponin, myosin, and smoothelin expression, an effect that was significantly attenuated after inhibition of Notch-mediated, CBF1/RBP-J[kappa]-dependent signaling by coexpression of RPMS-1 (Epstein-Barr virus-encoded gene product) and selective knockdown of basic helix-loop-helix factors [hairy enhancer of split (HES) gene and Hes-related transcription (Hrt) factors Hrt-1, Hrt-2, and Hrt-3] using targeted small interfering RNA. Cells cultured under conditions of defined equibiaxial cyclic strain (10% strain, 60 cycles/min, 24 h) exhibited a significant reduction in Notch 1 IC and Notch 3 IC expression concomitant with a significant increase in VSMC differentiation marker expression. Moreover, this cyclic strain-induced increase was further enhanced after inhibition of CBF-1/RBP-J[kappa]-dependent signaling with RPMS-1. These findings suggest that Notch promotes changes in hVSMC phenotype via activation of CBF-1/ RBP-J[kappa]-dependent pathways in vitro and contributes to the phenotypic response of VSMCs to cyclic strain-induced changes in VSMC differentiation. basic helix-loop-helix; cyclic strain; myosin; smoothelin

In mouse melanocytes, myosin Va is recruited onto the surface of melanosomes by a receptor complex containing Rab27a that is present in the melanosome membrane and melanophilin (Mlp), which links myosin Va to Rab27a. In this study, we show that Mlp is also a microtubule plus end-tracking protein or +TIP. Moreover, myosin Va tracks the plus end in a Mlp-dependent manner. Data showing that overexpression and short inhibitory RNA knockdown of the +TIP EB1 have opposite effects on Mlp-microtubule interaction, that Mlp interacts directly with EB1, and that deletion from Mlp of a region similar to one in the adenomatous polyposis coli protein involved in EB1 binding blocks Mlp's ability to plus end track argue that Mlp tracks the plus end directly by hitch-hiking on EB1. These results identify a novel +TIP and indicate that vertebrate cells possess a +TIP complex that is similar to the Myo2p-Kar9p-Bim1p complex in yeast. We suggest that the +TIP complex identified in this study may serve to focus the transfer of melanosomes from microtubules to actin at the microtubule plus end.

Class V myosins are multifunctional molecular motors implicated in vesicular traffic, RNA transport, and mechanochemical coupling of the actin and microtubule-based cytoskeletons. To assess the function of the single myosin V gene in Drosophila (MyoV), we have characterized both deletion and truncation alleles. Mutant animals exhibit no detectable defects during embryogenesis but are delayed in larval development; most die prior to 3rd instar. MyoV protein is widely distributed; however, there are no obvious cytological defects in mutant larval tissues where MyoV was normally highly expressed. Of the few adult MyoV mutant escapers, females were fertile but males were not. We examined the expression of MyoV during spermatogenesis. MyoV was associated with membranes, microtubule, and actin structures required for spermatid maturation; MyoV was strongly associated with the sperm nuclei during the maturation of the actin-rich investment cones that package spermatids in individual membranes. In MyoV mutant escaper males, the early stages of spermatogenesis were normal; however, in the later stages, the investment cones stained weakly for actin and their registration was disrupted; no mature sperm were produced. Our results suggest that MyoV contributes to the formation of the actin-based investment cones and acts to coordinate and/or anchor these structures and other components of the individualization complex. Keywords: Actin; Microtubules; Investment cones; Individualization complex; Gut; Oogenesis; CNS

Myosin light chain 2 (LC2) phosphorylation is of both physiological and pathological importance to myocardial function. The phosphatase that directly dephosphorylates LC2 is a type 1 protein phosphatase (PP1) that contains a catalytic subunit that complexes with a myosin-binding phosphatase targeting subunit (MYPT). The goal of the present study was to examine the role of MYPT in the regulation of PPI in ventricular myocytes. In the first part of the study, regional distribution of MYPT expression and phosphorylation were determined in unstimulated hearts. The pattern of MYPT phosphorylation was inversely related to the LC2 phosphorylation spatial gradient as described by Epstein and colleagues (Davis JS, Hassanzadeh S, Winitsky S, Lin H, Satorius C, Vemuri R, Aletras AH, Wen H, and Epstein ND. Cell 107: 631-641, 2001). In the second part of the study, adult rat isolated ventricular myocytes were exposed to an [alpha]-adrenergic receptor agonist, and properties of MYPT, PP1, and LC2 were studied. We found MYPT associates with cardiac myofilaments, and this association increases upon [alpha]-adrenergic receptor stimulation. Activation of [alpha]-adrenergic receptors also led to a decrease in the PP1-myofilament association. Furthermore, [alpha]-adrenergic receptor stimulation results in phosphorylation of MYPT and LC2 and an increase in myocyte [Ca.sup.2+] sensitivity of tension that all depend on Rho kinase activation. These data support the hypothesis that [alpha]-adrenergic receptor activation works through Rho kinase to phosphorylate MYPT, and phosphorylated MYPT dissociates from PP1 so that PP1 is no longer physically associated with LC2. Hence, we propose a pathway for the dynamic modulation of LC2 phosphorylation through receptor-dependent phosphorylation of MYPT, and a spatial gradient of LC2 phosphorylation under basal conditions that occurs due to varied levels of phosphorylation of MYPT in ventricles. light chain 2; [alpha]-adrenergic receptor; Rho kinase; ventricular myocytes; isometric tension

Recently, we have shown that Rho and Rho-activated kinase (ROCK) may become activated by high-millimolar KC1, which had previously been widely assumed to act solely through opening of voltage-dependent [[Ca.sup.2+]] channels. In this study, we explored in more detail the relationship between membrane depolarization, [[Ca.sup.2+]] currents, and activation of Rho/ ROCK in bovine tracheal smooth muscle. [[Ca.sup.2+]] currents began to activate at membrane voltages more positive than -40 mV and were maximally activated above 0 mV; at the same time, these underwent time- and voltage-dependent inactivation. Depolarizing intact tissues by KC1 challenge evoked contractions that were blocked equally, and in a nonadditive fashion, by nifedipine or by the ROCK inhibitor Y-27632. Other agents that elevate intracellular calcium concentration ([[[Ca.sup.2+]].sub.i]) by pathways independent of G protein-coupled receptors, namely the SERCA-pump inhibitor cyclopiazonic acid and the [[Ca.sup.2+]] ionophore A-23187, evoked contractions that were also largely reduced by Y-27632. KC1 directly increased Rho and ROCK activities in a concentration-dependent fashion that paralleled closely the effect of KC1 on tone and [[[Ca.sup.2+]].sub.i], as well as the voltage-dependent [[Ca.sup.2+]] currents that were measured over the voltage ranges that are evoked by 0-120 mM KC1. Through the use of various pharmacological inhibitors, we ruled out roles for [[Ca.sup.2+]]/calmodulin-dependent CaM kinase II, protein kinase C, and protein kinase A in mediating the KC1-stimulated changes in tone and Rho/ROCK activities. In conclusion, Rho is activated by elevation of [[[Ca.sup.2+]].sub.i] (although the signal transduction pathway underlying this [[Ca.sup.2+]] dependence is still unclear) and possibly also by membrane depolarization per se. myosin light chain phosphatase; RhoA; Rho-activated kinase; voltage-dependent [[Ca.sup.2+]] channels

Muscle contraction is due to myosin motors that transiently attach with their globular head to an actin filament and generate force. After a sudden reduction of the load below the maximum isometric force ([T.sub.0]), the attached myosin heads execute an axial movement (the working stroke) that drives the sliding of the actin filament toward the center of the sarcomere by an amount that is larger at lower load and is 11 nm near zero load. Here, we show that an increase in temperature from 2 to 17[degrees]C, which increases the average isometric force per attached myosin head by 60%, does not affect the amount of filament sliding promoted by a reduction in force from To to 0.7[T.sub.0], whereas it reduces the sliding under low load by 2.5 nm. These results exclude the possibility that the myosin working stroke is due to the release of the mechanical energy stored in the initial endothermic force-generating process and show that, at higher temperatures, the working stroke energy is greater because of higher force, although the stroke length is smaller at low load. We conclude the following: (i) the working stroke is made by a series of state transitions in the attached myosin head; (ii) the temperature increases the probability for the first transition, competent for isometric force generation; and (iii) the temperature-dependent rise in work at high load can be accounted for by the larger free energy drop that explains the rise in isometric force. muscle contraction | muscle energetics | myosin working stroke

Myosin V is an efficient processive molecular motor. Recent experiments have shown how the structure and kinetics of myosin V are specialized to produce a highly processive motor capable of taking multiple 36-nm steps on an actin filament track. Here, we examine how two identical heads coordinate their activity to produce efficient hand-over-hand stepping. We have used a modified laser-trap microscope to apply a [approximately equal to] 2-pN forward or backward force on a single-headed myosin V molecule, hypothesized to simulate forces experienced by the rear or lead head, respectively. We found that pulling forward produces only a small change in the kinetics, whereas pulling backward induces a large reduction in the cycling of the head. These results support a model in which the coordination of myosin V stepping is mediated by strain-generated inhibition of the lead head. load dependence | molecular motor | processivity | optical trap

Discoveries from human and mouse genetics have identified cytoskeletal and signaling proteins that are essential for neuronal migration in the developing brain. To provide a meaningful context for these studies, we took an unbiased approach of correlative electron microscopy of neurons migrating through a three-dimensional matrix, and characterized the cytoskeletal events that occur as migrating neurons initiate saltatory forward movements of the cell nucleus. The formation of a cytoplasmic dilation in the proximal leading process precedes nuclear translocation. Cell nuclei translocate into these dilations in saltatory movements. Time-lapse imaging and pharmacological perturbation suggest that nucleokinesis requires stepwise or hierarchical interactions between microtubules, myosin II, and cell adhesion. We hypothesize that these interactions couple leading process extension to nuclear translocation during neuronal migration. actin | microtubules | myosin II | blebbistatin | motility

The correct localization of myosin II to the equatorial cortex is crucial for proper cell division. Here, we examine a collection of genes that cause defects in cytokinesis and reveal with live cell imaging two distinct phases of myosin II localization. Three genes in the rho1 signaling pathway, pebble (a Rho guanidine nucleotide exchange factor), rho1, and rho kinase, are required for the initial recruitment of myosin II to the equatorial cortex. This initial localization mechanism does not require F-actin or the two components of the centralspindlin complex, the mitotic kinesin pavarotti/MKLP1 and racGAP50c/CYK-4. However, F-actin, the centralspindlin complex, formin (diaphanous), and profilin (chickadee) are required to stably maintain myosin II at the furrow. In the absence of these latter genes, myosin II delocalizes from the equatorial cortex and undergoes highly dynamic appearances and disappearances around the entire cell cortex, sometimes associated with abnormal contractions or blebbing. Our findings support a model in which a rho kinase-dependent event, possibly myosin II regulatory light chain phosphorylation, is required for the initial recruitment to the furrow, whereas the assembly of parallel, unbranched actin filaments, generated by formin-mediated actin nucleation, is required for maintaining myosin II exclusively at the equatorial cortex. contractile ring | rho1

A key part in the cytokinetic mechanism of Schizosaccharomyces pombe is the contractile ring myosin, which consists of two heavy chains (Myo2p), two essential light chains (Cdc4p) and two regulatory light chains. NMR spectroscopy is used to compare the structure, stability, and dynamics of the isolated N- and C-terminal domains with one another and with native Cdc4p.

Initiation of cytokinesis requires the establishment of the cleavage plane, the assembly of the contractile ring, and the ingression of the cleavage furrow. MgcRacGAP, a GTPase-activating protein for RhoA, is required for cytokinesis, but the mechanism of its action remains unknown. We report here that MgcRacGAP is required for the assembly of anillin and myosin into the contractile ring. In addition, MgcRacGAP is required for the localized activation of myosin through the RhoA-mediated phosphorylation of the myosin regulatory light chain. Cells with MgcRacGAP RNA interference (RNAi) failed cytokinesis without any ingression of the cleavage furrow. Paradoxically, MgcRacGAP, a GTPase-activating protein, associates during cytokinesis with ECT2, a guanine nucleotide exchange factor for RhoA, and the localization of ECT2 to both the central spindle and the contractile ring depends on MgcRacGAP. Knockdown of ECT2 phenocopies that of MgcRacGAP. We conclude that MgcRacGAP controls the initiation of cytokinesis by regulating ECT2, which in turn induces the assembly of the contractile ring and triggers the ingression of the cleavage furrow. ECT2 | MKLP1 | myosin | RhoA | cell division

Myosin V is a double-headed molecular motor involved in organelle transport. Two distinctive features of this motor, processivity and the ability to take extended linear steps of [approximately equal to] 36 nm along the actin helical track, depend on its unusually long light chainbinding domain (LCBD). The LCBD of myosin V consists of six tandem IQ motifs, which constitute the binding sites for calmodulin (CAM) and CaM-like light chains. Here, we report the 2-[Angstrom] resolution crystal structure of myosin light chain 1 (Mlc1p) bound to the IQ2-IQ3 fragment of Myo2p, a myosin V from Saccharomyces cerevisiae. This structure, combined with FRET distance measurements between probes in various CaM--IQ complexes, comparative sequence analysis, and the previously determined structures of Mlc1p-IQ2 and Mlc1p-IQ4, allowed building a model of the LCBD of myosin V. The IQs of myosin V are distributed into three pairs. There appear to be specific cooperative interactions between light chains within each IQ pair, but little or no interaction between pairs, providing flexibility at their junctions. The second and third IQ pairs each present a light chain, whether CaM or a CaM-related molecule, bound in a noncanonical extended conformation in which the N-lobe does not interact with the IQ motif. The resulting free N-lobes may engage in protein--protein interactions. The extended conformation is characteristic of the single IQ of myosin VI and is common throughout the myosin superfamily. The model points to a prominent role of the LCBD in the function, regulation, and molecular interactions of myosin V. calmodulin | IQ motif | x-ray crystallography | FRET

Myosin-based contractility plays important roles in the regulation of epithelial functions, particularly paracellular permeability. However, the triggering factors and the signaling pathways that control epithelial myosin light chain (MLC) phosphorylation have not been elucidated. Herein we show that plasma membrane depolarization provoked by distinct means, including high extracellular [K.sup.+], the lipophilic cation tetraphenylphosphonium, or the ionophore nystatin, induced strong diphosphorylation of MLC in kidney epithelial cells. In sharp contrast to smooth muscle, depolarization of epithelial cells did not provoke a [Ca.sup.2+] signal, and removal of external [Ca.sup.2+] promoted rather than inhibited MLC phosphorylation. Moreover, elevation of intracellular [Ca.sup.2+] did not induce significant MLC phosphorylation, and the myosin light chain kinase (MLCK) inhibitor ML-7 did not prevent the depolarization-induced MLC response, suggesting that MLCK is not a regulated element in this process. Instead, the Rho-Rho kinase (ROK) pathway is the key mediator because 1) depolarization stimulated Rho and induced its peripheral translocation, 2) inhibition of Rho by Clostridium difficile toxin B or C3 transferase abolished MLC phosphorylation, and 3) the ROK inhibitor Y-27632 suppressed the effect. Importantly, physiological depolarizing stimuli were able to activate the same pathway: L-alanine, the substrate of the electrogenic [Na.sup.+]-alanine cotransporter, stimulated Rho and induced Y-27632-sensitive MLC phosphorylation in a [Na.sup.+]-dependent manner. Together, our results define a novel mode of the regulation of MLC phosphorylation in epithelial cells, which is depolarization triggered and Rho-ROK-mediated but [Ca.sup.2+] signal independent. This pathway may be a central mechanism whereby electrogenic transmembrane transport processes control myosin phosphorylation and thereby regulate paracellular transport. membrane potential; [Na.sup.+]-alanine cotransport; epithelium; phosphatidylinositol 3-kinase; LLC-P[K.sub.1] cells

Myosin light-chain (MLC) kinase (MLCK)-dependent increase in MLC phosphorylation has been proposed to be a key mediator of the hyperosmotic activation of the [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter (NKCC). To address this hypothesis and to assess whether MLC phosphorylation plays a signaling or permissive role in NKCC regulation, we used pharmacological and genetic means to manipulate MLCK, MLC phosphorylation, or myosin ATPase activity and followed the impact of these alterations on the hypertonic stimulation of NKCC in porcine kidney tubular LLCPK1 epithelial cells. We found that the MLCK inhibitor ML-7 suppressed NKCC activity independently of MLC phosphorylation. Notably, ML-7 reduced both basal and hypertonically stimulated NKCC activity without influencing MLC phosphorylation under these conditions, and it inhibited NKCC activation by [Cl.sup.-] depletion, a treatment that did not increase MLC phosphorylation. Furthermore, prevention of the osmotically induced increase in MLC phosphorylation by viral induction of cells with a nonphosphorylatable, dominant negative MLC mutant (AA-MLC) did not affect the hypertonic activation of NKCC. Conversely, a constitutively active MLC mutant (DD-MLC) that mimics the diphosphorylated form neither stimulated isotonic nor potentiated hypertonic NKCC activity. Furthermore, a depolarization-induced increase in endogenous MLC phosphorylation failed to activate NKCC. However, complete abolition of basal MLC phosphorylation by K252a or the inhibition of myosin ATPase by blebbistatin significantly reduced the osmotic stimulation of NKCC without suppressing its basal or [Cl.sup.-] depletion-triggered activity. These results indicate that an increase in MLC phosphorylation is neither a sufficient nor a necessary signal to stimulate NKCC in tubular cells. However, basal myosin activity plays a permissive role in the optimal osmotic responsiveness of NKCC. proline-alanine-rich STE20-related kinase

The power stroke pulling myosin along actin filaments during muscle contraction is achieved by a large rotation ([approximately equal to] 60[degrees]) of the myosin lever arm after ATP hydrolysis. Upon binding the next ATP, myosin dissociates from actin, but its ATPase site is still partially open and catalytically off. Myosin must then close and activate its ATPase site while returning the lever arm for the next power stroke. A mechanism for this coupling between the ATPase site and the distant lever arm is determined here by generating a continuous series of optimized intermediates between the crystallographic end-states of the recovery stroke. This yields a detailed structural model for communication between the catalytic and the force-generating regions that is consistent with experimental observations. The coupling is achieved by an amplifying cascade of conformational changes along the relay helix lying between the ATPase and the domain carrying the lever arm. chemo-mechanical coupling | conformational transition | Conjugate Peak Refinement | muscle contraction | power stroke

The direct imaging of wound-edge fibroblasts after triggering microtubule-organizing center (MTOC) reorientation with soluble factors showed that the nucleus moved away from the leading edge to reorient the MTOC, while the MTOC remained stationary. The nuclear repositioning is an initial polarizing event in migrating cells and separate regulatory pathways establish the positions of the nucleus and the MTOC.

This investigation estimated the amount of variance in voluntary in vivo muscle performance that can be explained by relative myosin heavy chain (MHC) isoform expression. The role of the relative expression of these proteins in relation to in vitro force and velocity performance is well understood, but the in vivo model is less clear. Twenty-two men and women (mean [+ or -] SD age, 27 [+ or -] 6 years) performed isometric knee extensor actions in which peak force and rate of force development (RFD) were measured. The results of regression analysis showed that the inclusion of MHC IIb explained a significant (19.9%, p < 0.05) amount of variance in relative peak force (adjusted for muscle mass) and 14.1% of the variance in the first half of the rise phase of the force-time curve ([RFD.sub.0-50%],) (p < 0.1). The addition of MHC I into this model explained a significant (p < 0.05) amount of variance above that accounted for by MHC IIb in RFD (45.4%), [RFD.sub.0-50%], (50.8%), and [RFD.sub.50-100%] (second half of the rise phase of the force-time curve) (37.4%). Since the percentage of MHC IIb is reduced rather quickly with training, these data suggest that peak force may also be affected quickly by training. The percentage of MHC I has a longer course for change with training; therefore, it may be inferred that the greatest changes in RFD variables will likely occur during a longer course. KEY WORDS. explained variance, [r.sup.2] change, skeletal muscle protein expression

We examined the effects of thyroid hormones (THs) on left ventricular (LV) function and myocyte remodeling in rats with spontaneously hypertensive heart failure (SHHF). SHHF rats were treated with three different TH doses from 20-21 mo of age. In terminal experiments, LV function (as determined by echocardiography and catheterization) and isolated myocyte shape were examined in SHHF rat groups and age-matched Wistar-Furth control animals. Compared with Wistar-Furth rats, the ratio of [alpha]- to [beta]-myosin was reduced in untreated SHHF rats. The [alpha]-to-[beta]-myosin ratio increased in all TH groups, which suggests a reversal of the fetal gene program. Low-dose TH produced no changes in LV myocyte size or function, but high-dose TH produced signs of hyperthyroidism (e.g., increased heart weight, tachycardia). The chamber diameter-to-wall thickness ratio declined with increasing dose due to reduced chamber diameter and increased wall thickness. This resulted in a 38% reduction in LV systolic wall stress in the middle- and high-dose groups despite sustained hypertension. Isolated myocyte data indicated that chamber remodeling and reduced wall stress were due to a unique alteration in myocyte transverse shape (e.g., reduced major diameter and increased minor diameter). Based on our present understanding of ventricular remodeling and wall stress, we believe these changes are likely beneficial. Results suggest that TH may be an important regulator of myocyte transverse shape in heart disease. shape; ventricular remodeling; wall stress; myosin ratio

Myosin VI plays a role in the maintenance of Golgi morphology and in exocytosis. In a yeast 2-hybrid screen we identified optineurin as a binding partner for myosin VI at the Golgi complex and confirmed this interaction in a range of protein interaction studies. Both proteins colocalize at the Golgi complex and in vesicles at the plasma membrane. When optineurin is depleted from cells using RNA interference, myosin VI is lost from the Golgi complex, the Golgi is fragmented and exocytosis of vesicular stomatitis virus G-protein to the plasma membrane is dramatically reduced. Two further binding partners for optineurin have been identified: huntingtin and Rab8. We show that myosin VI and Rab8 colocalize around the Golgi complex and in vesicles at the plasma membrane and overexpression of constitutively active Rab8-Q67L recruits myosin VI onto Rab8-positive structures. These results show that optineurin links myosin VI to the Golgi complex and plays a central role in Golgi ribbon formation and exocytosis.

The purpose of the present study was to examine the effects of portal hypertension on agonist-induced myosin phosphorylation and RhoA expression in vascular smooth muscle. A possible link to cAMP-dependent events was also examined. Portal hypertension was produced by stenosis of the portal vein. Vessel segments were treated with or without 50 [micro]M of the PKA inhibitor Rp-cAMPS for 30 min and subsequently stimulated with [10.sup.4] M phenylephrine. Myosin regulatory light-chain phosphorylation was detected by immunoblotting. Total RNA from first-order mesenteric arteries and portal veins was isolated and amplified by RT-PCR using RhoA and GAPDH primers. RhoA protein expression was also measured in first-order mesenteric arteries using Western blot analysis. Myosin phosphorylation in maximally stimulated first-order mesenteric arteries was significantly lower in portal hypertensive animals (19.9 [+ or -] 2.86%) when compared with sham-operated control (43.8 [+ or -] 3.53%). Inhibition of PKA selectively increased myosin phosphorylation to 34.7 [+ or -] 4.18%. Rp-cAMPS did not affect the phosphorylation of the portal veins or superior mesenteric arteries. RhoA mRNA and membrane-associated RhoA protein expression in portal hypertensive first-order mesenteric arteries were significantly lower when compared with controls. Acute inhibition of PKA had no effect on RhoA mRNA expression. However, it restored membrane-associated RhoA protein expression in portal hypertensive vessels to control levels. The results suggest that reductions in membrane-associated RhoA expression, which appear to be regulated by cAMP-dependent events, lead to reduced myosin phosphorylation and may underlie the reduced vasoconstrictor effectiveness in the resistance vasculature of portal hypertensive intestine. myosin regulatory light chain; vascular smooth muscle; protein kinase A; Rp-cAMPS