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Smooth muscle contraction involves phosphorylation of the regulatory myosin light chain. However, this thick-filament system of regulation cannot account for all aspects of a smooth muscle contraction. An alternate site of contractile regulation may be in the thin-filament-associated proteins, in particular caldesmon. Caldesmon has been proposed to be an inhibitory protein that acts either as a brake to stop any increase in resting or basal tone, or as a modulatory protein during contraction. The goal of this study was to use short interfering RNA technology to decrease the levels of the smooth muscle-specific isoform of caldesmon in intact vascular smooth muscle tissue to determine more carefully what role(s) caldesmon has in smooth muscle regulation. Intact strips of vascular tissue depleted of caldesmon produced significant levels of shortening velocity, indicative of cross-bridge cycling, in the unstimulated tissue and exhibited lower levels of contractile force to histamine. Our results also suggest that caldesmon does not play a role in the cooperative activation of unphosphorylated cross bridges by phosphorylated cross bridges. The velocity of shortening of the constitutively active tissue and the high basal values of myosin light chain phosphorylation suggest that h-caldesmon in vivo acts as a brake against contractions due to basally phosphorylated myosin. It is also possible that phosphorylation of h-caldesmon alone in the resting state may be a mechanism to produce increases in force without stimulation and increases in calcium. Disinhibition of h-caldesmon by phosphorylation would then allow force to be developed by activated myosin in the resting state. myosin light chain; shortening velocity; cooperativity; carotid artery; cross-bridge cycling doi: 10.1152/ajpheart.00129.2009.

In Saccharomyces cerevisiae, peroxisomal inheritance from mother cell to bud is conducted by the class V myosin motor, Myo2p. However, homologues of S. cerevisiae Myo2p peroxisomal receptor, Inp2p, are not readily identifiable outside the Saccharomycetaceae family. Here, we demonstrate an unexpected role for Pex3 proteins in peroxisome inheritance. Both Pex3p and Pex3Bp are peroxisomal integral membrane proteins that function as peroxisomal receptors for class V myosin through direct interaction with the myosin globular tail. In cells lacking Pex3Bp, peroxisomes are preferentially retained by the mother cell, whereas most peroxisomes gather and are transferred en masse to the bud in cells overexpressing Pex3Bp or Pex3p. Our results reveal an unprecedented role for members of the Pex3 protein family in peroxisome motilily and inheritance in addition to their well-established role in peroxisome biogenesis at the endoplasmic reticulum. Our results point to a temporal link between peroxisome formation and inheritance and delineate a general mechanism of peroxisome inheritance in eukaryotic cells. doi 10.1083/jcb.200902117

Unconventional myosins interact with the dense cortical actin network during processes such as membrane trafficking, cell migration, and mechanotransduction. Our understanding of unconventional myosin function is derived largely from assays that examine the interaction of a single myosin with a single actin filament. In this study, we have developed a model system to study the interaction between multiple tethered unconventional myosins and a model F-actin cortex, namely the lamellipodium of a migrating fish epidermal keratocyte. Using myosin VI, which moves toward the pointed end of actin filaments, we directly determine the polarity of the extracted keratocyte lamellipodium from the cell periphery to the cell nucleus. We use a combination of experimentation and simulation to demonstrate that multiple myosin VI molecules can coordinate to efficiently transport vesiclesize cargo over 10 [micro]m of the dense interlaced actin network. Furthermore, several molecules of monomeric myosin VI, which are nonprocessive in single molecule assays, can coordinate to transport cargo with similar speeds as dimers.

The effects of remodeling of airway smooth muscle (SM) by hyperplasia on airway SM contractility in vivo are poorly explored. The aim of this study was to investigate the relationship between allergen-induced airway SM hyperplasia and its contractile phenotype. Brown Norway rats were sensitized with ovalbumin (OVA) or saline on day 0 and then either OVA-challenged once on clay 14 and killed 24 h later or OVA-challenged 3 times (on days 14, 19, and 24) and killed 2 or 7 days later. Changes in SM mass, expression of total myosin, SM myosin heavy chain fast isoform (SM-B) and myosin light chain kinase (MLCK), tracheal contractions ex vivo, and airway responsiveness to methacholine (MCh) in vivo were assessed. One day after a single OVA challenge, the number of SM cells positive for PCNA was greater than for control animals, whereas the SM mass, contractile phenotype, and tracheal contractility were unchanged. Two days after three challenges, SM mass and PCNA immunoreactive cells were increased (3- and 10-fold, respectively; P < 0.05), but airway responsiveness to MCh was unaffected. Lower expression in total myosin, SM-B, and MLCK was observed at the mRNA level (P < 0.05), and total myosin and MLCK expression were lower at the protein level (P < 0.05) after normalization for SM mass. Normalized tracheal SM force generation was also significantly lower 2 days after repeated challenges (P < 0.05). Seven days after repeated challenges, features of remodeling were restored toward control levels. Allergen-induced hyperplasia of SM cells was associated with a loss of contractile phenotype, which was offset by the increase in mass. phenotype; animal model; myosin heavy chain isoform doi: 10.1152/ajplung.00142.2009

IGF-I increases skeletal muscle mass, but whether IGF-I increases type IIb myosin heavy chain (MyHC) transcriptional activity is not known. [C.sub.2][C.sub.12] myotubes were cultured with or without IGF-I to determine whether IGF-I increases type IIb MyHC promoter activity, and if so, what region of the promoter might IGF-I signaling regulate. At differentiation days 3 and 4, IGF-I increased type IIb MyHC mRNA and mouse 3.0-kb type IIb MyHC promoter activity. Deletion construct studies identified a potential IGF-I-responsive region between 1.25 and 1.2 kb of the type IIb MyHC promoter, which contained an exact 6-bp T-cell factor/lymphoid enhancer factor (Tcf/Lef) binding site at position--1206 to--1201. Site-specific mutation of the putative Tcf/Lef binding site reduced IGF-I-induced 1.3-kb type IIb MyHC promoter activity. To identify potential 1GF-I signaling molecules, the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY-294002 were both found to markedly attenuate IGF-I activation of the 1.3-kb type IIb MyHC promoter. Downstream signaling of IGF-I can phosphorylate and inactivate GSK-3[beta], thereby enhancing [beta]-catenin protein. The GSK-313 inhibitor, LiCI, dramatically enhanced IGF-I induction of the 1.3-kb type IIb MyHC promoter, and constitutively active GSK-3[beta] attenuated IGF-I-induced 1.3-kb type IIb MyHC promoter activity. Finally, IGF-I increased nuclear 13-catenin protein, and small interfering RNA knockdown of [3-catenin attenuated IGFI-induced 1.3-kb type IIb MyHC promoter activity and type IIb MyHC mRNA. In summary, IGF-I stimulation of [C.sub.2][C.sub.12] myotubes increases mouse type IIb MyHC promoter activity, likely through signaling of PI3K, GSK-3[beta], [beta]-catenin, and a Tcf/Lef binding site at -1,206 to -1,201 bp in the promoter. insulin-like growth factor I; [C.sub.2][C.sub.12] myocytes; [beta]-catenin doi: 10.1152/ajpcell.00169.2009

The effect of constitutively activated proto-oncogene H-ras (H-rasQ61L) on the regulation of myosin heavy chain (MHC) promoter activities was investigated in rabbit satellite cell-derived muscle cell culture during the proliferation stage and early and later stages of differentiation, respectively. During proliferation, overexpression of H-rasQ61L did not affect basal level of activity of the slow MHCI/[beta] or the fast MHCIId/x promoter luciferase reporter gene construct in transient transfection assays. By contrast, H-rasQ61L affected both MHC promoter activities during differentiation, and this effect changes from inactivation after 2 days to activation after 4 days of differentiation. The activating effect of H-rasQ61L on both MHC promoters after 4 days of differentiation was significantly reduced by LY-294002, a specific inhibitor of the phosphoinositol-3-kinase (PI3K), a downstream target of Ras. Furthermore, the protein kinase Akt (protein kinase B), a downstream target of PI3k, was activated 4 days after initiation of differentiation in myotubes overexpressing H-rasQ61L. By contrast, inhibition of another Ras downstream pathway, mitogen-activated protein kinase kinase 1/2-extracellular signal-regulated protein kinase 1/2 (MKK1/ 2-ERK1/2-MAPK), increased activities of both MHC promoters, indicating a suppressive role of this pathway. Moreover, the Ras-PI3K-Akt signaling pathway is involved in the activation of MHCI/[beta] and IId/x promoters in a later stage of differentiation of muscle cells, presumably by a known inhibiting effect of activated Akt on the MKK1/2-ERK1/2-MAPK pathway. The experiments demonstrate that during differentiation of muscle cells activated H-ras is an important regulator of MHC isoform promoter function with opposite effects during early and later stages. extracellular signal regulated protein kinase 1/2; mitogen-activated protein kinase; phosphosinositol-3-kinase; Akt; mammalian target of rapamycin doi: 10.1152/ajpcell.00567.2008

The role of calcineurin (Cn) in skeletal muscle fiber-type expression has been a subject of great interest because of reports indicating that it controls the slow muscle phenotype. To delineate the role of Cn in phenotype remodeling, particularly its role in driving expression of the type I myosin heavy chain (MHC) gene, we used a novel strategy whereby a profound transition from fast to slow fiber type is induced and examined in the absence and presence of cyclosporin A (CsA), a Cn inhibitor. To induce the fast-to-slow transition, we first subjected rats to 7 days of hindlimb suspension (HS) + thyroid hormone [triiodothyronine ([T.sub.3])] to suppress nearly all expression of type I MHC mRNA in the soleus muscle. HS + [T.sub.3] was then withdrawn, and rats resumed normal ambulation and thyroid state, during which vehicle or CsA (30 mg x [kg.sup.-1] x [day.sup.-1]) was administered for 7 or 14 days. The findings demonstrate that, despite significant inhibition of Cn, pre-mRNA, mRNA, and protein abundance of type I MHC increased markedly during reloading relative to HS + [T.sub.3] (P < 0.05). Type I MHC expression was, however, attenuated by CsA compared with vehicle treatment. In addition, type IIa and IIx MHC pre-mRNA, mRNA, and relative protein levels were increased in Cn-treated compared with vehicle-treated rats. These findings indicate that Cn has a modulatory role in MHC transcription, rather than a role as a primary regulator of slow MHC gene expression. hindlimb suspension; cyclosporin A; antisense RNA; myosin heavy chain SDS-PAGE; thyroid hormone doi: 10.1152/ajpregu.00349.2009

To preserve epithelial barrier function, dying cells are squeezed out of an epithelium by 'apoptotic cell extrusion.' Specifically, a cell destined for apoptosis signals its live neighboring epithelial cells to form and contract a ring of actin and myosin II that squeezes the dying cell out of the epithelial sheet. Although most apoptotic cells extrude apically, we find that some exit basally. Localization of actin and myosin IIA contraction dictates the extrusion direction: basal extrusion requires circumferential contraction of neigh boring cells at their apices, whereas apical extrusion also requires downward contraction along the basolateral surfaces. To activate actin/myosin basolaterally, microtubules in neighboring cells reorient and target p115 RhoGEF to this site. Preventing microtubule reorientation restricts contraction to the apex, driving extrusion basally. Extrusion polarity has important implications for tumors where apoptosis is blocked but extrusion is not, as basal extrusion could enable these cells to initiate metastasis.

We performed 2 wk of mechanical overload by synergist ablation on plantaris muscles from a small rodent hibernator, Spermophilus lateralis. While this muscle displays prominent myosin heavy-chain (MyHC) isoform shifts during hibemation, sensitivity to mechanical loading as a stimulus for muscle mass and isoform plasticity has not been demonstrated. Squirrel muscles, whether during hibemation or not, potentially are less sensitive to mechanical unloading, but we hypothesized that increased loading would produce the typical mammalian response of greater plantaris mass and MyHC shifts. Mechanical overload produced a 50% increase in muscle mass but, surprisingly, no changes in MyHC isoform protein or mRNA expression, despite previously observed fast-to-slow MyHC isoform switching during hibernation. Citrate synthase enzyme activity, as well as mRNA expression of creatine kinase and the muscle growth factor myostatin, were all unchanged. The mRNA expression of critical muscle atrophy genes decreased by 50% during hypertrophy, including ubiquitin ligases MuRF1 and MAFbx, and the related transcription factor FOXO-1a. Insulin-like growth factor (IGF-1) and hypoxia-inducihle factor (HIF-1[alpha]) mRNA expression was elevated by 400% and 150%. Fast-to-slow MyHC isoform shifts appear unnecessary to support the increased recruitment of the plantaris muscle, shifts which are seen in other rodent models. Our results are consistent with muscular activity during interbout arousals as a potential mechanism to preserve muscle mass, but illustrate the primary importance of other seasonal factors besides patterns of muscle activation which must act in concert to alter MyHC isoforms and muscle fiber type during hibernation. myosin heavy-chain isoforms; hibernation; skeletal muscle hypertrophy; FOXO; ubiquitin ligases

The intracellular signals that convert fast and slow motor neuron activity into muscle fiber type specific transcriptional programs have only been partially defined. The calcium/calmodulin-dependent phosphatase calcineurin (Cn) has been shown to mediate the transcriptional effects of motor neuron activity, but precisely how 4 distinct muscle fiber types are composed and maintained in response to activity is largely unknown. Here, we show that 4 nuclear factor of activated T cell (NFAT) family members act coordinately downstream of Cn in the specification of muscle fiber types. We analyzed the role of NFAT family members in vivo by transient transfection in skeletal muscle using a loss-of-function approach by RNAi. Our results show that, depending on the applied activity pattern, different combinations of NFAT family members translocate to the nucleus contributing to the transcription of fiber type specific genes. We provide evidence that the transcription of slow and fast myosin heavy chain (MyHC) genes uses different combinations of NFAT family members, ranging from MyHC-slow, which uses all 4 NFAT isoforms, to MyHC-2B, which only uses NFATc4. Our data contribute to the elucidation of the mechanisms whereby activity can modulate the phenotype and performance of skeletal muscle. skeletal muscle | calcineurin | myosin | gene regulation

Phosphorylation of the myosin regulatory light chain (RLC) in skeletal muscle has been proposed to act as a molecular memory of recent activation by increasing the rate of force development, ATPase activity, and isometric force at submaximal activation in fibers. It has been proposed that these effects stem from phosphorylation-induced movement of myosin heads away from the thick filament backbone. In this study, we examined the molecular effects of skeletal muscle myosin RLC phosphorylation using in vitro motility assays. We showed that, independently of the thick filament backbone, the velocity of skeletal muscle myosin is decreased upon phosphorylation due to an increase in the myosin duty cycle. Furthermore, we did not observe a phosphorylation-dependent shift in calcium sensitivity in the absence of the myosin thick filament. These data suggest that phosphorylation-induced movement of myosin heads away from the thick filament backbone explains only part of the observed phosphorylation-induced changes in myosin mechanics. Last, we showed that the duty cycle of skeletal muscle myosin is strain dependent, consistent with the notion that strain slows the rate of ADP release in striated muscle. in vitro motility assay; mechanics

Muscular lymphatics use both phasic and tonic contractions to transport lymph for conducting their vital functions. The molecular mechanisms regulating lymphatic muscle contractions are not well understood. Based on the well-established finding that the phosphorylation of myosin light chain 20 ([MLC.sub.20]) plays an essential role in blood vessel smooth muscle contraction, we investigated if phosphorylated [MLC.sub.20] (p[MLC.sub.20]) would modulate the tonic and/or phasic contractions of lymphatic muscle. The effects of ML-7, a MLC kinase inhibitor (1-10 [micro]M), were tested on the contractile parameters of isolated and cannulated rat mesenteric lymphatics during their responses to the known modulators, pressure (1-5 cm[H.sub.2]O) and substance P (SP; [10.sup.-7] M). Immunohistochemical and Western blot analyses of p[MLC.sub.20] were also performed on isolated lymphatics. The results showed that 1) increasing pressure decreased both the lymphatic tonic contraction strength and p[MLC.sub.20]-to-[MLC.sub.20] ratio; 2) SP increased both the tonic contraction strength and phosphorylation of [MLC.sub.20]; 3) ML-7 decreased both the lymphatic tonic contraction strength and p[MLC.sub.20]-to-[MLC.sub.20] ratio; and 4) the increase in lymphatic phasic contraction frequency in response to increasing pressure was diminished by ML-7; however, the phasic contraction amplitude was not significantly altered by ML-7 either in the absence or presence of SP. These data provide the first evidence that tonic contraction strength and phasic contraction amplitude of the lymphatics can be differentially regulated, whereby the increase in [MLC.sub.20] phosphorylation produces an activation in the tonic contraction without significant changes in the phasic contraction amplitude. Thus, tonic contraction of rat mesenteric lymphatics appears to be MLC kinase dependent. ML-7; lymphatic contraction; tonic contraction; phasic contraction

The P2[X.sub.7] receptor is a ligand-gated cation channel that is highly expressed on monocyte-macrophages and that mediates the pro-inflammatory effects of extracellular ATP. Dilation of the P2[X.sub.7] channel and massive [K.sup.+] efflux follows initial channel opening, but the mechanism of secondary pore formation is unclear. The proteins associated with P2[X.sub.7] were isolated by using anti-P2[X.sub.7] monoclonal antibody-coated Dynabeads from both interferon-[gamma] plus LPS-stimulated monocytic THP-1 cells and P2[X.sub.7]-transfected HEK-293 cells. Two nonmuscle myosins, NMMHC-IIA and myosin Va, were found to associate with P2[X.sub.7] in THP-1 cells and HEK-293 cells, respectively. Activation of the P2[X.sub.7] receptor by ATP caused dissociation of P2[X.sub.7] from nonmuscle myosin in both cell types. The interaction of P2[X.sub.7] and NMMHC-IIA molecules was confirmed by fluorescent life time measurements and fluorescent resonance of energy transfer-based time-resolved flow cytometry assay. Reducing the expression of NMMHC-IIA or myosin Va by small interfering RNA or short hairpin RNA led to a significant increase of P2[X.sub.7] pore function without any increase in surface expression or ion channel function of P2[X.sub.7] receptors. S-l-blebbistatin, a specific inhibitor of NMMHC-IIA ATPase, inhibited both ATP-induced ethidium uptake and ATP-induced dissociation of P2[X.sub.7]-NMMHC-IIA complex. In both cell types nonmuscle myosin closely interacts with P2[X.sub.7] and is dissociated from the complex by extracellular ATP. Dissociation of this anchoring protein may be required for the transition of P2[X.sub.7] channel to a pore. short hairpin RNA; blebbistatin; THP-1 cells; fluorescent resonance energy transfer; ethidium uptake; myosin IIA; myosin Va; phagocytosis

The actin-myosin interaction of vascular smooth muscle cells (VSMCs) is regulated by myosin light chain kinase (MLCK), which is a fusion protein of the central catalytic domain with the N-terminal actin-binding and C-terminal myosin-binding domains. In addition to the regulatory role of kinase activity mediated by the catalytic domain, nonkinase activity that derives from both terminals is able to exert a regulatory role as reviewed by Nakamura et al. (32). We previously showed that nonkinase activity mediated the filopodia upon the stimulation by sphingosylphosphorylcholine (SPC) (25). To explore the regulatory role of nonkinase activity in chemotaxis, we constructed VSMCs where the expression of MLCK was totally abolished by using a lentivirus-mediated RNAi system. We hypothesized that the MLCK-downregulated VSMCs were unable to form filopodia and to migrate upon SPC stimulation and confirmed the hypothesis. We further constructed a kinase-inactive mutant from bovine cDNA coding wild-type (WT) MLCK by mutating the ATP-binding sites located in the catalytic domain, followed by confirming the presence (absence) of the kinase activity of WT (kinase-inactive mutant). We transfected WT and the mutant into MLCK-downregulated VSMCs. We expected that the transfected VSMCs will recover the ability to induce filopodia and chemotaxis toward SPC and found both constructs rescued the ability. Because they share the actin- and myosin-binding domains, we concluded nonkinase activity plays a major role for SPC-induced migration. chemotaxis; myosin light chain kinase

The Frank-Starling relationship of the heart yields increased stroke volume with greater end-diastolic volume, and this relationship is steeper after [beta]-adrenergic stimulation. The underlying basis for the Frank-Starling mechanism involves length-dependent changes in both [Ca.sup.2+] sensitivity of myofibrillar force and power output. In this study, we tested the hypothesis that PKA-induced phosphorylation of myofibrillar proteins would increase the length dependence of myofibrillar power output, which would provide a myofibrillar basis to, in part, explain the steeper Frank-Starling relations after [beta]-adrenergic stimulation. For these experiments, adult rat left ventricles were mechanically disrupted, permeabilized cardiac myocyte preparations were attached between a force transducer and position motor, and the length dependence of loaded shortening and power output were measured before and after treatment with PKA. PKA increased the phosphorylation of myosin binding protein C and cardiac troponin I, as assessed by autoradiography. In terms of myocyte mechanics, PKA decreased the [Ca.sup.2+] sensitivity of force and increased loaded shortening and power output at all relative loads when the myocyte preparations were at long sarcomere length (~2.30 [micro]m). PKA had less of an effect on loaded shortening and power output at short sarcomere length (~2.0 [micro]m). These changes resulted in a greater length dependence of myocyte power output after PKA treatment; peak normalized power output increased ~20% with length before PKA and ~40% after PKA. These results suggest that PKA-induced phosphorylation of myofibrillar proteins explains, in part, the steeper ventricular function curves (i.e., Frank-Starling relationship) after [beta]-adrenergic stimulation of the left ventricle. Frank-Starling; protein kinase A; myosin binding protein C; cardiac troponin I

Peroxisome proliferator-activated receptor (PPAR)-[gamma] ligands, thiazolidinediones, have been demonstrated to regulate vascular reactivity. We examined the effect of pioglitazone (PIO; 20 [micro]M) in rat primary cultured aortic smooth muscle cells on constitutive phosphorylation of the regulatory subunit of myosin phosphatase (MYPT). PIO decreased the phosphorylation of [Thr.sup.697] on MYPT within 15 min, and the inhibition was maintained up to 6 h. The PPAR-[gamma] antagonist GW-9662 (5 [micro]M) abrogated the inhibition of [Thr.sup.697] phosphorylation mediated by PIO. Because longer-term PIO treatment inhibits RhoA/Rho kinase (ROCK) signaling and [Thr.sup.697] phosphorylation, we tested the effect of the ROCK inhibitor Y-27632 (10 [micro]M) on the inhibition of [Thr.sup.697] phosphorylation by PIO. Y-27632 alone inhibited [Thr.sup.697] phosphorylation, and there was an additive effect with PIO. In addition, up to 1 h of PIO treatment did not affect RhoA localization or decrease ROCK-dependent phosphorylation of [Thr.sup.855]. These results suggest that the effect of PIO is independent of inhibition of RhoA/ROCK. PIO increased the phosphorylation of [Ser.sup.696] in the same time course as its effect on [Thr.sup.697]. [Ser.sup.696] has been shown to be phosphorylated by PKA and PKG. PKA inhibitor H-89 (10 [micro]M) and PKG inhibitor KT-5823 (0.5 [micro]M) abrogated the effect of PIO on both [Thr.sup.697] and [Ser.sup.696] phosphorylation. The constitutive turnover of phosphorylation of [Thr.sup.697] is rapid, suggesting that the decreased phosphorylation of [Thr.sup.697] by PIO is due to enhanced phosphorylation of [Ser.sup.696]. This is supported by the finding that PIO blocks ANG II-stimulated phosphorylation of [Thr.sup.697] but not ANG II-stimulated RhoA translocation. Therefore, the effect of shorter-term PIO apparently is to increase myosin light chain phosphatase activity, thereby desensitizing the vascular smooth muscle to agonist signaling. myosin phosphatase regulatory subunit; peroxisome proliferator-activated receptor-[gamma]; vascular

The potential roles of the regulatory proteins actin, tropomyosin (Tm), and caldesmon (CaD), i.e., the components of the thin filament, in smooth muscle have been extensively studied in several types of smooth muscles. However, controversy remains on the putative physiological significance of these proteins. In this study, we intended to determine the functional roles of Tm and CaD in the regulation of smooth muscle contraction by using a reconstitution system of the thin filaments. At appropriate conditions, the thin (actin) filaments within skinned smooth muscle strips of taenia caeci in guinea pigs could be selectively removed by an actin-severing protein, gelsolin, without irreversible damage to the contractile apparatus, and then the thin filaments were reconstituted with purified components of thin filaments, i.e., actin, Tm, and CaD. We found that the structural remodeling of actin filaments or thin filaments was functionally linked to the [Ca.sup.2+]-induced force development and reduction in muscle cross-sectional area (CSA). That is, after the reconstitution of the gelsolin-treated skinned smooth muscle strips with pure actin, the [Ca.sup.2+]-dependent force development was partially restored, but the [Ca.sup.2+]-induced reduction in CSA occurred once. In contrast, the reconstitution with actin, followed by Tm and CaD, restored not only the force generation but also both its [Ca.sup.2+] sensitivity and the reversible [Ca.sup.2+]-dependent reduction in CSA. We confirmed that both removal of the thin filaments by gelsolin treatment and reconstitution of the actin (thin) filaments with Tm and CaD caused no significant changes in the level of myosin regulatory light chain phosphorylation. We thus conclude that Tm and CaD are necessary for the full regulation of smooth muscle contraction in addition to the other regulatory systems, including the myosin-linked one. confocal fluorescence microscopy; force development; reduction in muscle cross-sectional area; thin filament-linked regulation; phalloidin; phosphorylation of myosin regulatory light chain

Roles of basolateral solute uptake via NKCC1 and of myosin II in vasopressin-induced cell swelling in inner medullary collecting duct. AM J Physiol Renal Physiol 292: F192-F201, 2008. Fist published April 16, 2008: doi:10.1152/ajprenal.00011-2008.--Collecting duct cells swell when exposed to argininge vasopressin (AVP) in the presence of a transepithelial osmolality gradient. We investigated the mechanisms of AVP-induced cell swelling in isolated , perfused rate inner medullary collecting ducts (IMCDs) using quantitative video microscopy and fluorescence-based measurements of transepithelial water transport. We tested the roles of transepithelial water flow, basolateral solute entry, and the cytoskeleton (actomysosin). Whe a transepithelial osmolality gradient was imposed by addition of NaCl to the bath, AVP significantly increased both water flux and cell height. When the osmolality gradient was imposed by addition of mannitol, AVP increased water flux but not cell height, suggesting that AVP-induced cell swelling requires a NaCl gradien and is not merely dependent on the associated water flux. Bumetanide (Na-K-2Cl contransporter inhibitor) added to the bath markedly diminished the AVP-induced cell height increase. AVP-induced cell swelling was absent in IMCDs form NKCC1-knockout mice. In rat IMCDs, replacement of Na, K, or Cl in the peritubular bath cased significant cell shrinkage, consistent with a basolateral solute transport pathway dependent on all three ions. Immunocytochemistry using an antibody to NKCC1 confirmed basolateral expression in IMCD cells. The conventional nonmuscle myosin II inhibitor blebbistatin also diminished the AVP-induced cell height increase and cell shape change, consistent with a role for the actin cytoskeleton and myosin II. We conclude that the AVP-induced cell height increase is dependent on basolateral solute uptake via NKCC1 and changes in actin organization via myosin II, but is not dependent specifically on increased apical water entry. cell volume; blebbistatin; cytoskeleton; kidney; inner medulla

Acidosis (low pH) is the oldest putative agent of muscular fatigue, but the molecular mechanism underlying its depressive effect on muscular performance remains unresolved. Therefore, the effect of low pH on the molecular mechanics and kinetics of chicken skeletal muscle myosin was studied using in vitro motility (IVM) and single molecule laser trap assays. Decreasing pH from 7.4 to 6.4 at saturating ATP slowed actin filament velocity ([V.sub.actin]) in the IVM by 36%. Single molecule experiments, at 1 [micro]M ATP, decreased the average unitary step size of myosin (d) from 10 [+ or -] 2 nm (pH 7.4) to 2 [+ or -] 1 nm (pH 6.4). Individual binding events at low pH were consistent with the presence of a population of both productive (average d = 10 nm) and nonproductive (average d = 0 nm) actomyosin interactions. Raising the ATP concentration from 1 [micro]M to 1 mM at pH 6.4 restored d (9 [+ or -] 3 nm), suggesting that the lifetime of the nonproductive interactions is solely dependent on the [ATP]. [V.sub.actin], however, was not restored by raising the [ATP] (1-10 mM) in the IVM assay, suggesting that low pH also prolongs actin strong binding ([t.sub.on]). Measurement of ton as a function of the [ATP] in the single molecule assay suggested that acidosis prolongs ton by slowing the rate of ADP release. Thus, in a detachment limited model of motility (i.e., [V.sub.actin] ~ d/[t.sub.on]), a slowed rate of ADP release and the presence of nonproductive actomyosin interactions could account for the acidosis-induced decrease in [V.sub.actin], suggesting a molecular explanation for this component of muscular fatigue. acidosis; fatigue; velocity; laser trap

We investigated the role of the actin-based myosin motor, myosin 5c (Myo5c) in vesicle transport in exocrine secretion. Lacrimal gland acinar cells (LGAC) are the major source for the regulated secretion of proteins from the lacrimal gland into the tear film. Confocal fluorescence and immunogold electron microscopy revealed that Myo5c was associated with secretory vesicles in primary rabbit LGAC. Upon stimulation of secretion with the muscarinic agonist, carbachol, Myo5c was also detected in association with actin-coated fusion intermediates. Adenovirus-mediated expression of green fluorescent protein (GFP) fused to the tail domain of Myo5c (Ad-GFP-Myo5c-tail) showed that this protein was localized to secretory vesicles. Furthermore, its expression induced a significant (P [less than or equal to] 0.05) decrease in carbachol-stimulated release of two secretory vesicle content markers, secretory component and syncollin-GFP. Adenovirus-mediated expression of GFP appended to the full-length Myo5c (Ad-GFP-Myo5c-full) was used in parallel with adenovirus-mediated expression of GFP-Myo5c-tail in LGAC to compare various parameters of secretory vesicles labeled with either GFP-labeled protein in resting and stimulated LGAC. These studies revealed that the carbachol-stimulated increase in secretory vesicle diameter associated with compound fusion of secretory vesicles that was also exhibited by vesicles labeled with GFP-Myo5c-full was impaired in vesicles labeled with GFP-Myo5c-tail. A significant decrease in GFP labeling of actin-coated fusion intermediates was also seen in carbachol-stimulated LGAC transduced with GFPMyo5c-tail relative to LGAC transduced with GFP-Myo5c-full. These results suggest that Myo5c participates in apical exocytosis of secretory vesicles. actin; lacrimal gland; tear film

Phosphorylation of the 20-kDa myosin regulatory light chains ([LC.sub.20]) plays a key role in the regulation of smooth muscle contraction. The level of [LC.sub.20] phosphorylation is governed by the relative activities of myosin light chain kinase and phosphatase pathways. The regulation of these two pathways differs in different smooth muscle types and in the actions of different vasoactive stimuli. Little is known concerning the regulation of [LC.sub.20] phosphorylation in the renal microcirculation. The available pharmacological probes are often nonspecific, and current techniques to directly measure [LC.sub.20] phosphorylation are not sensitive enough for quantification in small arterioles. We describe here a novel approach to address this important issue. Using SDS-PAGE with polyacrylamide-bound [Mn.sup.2+]-phosphate-binding tag and enhanced Western blot analysis, we were able to detect [LC.sub.20] phosphorylation using as little as 5 pg (250 amol) of isolated [LC.sub.20]. Phosphorylated and unphosphorylated [LC.sub.20] were detected in single isolated afferent arterioles, and [LC.sub.20] phosphorylation levels could be accurately quantified in pooled samples of three arterioles ( afferent arteriole; Phos-tag SDS-PAGE

Myosin VI is an actin-based motor that has been implicated in many cellular processes. Studies in vertebrates have demonstrated that animals lacking this ubiquitously expressed myosin are viable. However in Drosophila, myosin VI loss of function has been thought to be lethal. We show here that complete loss of myosin VI is not lethal in flies and that the previously reported lethality of the null mutation ([jar.sup.322]) is most likely due to deletion of a neighboring gene. Maternally provided myosin VI does not account for the survival of myosin VI null animals. Mutant animals are recovered at a lower than expected Mendelian frequency, suggesting that myosin VI participates in processes which contribute to normal development, but its participation is not essential.

Male but not female mice carrying a single R403Q missense allele for cardiac [alpha]-myosin heavy chain (M-[alpha].sup.MH[C.sup.R403Q/+] and F-[alpha]MH[C.sup.R403Q/+], respectively) develop significant hypertrophic cardiomyopathy (HCM) compared with male and female wild-type mice (M-[alpha]MH[C.sup.+/+] and F-[alpha]MH[C.sup.+/+], respectively) after Tilde 30 wk of age. We tested the hypothesis that myofilament mechanical performance differs between M-[alpha]MH [C.sup.403Q/+] and F-[alpha]MH[C.sup.R403Q/+ ] at younger ages (10-20 wk) and could account for sex differences in HCM development. The sensitivity of chemically skinned myocardial strips to [Ca.sup.2+] activation (p[Ca.sub.50]) was signifcantly (P < 0.05) enhanced in male mice independent of genotype (M-[alpha]MH[C.sup.R403Q/+] 5.70 [+ or -] 0.06, M-[alpha]MH[C.sup.+/+]: 5.63 [+ or -] + 0.05, F-[alpha]MH [C.sup.R403Q/+]: 5.57 [+ or -] 0.03, F-[alpha] MH[C.sup.+/+]: 5.54 [+ or -] 0.04) by two-way ANOVA, whereas maximum developed tension was significantly enhanced in [alpha]-MH[C.sup.R403Q/+] independent of sex (M-[alpha]MH[C.sup.R403Q/+]: 29.3 [+ or -] 2.3, M-[alpha]MH [C.sup.+/+]: 26.0 [+ or -] 1.4, F-[alpha]MH[C.sup.R403Q/+]: 30.2 [+ or -] 2.1, F-[alpha]MH[C.sup.+/+]: 26.2 [+ or -] 1.2 mN/[mm.sup.2]). The frequency of maximum work generated by sinusoidal length perturbation was significantly higher in [alpha]MH[C.sup.R403Q/+] mice than in sex-matched controls (M-[alpha]MH[CR403Q/+]: 2.26 [+ or -] 0.47, M-[alpha]MH[C.sup.+/+]: 1.29[+ or -] 0.18, F-[alpha]MH [C.sup.R403Q/+]: 3.21 [+ or -] 0.33, F-[alpha]MH[C.sup.+/+]: 2.52 [+ or -] 0.36 Hz). Unloaded shortening velocity was significantly enhanced in [alpha]MH[C.R403Q/+] and in female mice (M-[alpha]MH [C.sup.R403Q/+]: 2.26 [+ or -] 0.47, M-[alpha]MH[C.sup.+/+]: 1.29 [+ or -] 0.18, F-[alpha]MH[C.sup.R403Q/+]: 3.21 [+ or -] 0.33, F-[alpha]MH [C.sup.+/+]: 2.52 [+ or -] 0.36 muscle lengths/s), and normalized mechanical power, calculated from the tension-velocity relationship, was significantly enhanced in [alpha]MH[C.sup.R403Q/+] independent of sex (M[alpha]MH[C.sup.R403Q/+]: 60 [+ or -] 2 [10.sup.-3], M-[alpha]MH [C.sup.+/+]: 37 [+ or -] 3 [10.sup.- 3], F-[alpha]MH[C.sup.R403Q/+]: 57 [+ or -] 3 [10.sup.-3], F-[alpha]MH [C.sup.+/+] 25 [+ or -] 3 [10.sup.-3] muscle lengths/s x normalized tension). We did not find a statistically significant sex x mutation interaction for any measure of myofilament performance. Therefore, sarcomeric incorporation of the R403Q myosin similarly enhanced left ventricular myofilament mechanical performance in both male and female mice. The sex-dependent development of HCM due to the R403Q myosin may then be inhibited by female sex hormones, which may additionally underlie the observed sex differences for p[Ca.sub.50] and unloaded shortening velocity. hypertrophic cardiomyopathy; myosin heavy chain; isometric tension; calcium sensitivity; force-velocity

The study result which emphasizes the generic and specific features of the tropomyosin molecule is presented. The findings confirm the quasi-equivalent periodic surface residues for actin binding.

Myosin-motors are conserved from yeast to human and transport a great variety of cargoes. Most plus-end directed myosins, which constitute the vast majority of all myosin motors, form stable dimers and interact constitutively with their cargo complexes. To date, little is known about regulatory mechanisms for cargo-complex assembly. In this study, we show that the type V myosin Myo4p binds to its cargo via two distinct binding regions, the C-terminal tail and a coiled-coil domain-containing fragment. Furthermore, we find that Myo4p is strictly monomeric at physiologic concentrations. Because type V myosins are thought to require dimerization for processive movement, a mechanism must be in place to ensure that oligomeric Myo4p is incorporated into cargo-translocation complexes. Indeed, we find that artificial dimerization of the Myo4p C-terminal tail promotes stabilization of myosin-cargo complexes, suggesting that full-length Myo4p dimerizes in the cocomplex as well. We also combined the Myo4p C-terminal tail with the coiled-coil region, lever arm, and motor domain from a different myosin to form constitutively dimeric motor proteins. This heterologous motor successfully translocates its cargo in vivo, suggesting that wild-type Myo4p may also function as a dimer during cargo-complex transport. cell asymmetry | Myo4p | RNA localization | She3p | motor protein

Blebbistatin is a powerful inhibitor of actin-myosin interaction in isolated contractile proteins. To examine whether blebbistatin acts in a similar manner in the organized contractile system of striated muscle, the effects of blebbistatin on contraction of cardiac tissue from mouse were studied. The contraction of paced intact papillary muscle preparations and shortening of isolated cardiomyocytes were inhibited by blebbistatin with inhibitory constants in the micromolar range (1.3-2.8 [micro]M). The inhibition constants are similar to those previously reported for isolated cardiac myosin subfragments showing that blebbistatin action is similar in filamentous myosin of the cardiac contractile apparatus and isolated proteins. The inhibition was not associated with alterations in action potential duration or decreased influx through L-type [Ca.sup.2+] channels. Experiments on permeabilized cardiac muscle preparations showed that the inhibition was not due to alterations in [Ca.sup.2+] sensitivity of the contractile filaments. The maximal shortening velocity was not affected by 1 [micro]M blebbistatin. In conclusion, we show that blebbistatin is an inhibitor of the actin-myosin interaction in the organized contractile system of cardiac muscle and that its action is not due to effects on the [Ca.sup.2+] influx and activation systems. heart; electrophysiology; permeabilized muscle

Stopped-flow experiments result of the interaction of [gamma]-amido-ATP (ATPN) and its 2'(3')-O-methylanthraniloyl derivative (mantATPN) with skeletal myosin subfragment 1 (S1) and actomysin (actoS1) demonstrate that the use of ATPN provides experimental access to S1 and actoS1 states with myosin in the closed conformation which are not accessible by other nucleotide analogues.

Vascular smooth muscle (VSM) maturation is developmentally regulated and differs between vascular beds. The maturation and contribution of VSM function to tissue blood flow and blood pressure regulation during early gestation are unknown. The carotid artery (CA) contributes to fetal cerebral blood flow regulation and well being. We studied CA VSM contractility, protein contents, and phenotype beginning in the midthird of ovine development. CAs were collected from early (88-101 day of gestation) and late (138-150 day; term = day 150) fetal (n = 14), newborn (6-8 day old; n = 7), and adult (n = 5) sheep to measure forces in endothelium-denuded rings with KC1, phenylephrine, and ANG II; changes in cellular proteins, including total and soluble protein, actin and myosin, myosin heavy chain isoforms (MHC), filamin, and proliferating cell nuclear antigen; and vascular remodeling. KC1 and phenylephrine elicited age- and dose-dependent contraction responses (P < 0.001) at all ages except early fetal, which were unresponsive. In contrast, ANG II elicited dose responses only in adults, with contractility increasing greater than fivefold vs. that shown in fetal or neonatal animals (P < 0.001). Increased contractility paralleled age-dependent increases (P < 0.01) in soluble protein, actin and myosin, filamin, adult smooth muscle MHC-2 (SM2) and medial wall thickness and reciprocal decreases (P < 0.001) in nonmuscle MHC-B, proliferating cell nuclear antigen and medial cellular density. VSM nonreceptor- and receptor-mediated contractions are absent or markedly attenuated in midgestation and increase age dependently, paralleling the transition from synthetic to contractile VSM phenotype and, in the case of ANG II, paralleling the switch to the [AT.sub.1] receptor. The mechanisms regulating VSM maturation and thus blood pressure and tissue perfusion in early development remain to be determined. myosin heavy chain isoforms; nonmuscle myosin; fetal development; receptor and nonreceptor function; smooth muscle growth; angiotensin-II doi:10.1152/ajpregu.00851.2006

Diaphragm weakness commonly occurs in patients with congestive heart failure (CHF) and is an independent predictor of mortality. However, the pathophysiology of diaphragm weakness is poorly understood. We hypothesized that CHF induces diaphragm weakness at the single-fiber level by decreasing myosin content. In addition, we hypothesized that myofibrillar [Ca.sup.2+] sensitivity is decreased and cross-bridge kinetics are slower in CHF diaphragm fibers. Finally, we hypothesized that loss of myosin in CHF diaphragm weakness is associated with increased proteolytic activities of caspase-3 and the proteasome. In skinned diaphragm single fibers of rats with CHF, induced by left coronary artery ligation, maximum force generation was reduced by ~35% (P < 0.01) compared with sham-operated animals for slow, 2a, and 2x fibers. In these CHF diaphragm fibers, myosin heavy chain content per half-sarcomere was concomitantly decreased (P < 0.01). [Ca.sup.2+] sensitivity of force generation and the rate constant of tension redevelopment were significantly reduced in CHF diaphragm fibers compared with sham-operated animals for all fiber types. The cleavage activity of the proteolytic enzyme caspase-3 and the proteasome were ~30% (P < 0.05) and ~60% (P < 0.05) higher, respectively, in diaphragm homogenates from CHF rats than from sham-operated rats. The present study demonstrates diaphragm weakness at the single-fiber level in a myocardial infarct model of CHF. The reduced maximal force generation can be explained by a loss of myosin content in all fiber types and is associated with activation of caspase-3 and the proteasome. Furthermore, CHF decreases myofibrillar [Ca.sup.2+] sensitivity and slows cross-bridge cycling kinetics in diaphragm fibers. skinned muscle single fibers; cross-bridge cycling kinetics; [Ca.sup.2+] sensitivity; proteasome; caspase-3 doi:10.1152/ajpregu.00085.2007

Point mutations in cardiac myosin, the heart's molecular motor, produce distinct clinical phenotypes: hypertrophic (HCM) and dilated (DCM) cardiomyopathy. Do mutations alter myosin's molecular mechanics in a manner that is predictive of the clinical outcome? We have directly characterized the maximal force-generating capacity ([F.sub.max]) of two HCM (R403Q, R453C) and two DCM (S532P, F764L) mutant myosins isolated from homozygous mouse models using a novel load-clamped laser trap assay. [F.sub.max] was 50% (R403Q) and 80% (R453C) greater for the HCM mutants compared with the wild type, whereas [F.sub.max] was severely depressed for one of the DCM mutants (65% S532P). Although [F.sub.max] was normal for the F764L DCM mutant, its actin-activated ATPase activity and actin filament velocity ([V.sub.actin]) in a motility assay were significantly reduced (Schmitt JP, Debold EP, Ahmad F, Armstrong A, Frederico A, Conner DA, Mende U, Lohse MJ, Warshaw D, Seidman CE, Seidman JG. Proc Natl Acad Sci USA 103: 14525-14530, 2006.). These [F.sub.max] data combined with previous [V.sub.actin] measurements suggest that HCM and DCM result from alterations to one or more of myosin's fundamental mechanical properties, with HCM-causing mutations leading to enhanced but DCM-causing mutations leading to depressed function. These mutation-specific changes in mechanical properties must initiate distinct signaling cascades that ultimately lead to the disparate phenotypic responses observed in HCM and DCM. familial hypertrophic cardiomyopathy; in vitro motility; force-velocity relationship; heart failure doi:10.1152/ajpheart.00128.2007

Previously, a small molecule, reversine, was identified that reverses lineage-committed murine myoblasts to a more primitive multipotent state. Here, we show that reversine can increase the plasticity of C2C12 myoblasts at the single-cell level and that reversine-treated cells gain the ability to differentiate into osteoblasts and adipocytes under lineage-specific inducing conditions. Moreover, reversine is active in multiple cell types, including 3T3E1 osteoblasts and human primary skeletal myoblasts. Biochemical and cellular experiments suggest that reversine functions as a dual inhibitor of nonmuscle myosin II heavy chain and MEK1, and that both activities are required for reversine's effect. Inhibition of MEK1 and nonmuscle myosin II heavy chain results in altered cell cycle and changes in histone acetylation status, but other factors also may contribute to the activity of reversine, including activation of the PI3K signaling pathway. myoblasts | multipotent | small molecule | osteoblast | adipocyte

Chronic hypoxia triggers pulmonary vascular remodeling, which is associated with a modulation of the vascular smooth muscle cell (SMC) phenotype from a contractile, differentiated to a synthetic, dedifferentiated state. We previously reported that acute hypoxia represses cGMP-dependent protein kinase (PKG) expression in ovine fetal pulmonary venous SMCs (FPVSMCs). Therefore, we tested if altered expression of PKG could explain SMC phenotype modulation after exposure to hypoxia. Hypoxia-induced reduction in PKG protein expression strongly correlated with the repressed expression of SMC phenotype markers, myosin heavy chain (MHC), calponin, vimentin, s-smooth muscle actin ([alpha]SMA), and thrombospondin (TSP), indicating that hypoxic exposure of SMC induced phenotype modulation to dedifferentiated state, and PKG may be involved in SMC phenotype modulation. PKG-specific small interfering RNA (siRNA) transfection in FPVSMCs significantly attenuated calponin, vimentin, and MHC expression, with no effect on [alpha]SMA and TSP. Treatment with 30 [micro]M Drosophila Antennapedia (DT-3), a membrane-permeable peptide inhibitor of PKG, attenuated the expression of TSP, MHC, [alpha]SMA, vimentin, and calponin. The results from PKG siRNA and DT-3 studies indicate that hypoxia-induced reduction in protein expression was also similarly impacted by PKG inhibition. Overexpression of PKG in FPVSMCs by transfection with a full-length PKG construct tagged with green fluorescent fusion protein (PKG-GFP) reversed the effect of hypoxia on the expression of SMC phenotype marker proteins. These results suggest that PKG could be one of the determinants for the expression of SMC phenotype marker proteins and may be involved in the maintenance of the differentiated phenotype in pulmonary vascular SMCs in hypoxia. vein; small interfering RNA doi: 10.1152/ajplung.00143.2006

This study was aimed to achieve a definitive and unambiguous identification of fiber types in canine skeletal muscles and of myosin isoforms that are expressed therein. Correspondence of canine myosin isoforms with orthologs in other species as assessed by base sequence comparison was the basis for primer preparation and for expression analysis with RT-PCR. Expression was confirmed at protein level with histochemistry, immunohistochemistry, and SDS-PAGE combined together and showed that limb and trunk muscles of the dog express myosin heavy chain (MHC) type 1, 2A, and 2X isoforms and the so-called 'type 2dog' fibers express the MHC-2X isoform. MHC-2A was found to be the most abundant isoform in the trunk and limb muscle. MHC-2X was expressed in most but not all muscles and more frequently in hybrid 2A-2X fibers than in pure 2X fibers. MHC-2B was restricted to specialized extraocular and laryngeal muscles, although 2B mRNA, but not 2B protein, was occasionally detected in the semimembranosus muscle. Isometric tension ([P.sub.o]) and maximum shortening velocity ([V.sub.o]) were measured in single fibers classified on the basis of their MHC isoform composition. Purified myosin isoforms were extracted from single muscle fibers and characterized by the speed ([V.sub.f]) of actin filament sliding on myosin in an in vitro motility assay. A close proportionality between [V.sub.o] and [V.sub.f] indicated that the diversity in [V.sub.o] was due to the different myosin isoform composition. [V.sub.o] increased progressively in the order 1/slow < 2A < 2X < 2B, thus confirming the identification of the myosin isoforms and providing their first functional characterization of canine muscle fibers. dog; muscle fiber types; single fiber mechanics; in vitro motility doi:10.1152/ajpcell.00601.2006

Muscle fiber types are classified based on contractile speed and type of metabolism. Fast-contracting fibers involve mainly glycolytic-based metabolism, whereas slow-contracting fibers involve a more oxidative type of energy metabolism. The relationship between expression of the genes controlling these functional characteristics and their relative protein abundance in porcine muscle is unknown. The objective of this study was to determine the expression of adult myosin heavy-chain (MyHC) genes and their corresponding protein content in various porcine muscles. Moreover, changes in expression of 2 genes involved in energy metabolism (glycogen synthase and citrate synthase) were determined on muscles varying in MyHC. Using real-time PCR, the relative transcript abundance was determined for the adult MyHC isoforms (types I, IIA, IIX, and IIB), glycogen synthase, and citrate synthase in the masseter (MAS), diaphragm, longissimus, cutaneous trunci, and red and white semitendinosus muscles of 7 pigs. Each muscle was subjected to SDS-PAGE analyses to determine the relative abundance of each MyHC. The relative transcript abundance of type IIB MyHC was greatest (P < 0.05) in the longissimus, white semitendinosus, and cutaneous trunci muscles, whereas type I MyHC expression was greatest (P < 0.05) in the MAS, diaphragm, and red semitendinosus muscles. Glycogen synthase gene expression was least in the MAS (P < 0.01) but exhibited a pattern similar to MyHC IIB expression across muscles. Citrate synthase transcript abundance, however, varied (P < 0.05) independently of MyHC gene expression. Expression of types I and IIB MyHC was correlated with their tissue protein content ([R.sup.2] = 0.76 and 0.78, respectively), whereas type IIA and X MyHC expression did not correlate with the SDS-PAGE-determined protein content. These data show differences in MyHC gene expression across various porcine muscles and suggest that expression of these genes is reflective of the type of myosin contained within the muscle. Moreover, these data show that expression of energyspecific genes differs greatly across porcine muscles with different functions. Key words: myosin heavy chain, porcine, skeletal muscle

The intrinsic chemical reaction of adenosine triphosphate (ATP) hydrolysis catalyzed by myosin is modeled by using a combined quantum mechanics and molecular mechanics (QM/MM) methodology that achieves a near ab initio representation of the entire model. Starting with coordinates derived from the heavy atoms of the crystal structure (Protein Data Bank ID code 1VOM) in which myosin is bound to the ATP analog ADP x V[O.sub.4.sup.-], a minimum-energy path is found for the transformation ATP + [H.sub.2]O [right arrow] ADP + [P.sub.i] that is characterized by two distinct events: (i) a low activation-energy cleavage of the [P.sub.[gamma]]--[O.sub.[beta][gamma]] bond and separation of the [gamma]-phosphate from ADP and (ii) the formation of the inorganic phosphate as a consequence of proton transfers mediated by two water molecules and assisted by the Glu-459-Arg-238 salt bridge of the protein. The minimum-energy model of the enzyme-substrate complex features a stable hydrogen-bonding network in which the lytic water is positioned favorably for a nucleophilic attack of the ATP [gamma]-phosphate and for the transfer of a proton to stably bound second water. In addition, the [P.sub.[gamma]]--[O.sub.[beta][gamma]] bond has become significantly longer than in the unbound state of the ATP and thus is predisposed to cleavage. The modeled transformation is viewed as the part of the overall hydrolysis reaction occurring in the closed enzyme pocket after ATP is bound tightly to myosin and before conformational changes preceding release of inorganic phosphate. ATP hydrolysis | enzymatic catalysis | energy profile | quantum mechanics and molecular mechanics simulations

The role of Limulus myosin III and its phosphorylation in photoreceptors is investigated. The sites that become phosphorylated in Limulus myosin III are determined and it is shown that Limulus myosin II exhibits kinase activity. A major site for both protein kinase A and autophosphorylation is located within loop 2 of the myosin domain, which suggests that Limulus myosin III is an actin-binding and signaling protein.

Drosophilaflapwing (flw) codes for serine/threonine protein phosphatase type 1[beta] (PP1[beta]). Regulation of nonmuscle myosin activity is the single essential flw function that is nonredundant with the three closely related PP1[alpha] genes. Flw is thought to dephosphorylate the nonmuscle myosin regulatory light chain, Spaghetti Squash (Sqh); this inactivates the nonmuscle myosin heavy chain, Zipper (Zip). Thus, strong flw mutants lead to hyperphosphorylation of Sqh and hyperactivation of nonmuscle myosin activity. Here, we show genetically that a Jun N-terminal kinase (JNK) mutant suppresses the semilethality of a strong flw allele. Alleles of the JNK phosphatase puckered (puc) genetically enhance the weak allele [flw.sup.1], leading to severe wing defects. Introducing a mutant of the nonmuscle myosin-binding subunit (Mbs) further enhances this genetic interaction to lethality. We show that puc expression is upregulated in wing imaginal discs mutant for [flw.sup.1] and [puc.sup.A251] and that this upregulation is modified by JNK and Zip. The level of phosphorylated (active) JNK is elevated in [flw.sup.1] enhanced by puc. Together, we show that disruption of nonmuscle myosin activates JNK and puc expression in wing imaginal discs.

We have shown that myosin light chain phosphorylation inhibits fiber shortening velocity at high temperatures, 30[degrees]C, in the presence of the phosphate analog vanadate. Vanadate inhibits tension by reversing the transition to force-generating states, thus mimicking a prepower stroke state. We have previously shown that at low temperatures vanadate also inhibits velocity, but at high temperatures it does not, with an abrupt transition in inhibition occurring near 25[degrees]C (E. Pate, G. Wilson, M. Bhimani, and R. Cooke. Biophys J 66: 1554-1562, 1994). Here we show that for fibers activated in the presence of 0.5 mM vanadate, at 30[degrees]C, shortening velocity is not inhibited in dephosphorylated fibers but is inhibited by 37 [+ or -] 10% in fibers with phosphorylated myosin light chains. There is no effect of phosphorylation on fiber velocity in the presence of vanadate at 10[degrees]C. The [K.sub.m] for ATP, defined by the maximum velocity of fibers partially inhibited by vanadate at 30[degrees]C, is 20 [+ or -] 4 [micro]M for phosphorylated fibers and 192 [+ or -] 40 [micro]M for dephosphorylated fibers, showing that phosphorylation also affects the binding of ATP. Fiber stiffness is not affected by phosphorylation. Inhibition of velocity by phosphorylation at 30[degrees]C depends on the phosphate analog, with ~12% inhibition in fibers activated in the presence of 5 mM Be[F.sub.3] and no inhibition in the presence of 0.25 mM A1[F.sub.4]. Our results show that myosin phosphorylation can inhibit shortening velocity in fibers with large populations of myosin heads trapped in prepower stroke states, such as occurs during muscle fatigue. skeletal muscle; cross bridge; phosphate analogs; vanadate; aluminum fluoride; beryllium fluoride; motor proteins

Cardiac myosin binding protein-C (cMyBP-C) is a thick filament-associated protein that binds tightly to myosin and has a potential role for modulating myocardial contraction. We tested the hypothesis that cMyBP-C 1) contributes to the enhanced in vivo contractile state following [beta]-adrenergic stimulation and 2) is necessary for myocardial adaptation to chronic increases in afterload. In vivo pressure-volume relations demonstrated that left ventricular (LV) systolic and diastolic function were compromised under basal conditions in cMyBP-[C.sup.-/-] compared with WT mice. Moreover, whereas [beta]-adrenergic treatment significantly improved ejection fraction, peak elastance, and the time to peak elastance in WT mice, these functional indexes remained unchanged in cMyBP-[C.sup.-/-] mice. Morphological and functional changes were measured through echocardiography in anesthetized mice following 5 wk of aortic banding. Adaptation to pressure overload was diminished in cMyBP-[C.sup.-/-] mice as characterized by a lack of an increase in posterior wall thickness, increased LV diameter, deterioration of fractional shortening, and prolonged isovolumic relaxation time. These results suggest that the absence of cMyBP-C significantly diminishes in vivo LV function and markedly attenuates the increase in LV contractility following [beta]-adrenergic stimulation or adaptation to pressure overload. aortic handing; pressure-volume relations; dobutamine

The essential light chain of myosin (ELC) is known to be important for structural stability of the [alpha]-helical lever arm domain of the myosin head, but its function in striated muscle contraction is poorly understood. Two ELC isoforms are expressed in fast skeletal muscle, a long isoform and its N[H.sub.2]-terminal ~40 amino acid shorter counterpart, whereas only the long ELC is observed in the heart. Biochemical and structural studies revealed that the N[H.sub.2]-terminus of the long ELC can make direct contacts with actin, but the effects of the ELC on the affinity of myosin for actin, ATPase, force, and the kinetics of force generating myosin cross-bridges are inconclusive. Myosin containing the long ELC has been shown to have slower cross-bridge kinetics than myosin with the short isoform. A difference was also reported among myosins with long isoforms. Increased shortening velocity was observed in atrial compared with ventricular muscle fibers. The common findings suggest that ELC provides the fine tuning of the myosin motor function, which is regulated in an isoform and tissue-dependent manner. The functional importance of the ELC is further implicated by the discovery of ELC mutations associated with Familial Hypertrophic Cardiomyopathy. The pathological phenotypes vary in severity, but more notably, almost all ELC mutations result in sudden cardiac death at a young age. This review summarizes the functional roles of striated muscle ELC in normal healthy muscle and in disease. Transgenic animal models and phenotypic characterization of ELC-mediated remodeling of the heart are also discussed. cross-bridge kinetics; striated muscle contraction; familial hypertrophic cardiomyopathy; sarcomeric mutations; failing heart; sudden cardiac death

We examined the temporal relationship between portacaval anastamosis (PCA), weight gain, changes in skeletal muscle mass and molecular markers of protein synthesis, protein breakdown, and satellite cell proliferation and differentiation. Male Sprague-Dawley rats with end to side PCA (n = 24) were compared with sham-operated pair-fed rats (n = 24). Whole body weight, lean body mass, and forelimb grip strength were determined at weekly intervals. The skeletal muscle expression of the ubiquitin proteasome system, myostatin, its receptor (the activin 2B receptor) and its signal, cyclin-dependant kinase inhibitor (CDKI) p21, insulin-like growth factor (IGF)-I and its receptor (IGF-I receptor-a), and markers of satellite cell proliferation and differentiation were quantified. PCA rats did not gain body weight and had lower lean body mass, forelimb grip strength, and gastrocnemius muscle weight. The skeletal muscle expression of the mRNA of ubiquitin proteasome components was higher in PCA rats in the first 2 wk followed by a lower expression in the subsequent 2 wk (P < 0.01). The mRNA and protein of myostatin, activin 2B receptor, and CDKI p21 were higher, whereas IGF-I and its receptor as well as markers of satellite cell function (proliferating nuclear cell antigen, myoD, myf5, and myogenin) were lower at weeks 3 and 4 following PCA (P < 0.05). We conclude that PCA resulted in uninhibited proteolysis in the initial 2 wk. This was followed by an adaptive response in the later 2 wk consisting of an increased expression of myostatin that may have contributed to reduced muscle protein synthesis, impaired satellite cell function, and lower skeletal muscle mass. lean body mass; satellite cell; myostatin

We have previously shown that polyamine depletion decreased migration, Rac activation, and protein serine threonine phosphatase 2A activity. We have also shown that polyamine depletion increased cortical F-actin and decreased lamellipodia and stress fibers. In this study, we used stanrosporine (STS), a potent, cell-permeable, and broad-spectrum serine/threonine kinase inhibitor, and studied migration. STS concentrations above 100 nM induced apoptosis. However, in polyamine-depleted cells, a lower concentration of STS (5 nM) increased attachment, spreading, Rac1 activation, and, subsequently, migration without causing apoptosis. STS-induced migration was completely prevented by a Rac1 inhibitor (NSC-23766) and dominant negative Rac1. These results imply that STS restores migration in polyamine-depleted cells through Rac1. The most important finding in this study was that polyamine depletion increased the association of phosphorylated myosin regulatory light chain (p[Thr.sup.18]/[Ser.sup.19]-MRLC) at the cell periphery, which coloealized with thick cortical F-actin. Localization of [pThr.sup.18]-and [pSer.sup.19]MRLC was found with stress fibers and nuclei, respectively. STS decreased the phosphorylation of cellular and peripheral [pThr.sup.18]-MRLC without any effect on nuclear [pSer.sup.19]-MRLC, dissolved thick cortical F-actin, and increased lamellipodia and stress fiber formation in polyamine-depleted cells. In control and polyamine-depleted cells, focal adhesion kinase (FAK) colocalized with stress fibers and the actin cortex, respectively. STS reorganized FAK, paxillin, and the cytoskeleton. These results suggest that polyamine depletion prevents the dephosphorylation of MRLC and thereby prevents the dynamic reorganization of the actin cytoskeleton and decreases lamellipodia formation resulting in the inhibition of migration. putrescine; [alpha]-difluoromethylornithine; F-actin; focal adhesion kinase; paxillin

Sphingosine 1-phosphate (Sph-1-P), a product of sphingomyelin metabolism, can act via a family of cognate G protein-coupled receptors or as an intracellular second messenger for agonists acting through their membrane receptors. In view of the general growth promoting and developmental effects of Sph-1-P on target cells, we hypothesized that it plays a role in adaptation of the uterus to pregnancy. We analyzed its potential role and that of the related lysophospholipid lysophosphatidic acid in the pregnant rat uterus by examining changes in mRNA levels of cognate receptors and enzymes involved in their turnover. Of these, only sphingosine kinase-1 (SphK1) was markedly changed (~30-fold increase), being localized in the glandular epithelium, vasculature, and the myometrium. Uterine SphK1 mRNA and protein levels paralleled those of serum progesterone, and treatment with progesterone or an antagonist elevated or reduced SphK1 mRNA expression, respectively. Progesterone also increased SphK1 mRNA steady-state levels in a rat myometrial/leiomyoma cell line (ELT3). Overexpressing human SphK1 in these cells resulted in increased levels of the cell cycle regulator cyclin D1 and increased myosin light-chain phosphorylation. Ectopic expression of SphK1 also resulted in increased proliferation rates, possibly in conjunction with increased cyclin D1 expression. These studies suggest that the uterine expression of SphK1 mediates processes involved in growth and differentiation of uterine tissues during pregnancy. ELT3 cells; myosin light-chain phosphorylation; cyclin D1; lysophospholipids; sphingosine 1-phosphate; sphingosine-1-phosphate lyase

Constitutive myostatin gene knockout in mice causes excessive muscle growth during development. To examine the effect of knocking out the myostatin gene after muscle has matured, we generated mice in which myostatin exon 3 was flanked by loxP sequences (Mstn[f/f]) and crossed them with mice bearing a tamoxifen-inducible, ubiquitously expressed Cre recombinase transgene. At 4 mo of age, Mstn[f/f]/Cre+ mice that had not received tamoxifen had a 50-90% reduction in myostatin expression due to basal Cre activity but were not hypermuscular relative to Mstn[w/w]/Cre+ mice (homozygous for wild-type myostatin gene). Three months after tamoxifen treatment (initiated at 4 mo of age), muscle mass had not changed from the pretreatment level in Mstn[w/w]/Cre+ control mice. Tamoxifen administration to 4-mo-old Mstn[f/f]/Cre+ mice reduced myostatin mRNA expression to less than 1% of normal, which increased muscle mass ~25% over the following 3 mo in both male and female mice (P < 0.005 vs. control). Fiber hypertrophy appeared to be sufficient to explain the increase in muscle mass. The pattern of expression of genes encoding the various myosin heavy-chain isoforms was unaffected by postdevelopmental myostatin knockout. We conclude that, even after developmental muscle growth has ceased, knockout of the myostatin gene induces a significant increase in muscle mass. Cre recombinase; tamoxifen; muscle fiber hypertrophy; myosin heavy chains; conditional knockout

The influence of semicrystalline order on the second-harmonic generation (SHG) efficiency in the anisotropic bands of Drosophila melanogaster sarcomeres from larval and adult muscle has been investigated. Differences in the semicrystalline order were obtained by using wild-type and mutant strains containing different amounts of headless myosin. The reduction in semicrystalline order without altering the chemical composition of myofibrils was achieved by observing highly stretched sarcomeres and by inducing a loss of viability in myocytes. In all cases the reduction of semicrystalline order in anisotropic bands of myocytes resulted in a substantial decrease in SHG. Second-harmonic imaging during periodic contractions of myocytes revealed higher intensities when sarcomeres were in the relaxed state compared with the contracted state. This study demonstrates that an ordered semicrystalline arrangement of anisotropic bands plays a determining role in the efficiency of SHG in myocytes. OCIS codes: 170.3880, 180.5810, 190.4160.

Cells known as melanophores contain melanosomes, which are membrane organelles filled with melanin, a dark, nonfluorescent pigment. Melanophores aggregate or disperse their melanosomes when the host needs to change its color in response to the environment (e.g., camouflage or social interactions). Melanosome transport in cultured Xenopus melanophores is mediated by myosin V, heterotrimeric kinesin-2, and cytoplasmic dynein. Here, we describe a technique for tracking individual motors of each type, both individually and in their interaction, with high spatial ([approximately equal to]2 nm) and temporal ([approximately equal to]1 msec) localization accuracy. This method enabled us to observe (i) stepwise movement of kinesin-2 with an average step size of 8 nm; (ii) smoother melanosome transport (with fewer pauses), in the absence of intermediate filaments (IFs); and (iii) motors of actin filaments and microtubules working on the same cargo nearly simultaneously, indicating that a diffusive step is not needed between the two systems of transport. In concert with our previous report, our results also show that dynein-driven retrograde movement occurs in 8-nm steps. Furthermore, previous studies have shown that melanosomes carried by myosin V move 35 nm in a stepwise fashion in which the step rise-times can be as long as 80 msec. We observed 35-nm myosin V steps in melanophores containing no IFs. We find that myosin V steps occur faster in the absence of IFs, indicating that the IF network physically hinders organelle transport. bright-field imaging with one-nanometer accuracy (bFIONA) | dynein | kinesin-2 | myosin V | intermediate filaments

Certain types of intracellular organelle transport to the cell periphery are thought to involve long-range movement on microtubules by kinesin with subsequent handoff to vertebrate myosin Va (myoVa) for local delivery on actin tracks. This process may involve direct interactions between these two processive motors. Here we demonstrate using single molecule in vitro techniques that myoVa is flexible enough to effectively maneuver its way through actin filament intersections and Arp2/3 branches. In addition, myoVa surprisingly undergoes a one-dimensional diffusive search along microtubules, which may allow it to scan efficiently for kinesin and/or its cargo. These features of myoVa may help ensure efficient cargo delivery from the cell center to the periphery. cytoskeleton | molecular motor | motility | processivity

Mouse telokin and SM22[alpha] promoters have previously been shown to direct smooth muscle cell-specific expression of transgenes in vivo in adult mice. However, the activity of these promoters is highly dependent on the integration site of the transgene. In the current study, we found that the ectopic expression of telokin promoter transgenes could be abolished by flanking the transgene with insulator elements from the H19 gene. However, the insulator elements did not increase the proportion of mouse lines that exhibited consistent, detectable levels of transgene expression. In contrast, when transgenes were targeted to the hprt locus, both telokin and SM22[alpha] promoters resulted in reproducible patterns and levels of transgene expression in all lines of mice examined. Telokin promoter transgene expression was restricted to smooth muscle tissues in adult and embryonic mice. As reported previously, SM22[alpha] transgenes were expressed at high levels specifically in arterial smooth muscle cells; however, in contrast to randomly integrated transgenes, the hprt-targeted SM22[alpha] transgenes were also expressed at high levels in smooth muscle cells in veins, bladder, and gallbladder. Using hprt-targeted transgenes, we further analyzed elements within the telokin promoter required for tissue specific activity in vivo. Analysis of these transgenes revealed that the CArG element in the telokin promoter is required for promoter activity in all tissues and that the CArG element and adjacent AT-rich region are sufficient to drive transgene expression in bladder but not intestinal smooth muscle cells. visceral smooth muscle; development; myosin light chain kinase; embryos; CArG element