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Microvillus inclusion disease (MVID) is a severe form of congenital diarrhea that arises from inactivating mutations in the gene encoding myosin Vb (MYO5B). We have examined the association of mutations in MYO5B and disruption of microvillar assembly and polarity in enterocytes. Stable MYO5B knockdown (MYO5B-KD) in CaCo2-BBE cells elicited loss of microvilli, alterations in junctional claudins, and disruption of apical and basolateral trafficking; however, no microvillus inclusions were observed in MYO5B-KD cells. Expression of WT MYO5B in MYO5B-KD cells restored microvilli; however, expression of MYO5B-P660L, a MVID-associated mutation found within Navajo populations, did not rescue the MYO5B-KD phenotype but induced formation of microvillus inclusions. Microvilli establishment required interaction between RAB8A and MYO5B, while loss of the interaction between RAB11A and MYO5B induced microvillus inclusions. Using surface biotinylation and dual immunofluorescence staining in MYO5B-KD cells expressing mutant forms of MYO5B, we observed that early microvillus inclusions were positive for the sorting marker SNX18 and derived from apical membrane internalization. In patients with MVID, MYO5B-P660L results in global changes in polarity at the villus tips that could account for deficits in apical absorption, loss of microvilli, aberrant junctions, and losses in transcellular ion transport pathways, likely leading to the MVID clinical phenotype of neonatal secretory diarrhea., Introduction Microvillus inclusion disease (MVID) is a rare neonatal diarrheal disorder of the small intestine that arises mainly in European, Middle Eastern, and Navajo Native American cohorts (1, 2). Recent [...]

Background: The human tongue muscle genioglossus (GG) is active in speech, swallowing, respiration, and oral transport, behaviors encompassing a wide range of tongue shapes and movement speeds. Studies demonstrate substantial diversity in patterns of human GG motor unit activation, but whether this is accompanied by complex expression of muscle contractile proteins is not known. Purpose: The authors tested for conventional myosin heavy chain (MHC) MHCI, MHCIIA, MHCIIX, developmental MHCembryonic and MHCneonatal and unconventional MHCacardiac, MHCextraocular, and MHCslow tonic in antero-superior (GG-A) and posterior (GG-P) adult human GG. Method: SDS-PAGE, Western blot, and immunohistochemistry were used to describe MHC composition of GG-A and GG-P and the prevalence of muscle fiber MHC phenotypes in GG-A. Results: By SDS-PAGE, only conventional MHC are present with ranking from most to least prevalent MHCIIA > MHCI > MHCIIX in GG-A and MHCI > MHCIIA > MHCIIX in GG-P. By immunohistochemistry, many muscle fibers contain MHCI, MHCIIA, and MHCIIX, but few contain developmental or unconventional MHC. GG-A is composed of 5 phenotypes (MHCIIA > MHCI-IIX > MHCI > MHCI-IIA > MHCIIX). Phenotypes MHCI, MHCIIA, and MHCI-IIX account for 96% of muscle fibers. Conclusions: Despite activation of GG during kinematically diverse behaviors and complex patterns of GG motor unit activity, the human GG is composed of conventional MHC isoforms and 3 primary MHC phenotypes. Key Words: tongue, swallowing, speech, musculoskeletal system, normal respiration, The human tongue muscle genioglossus (GG) is active in speech, swallowing, respiration, and oral transport, behaviors that encompass a wide range of tongue shapes and movement speeds (Cheng, Peng, Chiou, [...]

Actin-myosin interactions provide the driving force underlying each heartbeat. The current view is that actin-bound regulatory proteins play a dominant role in the activation of calcium-dependent cardiac muscle contraction. In contrast, the relevance and nature of regulation by myosin regulatory proteins (for example, myosin light chain-2 [MLC2]) in cardiac muscle remain poorly understood. By integrating gene-targeted mouse and computational models, we have identified an indispensable role for ventricular Mlc2 (Mlc2v) phosphorylation in regulating cardiac muscle contraction. Cardiac myosin cycling kinetics, which directly control actin-myosin interactions, were directly affected, but surprisingly, Mlc2v phosphorylation also fed back to cooperatively influence calcium-dependent activation of the thin filament. Loss of these mechanisms produced early defects in the rate of cardiac muscle twitch relaxation and ventricular torsion. Strikingly, these defects preceded the left ventricular dysfunction of heart disease and failure in a mouse model with nonphosphorylatable Mlc2v. Thus, there is a direct and early role for Mlc2 phosphorylation in regulating actin-myosin interactions in striated muscle contraction, and dephosphorylation of Mlc2 or loss of these mechanisms can play a critical role in heart failure., Introduction Interactions between the contractile proteins myosin and actin provide the driving force for muscle contraction (1). However, the molecular mechanisms underlying the regulation of these interactions in striated muscle, [...]

ABSTRACT The physicochemical and functional properties of actomyosin from fins of frozen, stored squid caught either by jigging machines (AME 1) or by trawling (AME2) were investigated. At time 0 [...]

Autoimmunity has long been linked to myocarditis and its sequela, dilated cardiomyopathy, the leading causes of heart failure in young patients. However, the underlying mechanisms are poorly defined, with most clinical investigations focused on humoral autoimmunity as the target for intervention. Here, we show that the α-isoform of myosin heavy chain (α-MyHC, which is encoded by the gene Myh6) is the pathogenic autoantigen for [CD4.sup.+] T cells in a spontaneous mouse model of myocarditis. Further, we found that Myh6 transcripts were absent in mouse medullary thymic epithelial cells (mTECs) and peripheral lymphoid stromal cells, which have been implicated in mediating central and peripheral T cell tolerance, respectively. Transgenic expression of α-MyHC in thymic epithelium conferred tolerance to cardiac myosin and prevented myocarditis, demonstrating that α-MyHC is a primary autoantigen in this disease process. Remarkably, we found that humans also lacked α-MyHC in mTECs and had high frequencies of α-MyHC-specific T cells in peripheral blood, with markedly augmented T cell responses to α-MyHC in patients with myocarditis. Since α-MyHC constitutes a small fraction of MyHC in human heart, these findings challenge the longstanding notion that autoimmune targeting of My HC is due to its cardiac abundance and instead suggest that it is targeted as a result of impaired T cell tolerance mechanisms. These results thus support a role for T cell-specific therapies for myocarditis., Introduction Myocarditis is a major cause of heart disease in young patients and a common precursor of heart failure due to dilated cardiomyopathy (1-3). The prognosis for dilated cardiomyopathy is [...]

Cytokinesis in animal and fungal cells utilizes a contractile actomyosin ring (AMR). However, how myosin II is targeted to the division site and promotes AMR assembly, and how the AMR coordinates with membrane trafficking during cytokinesis, remains poorly understood. Here we show that Myo1 is a two-headed myosin II in Saccharomyces cerevisiae, and that Myo1 localizes to the division site via two distinct targeting signals in its tail that act sequentially during the cell cycle. Before cytokinesis, Myo1 localization depends on the septin-binding protein Bni5. During cytokinesis, Myo1 localization depends on the IQGAP Iqg1. We also show that the Myo1 tail is sufficient for promoting the assembly of a 'headless' AMR, which guides membrane deposition and extracellular matrix remodeling at the division site. Our study establishes a biphasic targeting mechanism for myosin II and highlights an underappreciated role of the AMR in cytokinesis beyond force generation. doi/ 10.1083/jcb.201005134

UNC-45/CRO1/She4p (UCS) proteins have variously been proposed to affect the folding, stability, and ATPase activity of myosins. They are the only proteins known to interact directly with the motor domain. To gain more insight into UCS function, we determined the atomic structure of the yeast UCS protein, She4p, at 2.9 [Angstrom] resolution. We found that 16 helical repeats are organized into an L-shaped superhelix with an amphipathic N-terminal helix dangling off the short arm of the L-shaped molecule. In the crystal, She4p forms a 193-[Angstrom]-long, zigzag-shaped dimer through three distinct and evolutionary conserved interfaces. We have identified She4p's C-terminal region as a ligand for a 27-residue-long epitope on the myosin motor domain. Remarkably, this region consists of two adjacent, but distinct, binding epitopes localized at the nucleotide-responsive cleft between the nucleotide- and actin-filament-binding sites. One epitope is situated inside the cleft, the other outside the cleft. After ATP hydrolysis and Pi ejection, the cleft narrows at its base from 20 to 12 [Angstrom] thereby occluding the inside the cleft epitope, while leaving the adjacent, outside the cleft binding epitope accessible to UCS binding. Hence, one cycle of higher and lower binding affinity would accompany one ATP hydrolysis cycle and a single step in the walk on an actin filament rope. We propose that a UCS dimer links two myosins at their motor domains and thereby functions as one of the determinants for step size of myosin on actin filaments. myosin stability | processivity | chaperone doi/ 10.1073/pnas.1013038107

The antithetical regulation of cardiac [alpha]- and [beta]-myosin heavy chain (MHC) genes by thyroid hormone ([T.sub.3]) is not well understood but appears to involve thyroid hormone interaction with its nuclear receptor and MHC promoters as well as cis-acting noncoding regulatory RNA (ncRNA). Both of these phenomena involve epigenetic regulations. This study investigated the extent that altered thyroid state induces histone modifications in the chromatin associated with the cardiac MHC genes. We hypothesized that specific epigenetic events could be identified and linked to cardiac MHC gene switching in response to a hypothyroid or hyperthyroid state. A hypothyroid state was induced in rats by propylthiouracil treatment (PTU), whereas a hyperthyroid ([T.sub.3]) was induced by [T.sub.3] treatment. The left ventricle was analyzed after 7 days for MHC pre-mRNA expression, and the chromatin was assessed for enrichment in specific histone modifications using chromatin immunoprecipitation quantitative PCR assays. At both the [alpha]-MHC promoter and the intergenic region, the enrichment in acetyl histone H3 at K9/14 (H3K9/14ac) and trimethyl histone H3 at K4 (H3K4me3) changed in a similar fashion. They were both decreased with PTU treatment but did not change under [T.sub.3], except at a location situated 5' to the antisense intergenic transcription start site. These same marks varied differently on the [beta]-MHC promoter. For example, H3K4me3 enrichment correlated with the [beta]-promoter activity in PTU and [T.sub.3] groups, whereas H3K9/14ac was repressed in the [T.sub.3] group but did not change under PTU. Histone H3K9me was enriched in chromatin of both the intergenic and [alpha]-MHC promoters in the PTU group, whereas histone H4K20mel was enriched in chromatin of [beta]-MHC promoter in the normal control and [T.sub.3] groups. Collectively, these findings provide evidence that specific epigenetic phenomena modulate MHC gene expression in altered thyroid states. gene transcription; epigenetic regulation; chromatin immunoprecipitation quantitative polymerase chain reaction; noncoding RNA; Sprague-Dawley rats doi: 10.1152/ajpheart.00644.2010.

Asymmetric positioning of the mitotic spindle before cytoldnesis can produce different-sized daughter cells that have distinct fates. Here, we found an asymmetric division in the Coenorhabditis elegans Q neuroblast lineage that began with a centered spindle but generated different-sized daughters, the smaller (anterior) of which underwent apoptosis. During this division, more myosin II accomulated anteriorly, suggesting that asymmetric contractile forces might produce different-sized daughters. Indeed, partial inactivation of anterior myosin by chromophore-assisted laser inactivation created a more symmetric division and allowed the survival and differentiation of the anterior daughter. Thus, the balance of myosin activity on the two sides of a dividing cell can govern the size and fate of the daughters. 10.1126/science.1196112

Familial hypertrophic cardiomyopathy (FHC) is caused by mutations in sarcomeric proteins including the myosin regulatory light chain (RLC). Two such FHC mutations, R58Q and N47K, located near the cationic binding site of the RLC, have been identified from population studies. To examine the molecular basis for the observed phenotypes, we exchanged endogenous RLC from native porcine cardiac myosin with recombinant human ventricular wild type (WT) or FHC mutant RLC and examined the ability of the reconstituted myosin to propel actin filament sliding using the in vitro motility assay. We find that, whereas the mutant myosins are indistinguishable from the controls (WT or native myosin) under unloaded conditions, both R58Q-and N47K-exchanged myosins show reductions in force and power output compared with WT or native myosin. We also show that the changes in loaded kinetics are a result of mutation-induced loss of myosin strain sensitivity of ADP affinity. We propose that the R58Q and N47K mutations alter the mechanical properties of the myosin neck region, leading to altered load-dependent kinetics that may explain the observed mutant-induced FHC phenotypes. in vitro motility | load-dependent kinetics | familial hypertrophic cardiornyopathy | R58Q | N47K doi/ 10.1073/pnas.1009619107

Post-tetanic potentiation (PTP) at the calyx of Held synapse is caused by increases not only in release probability ([P.sub.r]) but also in the readily releasable pool size estimated from a cumulative plot of excitatory post-synaptic current amplitudes ([RRP.sub.cum]), which contribute to the augmentation phase and the late phase of PTP, respectively. The vesicle pool dynamics underlying the latter has not been investigated, because PTP is abolished by presynaptic whole-cell patch clamp. We found that supplement of recombinant calmodulin to the presynaptic pipette solution rescued the increase in the [RRP.sub.cum] after high-frequency stimulation (100 Hz for 4-s duration, HFS), but not the increase in [P.sub.r]. Release-competent synaptic vesicles (SVs) are heterogeneous in their releasing kinetics. To investigate posttetanic changes of fast and slowly releasing SV pool (FRP and SRP) sizes, we estimated quantal release rates before and 40 s after HFS using the deconvolution method. After HFS, the FRP size increased by 19.1% and the SRP size decreased by 25.4%, whereas the sum of FRP and SRP sizes did not increase. Similar changes in the RRP were induced by a single long depolarizing pulse (100 ms). The post-tetanic complementary changes of FRP and SRP sizes were abolished by inhibitors of myosin II or myosin light chain kinase. The post-tetanic increase in the FRP size coupled to a decrease in the SRP size provides the first line of evidence for the idea that a slowly releasing SV can be converted to a fast releasing one. doi/10.1085/jgp.201010437

Nonmuscle myosins (NMs) II-A and II-B are essential for embryonic mouse development, but their specific roles are not completely defined. Here we examine the isoforms and their domain specifically in vivo and in vitro by studying mice and cells in which nonmuscle myosin heavy chain (NMHC) II-A is genetically replaced by NMHC II-B or chimeric NMHC IIs that exchange the rod and head domains of NM II-A and II-B. In contrast with the failure of visceral endoderm formation resulting in embryonic day (E)6.5 lethality of [A.sup.-]/[A.sup.-] mice, replacement with NM II-B or chimeric NM IIs restores a normal visceral endoderm. This finding is consistent with NM II's role in cell adhesion and also confirms an essential, isoform-independent requirement for NM II in visceral endoderm function. The knock-in mice die between E9.5 and 12.5 because of defects in placenta formation associated with abnormal angiogenesis and cell migration, revealing a unique function for NM II-A in placenta development. In vitro results further support a requirement for NM II-A in directed cell migration and focal adhesion formation. These findings demonstrate an isoform-specific role for NM II-A during these processes, making replacement by another isoform, or chimeric NM II isoforms, less successful. The failure of these substitutions is not only related to the different kinetic properties of NM II-A and II-B, but also to their subcellular localization determined by the C-terminal domain. These results highlight the functions of the N-terminal motor and C-terminal rod domains of NM II and their different roles in cell-cell and cell-matrix adhesion. cell migration | genetic substitution | placenta development | visceral endoderm formation | chimeric myosin II doi/ 10.1073/pnas.1004023107

In order to understand the mechanism of muscle contraction at the atomic level, it is necessary to understand how myosin binds to actin in a reversible way. We have used a novel molecular dynamics technique constrained by an EM map of the actin-myosin complex at 13-[Angstrom] resolution to obtain an atomic model of the strong-binding (rigor) actin-myosin interface. The constraining force resulting from the EM map during the molecular dynamics simulation was sufficient to convert the myosin head from the initial weak-binding state to the strong-binding (rigor) state. Our actin-myosin model suggests extensive contacts between actin and the myosin head (S1). S1 binds to two actin monomers. The contact surface between actin and S1 has increased dramatically compared with previous models. A number of loops in S1 and actin are involved in establishing the interface. Our model also suggests how the loop carrying the critical Arg 405 Glu mutation in S1 found in a familial cardiomyopathy might be functionally involved. cryoelectron microscopy | myosin II | myosin V doi/10.1073/pnas.1003604107

NGF, the principal neurotrophic factor for basal forebrain cholinergic neurons (BFCNs), has been correlated to Alzheimer's disease (AD) because of the selective vulnerability of BFCNs in AD. These correlative links do not substantiate a comprehensive cause--effect mechanism connecting NGF deficit to overall AD neurodegeneration. A demonstration that neutralizing NGF activity could have consequences beyond a direct interference with the cholinergic system came from studies in the AD11 mouse model, in which the expression of a highly specific anti-NGF antibody determines a neurodegeneration that encompasses several features of human AD. Because the transgenic antibody binds to mature NGF much more strongly than to proNGF and prevents binding of mature NGF to the tropomyosin-related kinase A (TrkA) receptor and to p75 neurotrophin receptor (p75NTR), we postulated that neurodegeneration in AD11 mice is provoked by an imbalance of proNGF/NGF signaling and, consequently, of TrkA/p75NTR signaling. To test this hypothesis, in this study we characterize the phenotype of two lines of transgenic mice, one in which TrkA signaling is inhibited by neutralizing anti-TrkA antibodies and a second one in which anti-NGF mice were crossed to [p75NTR.sup.exoIII(-/-)] mice to abrogate p75NTR signaling. TrkA neutralization determines a strong cholinergic deficit and the appearance of [beta]-amyloid peptide (A[beta]) but no tau-related pathology. In contrast, abrogating p75NTR signaling determines a full rescue of the cholinergic and A[beta] phenotype of anti-NGF mice, but tau hyperphosphorylation is exacerbated. Thus, we demonstrate that inhibiting TrkA signaling activates A[beta] accumulation and that different streams of AD neurodegeneration are related in complex ways to TrkA versus p75NTR signaling. Alzheimer | [beta]-amyloid | proNGF | signaling unbalance doi/10.1073/pnas.1007181107

Directional motility is a fundamental function of immune cells, which are recruited to sites of pathogen invasion or tissue damage by chemoattractant signals. To move, cells need to generate lamellipodial membrane protrusions at the front and retract the trailing end. These elementary events are initiated by Rho-family GTPases, which cycle between active GTP-bound and inactive GDP-bound states. How the activity of these 'molecular switches' is spatially coordinated is only beginning to be understood. Here, we show that myosin IXb (Myo9b), a Rho GTPase-activating protein (RhoGAP) expressed in immune cells, is essential for coordinating the activity of Rho. We generated Myo9b-deficient mice and show that [Myo9b.sup.-/-] macrophages have strikingly defective spreading and polarization. Furthermore, [Myo9b.sup.-/-] macrophages fail to generate lamellipodia in response to a chemoattractant, and migration in a chemotactic gradient is severely impaired. Inhibition of Rho rescues the [Myo9b.sub.-/-] phenotype, but impairs tail retraction. We also found that Myo9b is important in vivo. Chemoattractant-induced monocyte recruitment to the peritoneal cavity is substantially reduced in [Myo9b.sup.-/-] mice. Thus, we identify the 'motorized Rho inhibitor' Myo9b as a key molecular component required for spatially coordinated cell shape changes and motility. cell migration | knockout | Rho-family GTPases | signal transduction doi/10.1073/pnas.0911986107

Myosin phosphatase target subunit 1 (MYPT1) is the regulatory subunit of myosin light chain phosphatase (MLCP). It plays a critical role in vasodilatation induced by cGMP-elevating agents such as nitric oxide (NO). The present study was performed to determine the role of MYPT1 in the development of tolerance of the pulmonary artery to NO. Incubation of isolated porcine pulmonary arteries for 24 or 48 h with DETA NONOate (DETA NO) significantly reduced protein levels of MYPT1 and the leucine zipper-positive (LZ+) isoform of MYPTI but not that of PPlcS. The extent of reduction in total MYPT1 protein level was comparable to that of MYPTI (LZ+). The decrease in MYPT1 protein caused by 48-h DETA NO incubation was prevented by ODQ, an inhibitor of guanylyl cyclase, and by inhibitors of proteasomes (MG-132 and lactacystin) but was not affected by the inhibitor of protein synthesis, cycloheximide. A reduction in MYPT1 protein was also obtained with 8-bromo-cGMP, but this was prevented by Rp-8bromo-PET-cGMP [inhibitor of cGMP-dependent protein kinase (PKG)]. Incubation for 48 h with DETA NO also reduced dephosphorylation of myosin light chain and relaxation of the artery in response to DETA NO, which was prevented by MG-132. These results suggest that the reduction in MYPT1 protein contributes to the development of tolerance of pulmonary arteries to NO. This may result from increased degradation of MYPT1 after prolonged PKG activation. myosin light chain phosphatase; guanosine 3',5'-cyclic monophosphate-dependent protein kinase; vasodilatation doi: 10.1152/ajplung.00340.2009.

Actomyosin contractility affects cellular organization within tissues in part through the generation of mechanical forces at sites of cell--matrix and cell--cell contact. While increased mechanical loading at cell--matrix adhesions results in focal adhesion growth, whether forces drive changes in the size of cell--cell adhesions remains an open question. To investigate the responsiveness of adherens junctions (AJ) to force, we adapted a system of microfabricated force sensors to quantitatively report cell--cell tugging force and AJ size. We observed that AJ size was modulated by endothelial cell--cell tugging forces: AJs and tugging force grew or decayed with myosin activation or inhibition, respectively. Myosin-dependent regulation of AJs operated in concert with a Rac1, and this coordinated regulation was illustrated by showing that the effects of vascular permeability agents (S1P, thrombin) on junctional stability were reversed by changing the extent to which these agents coupled to the Rac and myosin-dependent pathways. Furthermore, direct application of mechanical tugging force, rather than myosin activity per se, was sufficient to trigger AJ growth. These findings demonstrate that the dynamic coordination of mechanical forces and cell-cell adhesive interactions likely is critical to the maintenance of multicellular integrity and highlight the need for new approaches to study tugging forces. adherens junction | mechanotransduction | myosin | PDMS | traction force doi/ 10.1073/pnas.0914547107

The perijunctional actomyosin ring contributes to myosin light chain kinase (MLCK)-dependent tight junction regulation. However, the specific protein interactions involved in this process are unknown. To test the hypothesis that molecular remodeling contributes to barrier regulation, tight junction protein dynamic behavior was assessed by fluorescence recovery after photobleaching (FRAP). M LCK inhibition increased barrier function and stabilized ZO-1 at the tight junction but did not affect claudin-1, occludin, or actin exchange in vitro. Pharmacologic MLCK inhibition also blocked in vivo ZO-1 exchange in wild-type, but not long [MLCK.sup.-/-], mice. Conversely, ZO-1 exchange was accelerated in transgenic mice expressing constitutively active MLCK. In vitro, ZO-1 lacking the actin binding region (ABR) was not stabilized by MLCK inhibition, either in the presence or absence of endogenous ZO-1. Moreover, the free ABR interfered with full-length ZO-1 exchange and reduced basal barrier function. The free ABR also prevented increases in barrier function following MLCK inhibition in a manner that required endogenous ZO-1 expression. In silico modeling of the FRAP data suggests that tight junction-associated ZO-1 exists in three pools, two of which exchange with cytosolic ZO-1. Transport of the ABR-anchored exchangeable pool is regulated by MLCK. These data demonstrate a critical role for the ZO-1 ABR in barrier function and suggest that MLCK-dependent ZO-1 exchange is essential to this mechanism of barrier regulation. fluorescence recovery after photobleaching | mathematical models | myosin light chain kinase | paracellular permeability | intestinal epithelium doi/ 10.1073/pnas.0908869107

We have performed complementary time-resolved fluorescence resonance energy transfer (TR-FRET) experiments and molecular dynamics (MD) simulations to elucidate structural changes in the phosphorylation domain (PD) of smooth muscle regulatory light chain (RLC) bound to myosin. PD is absent in crystal structures, leaving uncertainty about the mechanism of regulation. Donor-acceptor pairs of probes were attached to three site-directed di-Cys mutants of RLC, each having one Cys at position 129 in the C-terminal lobe and the other at position 2, 3, or 7 in the N-terminal PD. Labeled RLC was reconstituted onto myosin subfragment 1 (S1). TR-FRET resolved two simultaneously populated structural states of RLC, closed and open, in both unphosphorylated and phosphorylated biochemical states. All three FRET pairs show that phosphorylation shifts the equilibrium toward the open state, increasing its mol fraction by ~20%. MD simulations agree with experiments in remarkable detail, confirming the coexistence of two structural states, with phosphorylation shifting the system toward the more dynamic open structural state. This agreement between experiment and simulation validates the additional structural details provided by MD simulations: In the closed state, PD is bent onto the surface of the C-terminal lobe, stabilized by interdomain salt bridges. In the open state, PD is more helical and straight, resides farther from the C-terminal lobe, and is stabilized by an intradomain salt bridge. The result is a vivid atomic-resolution visualization of the first step in the molecular mechanism by which phosphorylation activates smooth muscle. fluorescence resonance energy transfer | FRET | molecular dynamics | molecular dynamics simulation | time-resolved fluorescence doi/ 10.1073/pnas.1001941107

Type V myosin (MyoV)-dependent transport of cargo is an essential process in eukaryotes. Studies on yeast and vertebrate MyoV showed that their globular tails mediate binding to the cargo complexes. In Saccharomyces cerevisiae, the MyoV motor Myo4p interacts with She3p to localize asymmetric synthesis of HO 1 (ASH1) mRNA into the bud of dividing cells. A recent study showed that localization of GFP-MS2-tethered ASH1 particles does not require the Myo4p globular tail, challenging the supposed role of this domain. We assessed ASH1 mRNA and Myo4p distribution more directly and found that their localization is impaired in cells expressing globular tail-lacking Myo4p. In vitro studies further show that the globular tail together with a more N-terminal linker region is required for efficient She3p binding. We also determined the x-ray structure of the Myo4p globular tail and identify a conserved surface patch important for She3p binding. The structure shows pronounced similarities to membrane-tethering complexes and indicates that Myo4p may not undergo auto-inhibition of its motor domain. doi/ 10.1083/jcb.201002076

Myosin VI is proposed to act as both a molecular transporter and as an anchor in vivo. A portion of the molecule C-terminal to the canonical lever arm, termed the medial tail (MT), has been proposed to act as either a lever arm extension or as a dimerization motif. We describe constructs in which the MT is interrupted by a glycine-rich molecular swivel. Disruption of the MT results in decreased processive run lengths measured using single-molecule fluorescence microscopy and a decreased step size under applied load as measured in an optical trap. We used single-molecule gold nanoparticle tracking and optical trapping to examine the mechanism of coordination between the heads of dimeric myosin VI. We detect two rate-limiting kinetic processes at low ( gating | myo6 | TIRF | tweezers | gold nanoparticle tracking doi/ 10.1073/pnas.1002430107

MyTH/FERM (myosin tail homology 4/band 4.1, ezrin, radixin, and moesin) myosins have roles in cellular adhesion, extension of actin-filled projections such as filopodia and stereocilia, and directional migration. The amoeba Dictyostelium discoideum expresses a simple complement of MyTH/FERM myosins, a class VII (M7) myosin required for cell-substrate adhesion and a unique myosin named MyoG. Mutants lacking MyoG exhibit a wide range of normal actin-based behaviors, including chemotaxis to folic acid, but have a striking defect in polarization and chemotaxis to cAMP. Although the myoG mutants respond to cAMP stimulation by increasing persistence and weakly increasing levels of cortical F-actin, they do not polarize; instead, they maintain a round shape and move slowly and randomly when exposed to a chemotactic gradient. The mutants also fail to activate and localize PI3K to the membrane closest to the source of chemoattractant. These data reveal a role for a MyTH/FERM myosin in mediating early chemotactic signaling and suggest that MyTH/FERM proteins have conserved roles in signaling and the generation of cell polarity. actin cytoskeleton | cell signaling | cell motility | cytoskeletal dynamics doi/ 10.1073/pnas.0909796107

Plants exhibit an ultimate case of the intracellular motility involving rapid organelle trafficking and continuous streaming of the endoplasmic reticulum (ER). Although it was long assumed that the ER dynamics is actomyosin-driven, the responsible myosins were not identified, and the ER streaming was not characterized quantitatively. Here we developed software to generate a detailed velocity-distribution map for the GFP-labeled ER. This map revealed that the ER in the most peripheral plane was relatively static, whereas the ER in the inner plane was rapidly streaming with the velocities of up to ~3.5 [micro]m/sec. Similar patterns were observed when the cytosolic GFP was used to evaluate the cytoplasmic streaming. Using gene knockouts, we demonstrate that the ER dynamics is driven primarily by the ER-associated myosin XI-K, a member of a plant-specific myosin class XI. Furthermore, we show that the myosin XI deficiency affects organization of the ER network and orientation of the actin filament bundles. Collectively, our findings suggest a model whereby dynamic three-way interactions between ER, F-actin, and myosins determine the architecture and movement patterns of the ER strands, and cause cytosol hauling traditionally defined as cytoplasmic streaming. myosin XI | actin filament | cytoplasmic streaming | velocity distribution map | Arabidopsis thaliana doi/ 10.1073/pnas.0911482107

We have studied the mechanism by which a previously described primary muscle culture growing on microcarriers predominantly expresses fast myosin heavy chain (MHC) IId/x. We have measured MHC IId/x mRNA and protein levels, mRNA of MHC I and markers of muscle metabolism, insulin-like growth factor (IGF)-1 and mechano-growth factor (MGF) transcripts, indicators of the activation of the Akt-mammalian target of rapamycin (mTOR) axis, the p38-, ERKI/2-, and JNK-mitogen-activated protein kinase (MAP) kinase pathways, and of protein phosphatase PP2A, and we have assessed the involvement of integrin. By placing the culture flasks on a rotary shaker, we induce a continuous motion of the culture medium in which the carrier-myotube aggregates are suspended. This motion exerts passive forces on the myotubes that are decisive for the predominance of MHC II expression. These forces act via integrin, which transduces the mechanical signal into activation of PP2A and of p38 MAP-Kinase. The latter presumably is directly responsible for a drastic upregulation of MHC IId/x, whereas MHC I and metabolic markers remain unaffected. At the same time, despite an elevated level of IGF-1 transcription under passive forces, the IGF-1 receptor-Akt-mTOR axis is switched off as evident from the lack of an effect of inhibition of the IGF- 1 receptor and from the PP2A-mediated low degree of phosphorylation of Akt and 4E-BP1. Similarly, the ERK1/2- and JNK-MAP kinase pathways are repressed. We conclude that passive stretch exerted on the myotubes by the rotary fluid motion induces a rather selective upregulation of fast MHC II, which goes along with a mild muscle hypertrophy as judged from the amount of protein per cell and is caused by p38 MAP kinase activity elevated via integrin sensing. The direct link between passive stretch and MHC II expression constitutes a novel mechanism, which is expected to become effective physiologically under passive stretch and eccentric contractions of skeletal muscles. passive stretch; protein phosphatase; Akt doi: 10.1152/ajpcell.00265.2009.

Focal adhesions (FAs) are mechanosensitive adhesion and signaling complexes that grow and change composition in response to myosin II--mediated cytoskeletal tension in a process known as FA maturation. To understand tension-mediated FA maturation, we sought to identify proteins that are recruited to FAs in a myosin II--dependent manner and to examine the mechanism for their myosin II--sensitive FA association. We find that FA recruitment of both the cytoskeletal adapter protein vinculin and the tyrosine kinase FA kinase (FAK) are myosin II and extracellular matrix (ECM) stiffness dependent. Myosin II activity promotes FAK/ Src-mediated phosphorylation of paxillin on tyrosines 31 and 118 and vinculin association with paxillin. We show that phosphomimic mutations of paxillin can specifically induce the recruitment of vinculin to adhesions independent of myosin II activity. These results reveal an important role for paxillin in adhesion mechanosensing via myosin II--mediated FAK phosphorylation of paxillin that promotes vinculin FA recruitment to reinforce the cytoskeletal ECM linkage and drive FA maturation. doi/10.1083/jcb.200906012

Contractile forces generated by the actomyosin cytoskeleton within individual cells collectively generate tissue-level force during epithelial morphogenesis. During Drosophila mesoderm invagination, pulsed actomyosin meshwork contractions and a ratchet-like stabilization of cell shape drive apical constriction. Here, we investigate how contractile forces are integrated across the tissue. Reducing adherens junction (AJ) levels or ablating actomyosin meshworks causes tissue-wide epithelial tears, which release tension that is predominantly oriented along the anterior--posterior (a-p) embryonic axis. Epithelial tears allow cells normally elongated along the a-p axis to constrict isotropically, which suggests that apical constriction generates anisotropic epithelial tension that feeds back to control cell shape. Epithelial tension requires the transcription factor Twist, which stabilizes apical myosin II, promoting the formation of a supracellular actomyosin meshwork in which radial actomyosin fibers are joined end-to-end at spot AJs. Thus, pulsed actomyosin contractions require a supracellular, tensile meshwork to transmit cellular forces to the tissue level during morphogenesis. doi/10.1083/jcb.200910099

Using electron microscopy and image processing, we have observed myosin 5a modified with lever arms of different lengths (four, six, and eight calmodulin-binding IQ domains) and orientations walking along actin filaments. Step lengths were dependent on lever length: 8IQ > 6IQ > 4IQ, which is consistent with myosin 5a having evolved to walk straight along actin. Lead heads were mostly in the prepowerstroke state, tethered there by the trail head. However, improved image processing showed that in 5-10% of molecules the lead motor was in the postpowerstroke state. This is a unique attached state of myosin, where the motor domain has completed its powerstroke at the expense of severe lever distortion, but with little cargo movement. Postpowerstroke lead heads were seen in both wild-type and modified lever molecules, mostly where there was least strain. These data allow the strain dependence of the equilibrium between pre- and postpowerstroke conformations to be measured. Slow rates of ADP dissociation observed from lead heads of these molecules can be explained by the unfavorable equilibrium between the pre- and postpowerstroke conformations preceding ADP loss. actin | powerstroke | electron microscopy | kinetics | molecular motor www.pnas.org/cgi/doi/10.1073/pnas.0906907107

Myosin V motor proteins facilitate recycling of synaptic receptors, including AMPA and acetylcholine receptors, in central and peripheral synapses, respectively. To shed light on the regulation of receptor recycling, we employed in vivo imaging of mouse neuromuscular synapses. We found that myosin Va cooperates with PKA on the postsynapse to maintain size and integrity of the synapse; this cooperation also regulated the lifetime of acetylcholine receptors. Myosin Va and PKA colocalized in subsynaptic enrichments. These accumulations were crucial for synaptic integrity and proper cAMP signaling, and were dependent on AKAP function, myosin Va, and an intact actin cytoskeleton. The neuropeptide and cAMP agonist, calcitonin-gene related peptide, rescued fragmentation of synapses upon denervation. We hypothesize that neuronal ligands trigger local activation of PKA, which in turn controls synaptic integrity and turnover of receptors. To this end, myosin Va mediates correct positioning of PKA in a postsynaptic micro-domain, presumably by tethering PKA to the actin cytoskeleton. cAMP | muscle | neuromuscular junction | microdomain | synaptic plasticity doi/10.1073/pnas.0914087107

Human skeletal muscle fibers express five highly conserved type-II myosin heavy chain (MyHC) genes in distinct spatial and temporal patterns. In addition, the human genome contains an intact sixth gene, MyHC-IIb, which is thought under most circumstances not to be expressed. The physiological and biochemical properties of individual muscle fibers correlate with the predominantly expressed MyHC isoform, but a functional analysis of homogenous skeletal muscle myosin isoforms has not been possible. This is due to the difficulties of separating the multiple isoforms usually coexpressed in muscle fibers, as well as the lack of an expression system that produces active recombinant type II skeletal muscle myosin. In this study we describe a mammalian muscle cell expression system and the functional analysis of all six recombinant human type II skeletal muscle myosin isoforms. The diverse biochemical activities and actin-filament velocities of these myosins indicate that they likely have distinct functions in muscle. Our data also show that ATPase activity and motility are generally correlated for human skeletal muscle myosins. The exception, MyHC-IIb, encodes a protein that is high in ATPase activity but slow in motility; this is the first functional analysis of the protein from this gene. In addition, the developmental isoforms, hypothesized to have low ATPase activity, were indistinguishable from adult-fast MyHC-Ila and the specialized MyHC-Extraocular isoform, that was predicted to be the fastest of all six isoforms but was functionally similar to the slower isoforms. in vitro motility | recombinant myosins | skeletal myosin isoforms | actin-activated ATPase doi/10.1073/pnas.0913527107

We have used two complementary time-resolved spectroscopic techniques, dipolar electron--electron resonance and fluorescence resonance energy transfer to determine conformational changes in a single structural element of the myosin motor domain, the relay helix, before and after the recovery stroke. Two double-Cys mutants were labeled with optical probes or spin labels, and interprobe distances were determined. Both methods resolved two distinct structural states of myosin, corresponding to straight and bent conformations of the relay helix. The bent state was occupied only upon nucleotide addition, indicating that relay helix, like the entire myosin head, bends in the recovery stroke. However, saturation of myosin with nucleotide, producing a single biochemical state, did not produce a single structural state. Both straight and bent structural states of the relay helix were occupied when either ATP (ADP.Be[F.sub.x]) or ADP.[P.sub.i] (ADP.Al[F.sub.4]) analogs were bound at the active site. A greater population was found in the bent structural state when the posthydrolysis analog ADP.Al[F.sub.4] was bound. We conclude that the bending of the relay helix in the recovery stroke does not require ATP hydrolysis but is favored by it. A narrower interprobe distance distribution shows ordering of the relay helix, despite its bending, during the recovery stroke, providing further insight into the dynamics of this energy-transducing structural transition. dipolar electron--electron resonance | DEER | molecular dynamics simulation | recovery stroke | disorder-to-order transition doi/10.1073/pnas.0909757106

The purpose of the present study was to determine if the combination of isometric knee extension strength and mechanomyographic (MMG) median frequency (MDF) could be used to estimate the percent (%) myosin heavy chain (MHC) Type II isoform content of the vastus lateralis. Five resistance-trained (mean [+ or -] SD age = 23.2 [+ or -] 3.7 years) and 5 aerobically trained (mean [+ or -] SD age = 32.6 [+ or -] 5.2 years) men volunteered to perform a 6-second isometric maximum voluntary contraction (MVC) of the dominant knee extensors at a joint angle of 90 degrees between the thigh and leg. During the isometric MVC, the surface MMG signal was detected from the vastus lateralis, and the MDF was calculated with the discrete Fourier transform. Following the isometric MVC and MMG measurements, muscle biopsies were taken from the vastus lateralis and analyzed for % MHC Type II isoform content. The results showed that neither isometric knee extension strength nor MMG MDF alone was significantly correlated with the % MHC Type II isoform content. The combination of isometric knee extension strength and MMG MDF, however, explained a significant proportion (i.e., 59.8%) of the variance in % MHC Type II isoform content, with a multiple correlation of R = 0.773 and a standard error of the estimate (SEE) of 15.4%. These findings indicated that a simple, time-efficient, and noninvasive test that simultaneously measures isometric strength and MMG MDF could be useful for estimating the % MHC Type II isoform content in well-trained men. KEY WORDS muscle fiber type, mechanomyography, force

A rigorous numerical test of a hypothetical mechanism of a molecular motor should model explicitly the diffusive motion of the motor's degrees of freedom as well as the transition rates between the motor's chemical states. We present such a Brownian dynamics, mechanochemcial model of the coarse-grain structure of the dimeric, linear motor myosin V. Compared with run-length data, our model provides strong support for a proposed strain-controlled gating mechanism that enhances processivity. We demonstrate that the diffusion rate of a detached motor head during motor stepping is self-consistent with known kinetic rate constants and can explain the motor's key performance features, such as speed and stall force. We present illustrative and realistic animations of motor stepping in the presence of thermal noise. The quantitative success and illustrative power of this type of model suggest that it will be useful in testing our understanding of a range of biological and synthetic motors. Brownian dynamics | molecular motor | strain-dependent gating doi/10.1073/pnas.0908192106

We simultaneously measure both the step size, via FIONA, and the 3-D orientation, via DOPI, of the light-chain domain of individual dimeric myosin VIs. This allows for the correlation of the change in orientation of the light chain domain to the stepping of the motor. Three different pairs of positions were tested using a rigid bifunctional rhodamine on the calmodulin of the IQ domain. The data for all three labeling positions support the model that the light chain domain undergoes a significant rotation of approximately 180[degrees]. Contrary to an earlier study [Sun, Y. et al. (2007) Mol Cell 28, 954-964], our data does not support a model of multiple angles of the lever arm of the lead head, nor 'wiggly' walking on actin. Instead, we propose that for the two heads of myosin VI to coordinate their processive movement, the lever arm of the lead head must be uncoupled from the converter until the rear head detaches. More specifically, intramolecular strain causes the myosin VI lever arm of the lead head to uncouple from the motor domain, allowing the motor domain to go through its product-release (phosphate and ADP) steps at an unstrained rate. The lever arm of the lead head rebinds to the motor and attains a rigor conformation when the rear head detaches. By coupling the orientation and position information with previously described kinetics, this allows us to explain how myosin VI coordinates its heads processively while maintaining the ability to move under load with a (semi-) rigid lever arm. FIONA | fluorescence | motility | single molecule assay | unconventional myosin doi/10.1073/pnas.0900005106

Living cells sense the rigidity of their environment and adapt their activity to it. In particular, cells cultured on elastic substrates align their shape and their traction forces along the direction of highest stiffness and preferably migrate towards stiffer regions. Although numerous studies investigated the role of adhesion complexes in rigidity sensing, less is known about the specific contribution of acto-myosin based contractility. Here we used a custom-made single-cell technique to measure the traction force as well as the speed of shortening of isolated myoblasts deflecting microplates of variable stiffness. The rate of force generation increased with increasing stiffness and followed a Hill force--velocity relationship. Hence, cell response to stiffness was similar to muscle adaptation to load, reflecting the force-dependent kinetics of myosin binding to actin. These results reveal an unexpected mechanism of rigidity sensing, whereby the contractile acto-myosin units themselves can act as sensors. This mechanism may translate anisotropy in substrate rigidity into anisotropy in cytoskeletal tension, and could thus coordinate local activity of adhesion complexes and guide cell migration along rigidity gradients. mechanosensing | adaptation to load | cell migration | cell spreading | cell mechanics doi/10.1073/pnas.0903994106

The actin cytoskeleton has been implicated in the intra- and intercellular movement of a growing number of plant and animal viruses. However, the range of viruses influenced by actin for movement and the mechanism of this transport are poorly understood. Here we determine the importance of microfilaments and myosins for the sustained intercellular movement of a group of RNA-based plant viruses. We demonstrate that the intercellular movement of viruses from different genera [tobacco mosaic virus (TMV), potato virus X (PVX), tomato bushy stunt virus (TBSV)], is inhibited by disruption of microfilaments. Surprisingly, turnip vein-clearing virus (TVCV), a virus from the same genus as TMV, did not require intact microfilaments for normal spread. To investigate the molecular basis for this difference we compared the subcellular location of GFP fusions to the 126-kDa protein and the homologous 125-kDa protein from TMV and TVCV, respectively. The 126-kDa protein formed numerous large cytoplasmic inclusions associated with microfilaments, whereas the 125-kDa protein formed few small possible inclusions, none associated with microfilaments. The dependence of TMV, PVX, and TBSV on intact microfilaments for intercellular movement led us to investigate the role of myosin motors in this process. Virus-induced gene silencing of the Nicotiana benthamiana myosin XI-2 gene, but not three other myosins, inhibited only TMV movement. These results indicate that RNA viruses have evolved differently in their requirements for microfilaments and the associated myosin motors, in a manner not correlated with predicted phylogeny. cytoskeleton | microfilaments | tobacco mosaic virus | potato virus X | tomato bushy stunt virus doi/ 10.1073/pnas.0909239106

Although myosin VI has properties that would allow it to function optimally as a dimer, full-length myosin VI exists as a monomer in isolation. Based on the ability of myosin VI monomers to dimerize when held in close proximity, we postulated that cargo binding normally regulates dimerization of myosin VI. We tested this hypothesis by expressing a known dimeric cargo adaptor protein of myosin VI, optineurin, and the myosin VI-binding segment from a monomeric cargo adaptor protein, Dab2. In the presence of these adaptor proteins, full-length myosin VI has ATPase properties of a dimer, appears as a dimer in electron micrographs, and moves processively on actin filaments. The results support a model in which cargo binding exposes internal dimerization sequences within full-length myosin VI. Because, unexpectedly, a monomeric fragment of Dab2 triggers dimerization, it would appear that myosin VI is designed to function as a dimer in cells. Dab2 | directionality | motility | optineurin | unconventional myosin doi/ 10.1073/pnas.0909748106

The purpose of this study was to determine the effect of 5'-AMP-activated protein kinase (AMPK) on energy metabolism and myosin heavy chain (MyHC) isoform expression in growing pigs using chronic treatment with 5-aminoimidazole-4-carboxamide-l[beta]-D-ribofuranoside (AICAR) as a model. Four-weekold pigs were given daily injections of AICAR or 0.9% saline for 10 d. Treatment with AICAR increased (P < 0.05) AMPK activity in semitendinosus muscles (STM). Expression of skeletal muscle specific glucose transporter 4 (GLUT4) was also enhanced (P < 0.05) by AICAR treatment. Using real-time PCR, electrophoresis, and Western blot analyses, we confirmed that AICAR treatment caused a decrease (P < 0.05) in type IIa MyHC isoform mRNA and protein levels and a con comitant increase (P < 0.05) in type IIx MyHC containing fibers. Consistent with a MyHC isoform shift from IIa to IIx, muscles from pigs treated with AICAR had greater (P < 0.05) lactate dehydrogenase (LDH) activity. Moreover, muscle of treated pigs expressed greater (P < 0.05) message for LDH. Administration of AICAR, however, did not alter expression of PPAR-[gamma] coactivator-l[alpha], fatty acid translocase, citrate synthase, or the activity of cytochrome c oxidase. Overall, these results indicate that activation of AMPK by AICAR causes muscle to assume a faster-contracting, more glycolytic nature. These data are in direct contrast to documented effects in rodent models, but these effects may be dependent on the time of administration and the overall growth status of the animal. Key words: 5'-AMP-activated protein kinase, 5-aminoimidazole-4-carboxamide-l-[beta]-D-ribofuranoside, myosin heavy chain, skeletal muscle

We describe an active polymer network in which processive molecular motors control network elasticity. This system consists of actin filaments cross-linked by filamin A (FLNa) and contracted by bipolar filaments of muscle myosin II. The myosin motors stiffen the network by more than two orders of magnitude by pulling on actin filaments anchored in the network by FLNa cross-links, thereby generating internal stress. The stiffening response closely mimics the effects of external stress applied by mechanical shear. Both internal and external stresses can drive the network into a highly nonlinear, stiffened regime. The active stress reaches values that are equivalent to an external stress of 14 Pa, consistent with a 1-pN force per myosin head. This active network mimics many mechanical properties of cells and suggests that adherent cells exert mechanical control by operating in a nonlinear regime where cell stiffness is sensitive to changes in motor activity. This design principle may be applicable to engineering novel biologically inspired, active materials that adjust their own stiffness by internal catalytic control. active soft matter | cytoskeleton | filamin A | rheology | myosin II

The contractile vacuole (CV) complex in Dictyostelium is a tubulovesicular osmoregulatory organelle that exhibits extensive motility along the actin-rich cortex, providing a useful model for investigating myos-independent membrane transport. Here, we show that the type V myosin myoJ localizes to CV membranes and is required for efficient osmoregulation, the normal accumulation of CV membranes in the cortex, and the conversion of collapsed bladder membranes into outwardly radiating cortical CV tubules. Complementation of myoJ-null cells with a version of myoJ containing a shorter lever arm causes these radiating tubules to move at a slower speed, confirming myoJ's role in translocating CV membranes along the cortex. MyoJ-null cells also exhibit a dramatic concentration of CV membranes around the microtubule-organizing center. Consistently, we demonstrate that CV membranes also move bi-directionally on microtubules between the cortex and the centrosome. Therefore, myoJ cooperates with plus and minus end-directed microtubule motors to drive the normal distribution and dynamics of the CV complex in Dictyostelium.

In Saccharomyces cerevisiae, the class V myosin motor Myo2p propels the movement of most organelles. We recently identified Inp2p as the peroxisome-specific receptor for Myo2p. In this study, we delineate the region of Myo2p devoted to binding peroxisomes. Using mutants of Myo2p specifically impaired in peroxisome binding, we dissect cell cycle-dependent and peroxisome partitioning-dependent mechanisms of Inp2p regulation. We find that although total Inp2p levels oscillate with the cell cycle, Inp2p levels on individual peroxisomes are controlled by peroxisome inheritance, as Inp2p aberrantly accumulates and decorates all peroxisomes in mother cells when peroxisome partitioning is abolished. We also find that Inp2p is a phosphoprotein whose level of phosphorylation is coupled to the cell cycle irrespective of peroxisome positioning in the cell. Our findings demonstrate that both organelle positioning and cell cycle progression control the levels of organelle-specific receptors for molecular motors to ultimately achieve an equidistribution of compartments between mother and daughter cells.

The participation of nonmuscle myosin in force maintenance is controversial. Furthermore, its regulation is difficult to examine in a cellular context, as the light chains of smooth muscle and nonmuscle myosin comigrate under native and denaturing electrophoresis techniques. Therefore, the regulatory light chains of smooth muscle myosin (SM-RLC) and nonmuscle myosin (NM-RLC) were purified, and these proteins were resolved by isoelectric focusing. Using this method, intact mouse aortic smooth muscle homogenates demonstrated four distinct RLC isoelectric variants. These spots were identified as phosphorylated NM-RLC (most acidic), nonphosphorylated NM-RLC, phosphorylated SM-RLC, and nonphosphorylated SM-RLC (most basic). During smooth muscle activation, NM-RLC phosphorylation increased. During depolarization, the increase in NM-RLC phosphorylation was unaffected by inhibition of either Rho kinase or PKC. However, inhibition of Rho kinase blocked the angiotensin II-induced increase in NM-RLC phosphorylation. Additionally, force for angiotensin II stimulation of aortic smooth muscle from heterozygous nonmuscle myosin IIB knockout mice was significantly less than that of wild-type littermates, suggesting that, in smooth muscle, activation of nonmuscle myosin is important for force maintenance. The data also demonstrate that, in smooth muscle, the activation of nonmuscle myosin is regulated by [Ca.sup.2+]-calmodulin-activated myosin light chain kinase during depolarization and a Rho kinase-dependent pathway during agonist stimulation. Rho kinase; agonist; depolarization; myosin light chain kinase

Eukaryotic cells have a self-organizing cytoskeleton where motors transport cargoes along cytoskeletal tracks. To understand the sorting process, we developed a system to observe single-molecule motility in a cellular context. We followed myosin classes V, VI, and X on triton-extracted actin cytoskeletons from Drosophila S2, mammalian COS-7, and mammalian U2OS cells. We find that these cells vary considerably in their global traffic patterns. The S2 and U2OS cells have regions of actin that either enhance or inhibit specific myosin classes. U2OS cells allow for 1 motor class, myosin Vl, to move along stress fiber bundles, while motility of myosin V and X are suppressed. Myosin X motors are recruited to filopodia and the lamellar edge in S2 cells, whereas myosin VI motility is excluded from the same regions. Furthermore, we also see different velocities of myosin V motors in central regions of S2 cells, suggesting regional control of motor motility by the actin cytoskeleton. We also find unexpected features of the actin cytoskeletal network, including a population of reversed filaments with the barbed-end toward the cell center. This myosin motor regulation demonstrates that native actin cytoskeletons are more than just a collection of filaments. actin cytoskeleton | myosin motors | single-molecule

Myosin VIIA is an unconventional myosin, responsible for human Usher syndrome type 1B, which causes hearing and visual loss. Here, we studied the molecular mechanism of regulation of myosin VlIA, which is currently unknown. Although it was originally thought that myosin VIIA is a dimeric myosin, our electron microscopic (EM) observations revealed that full-length Drosophila myosin VlIA (DM7A) is a monomer. Interestingly, the tail domain markedly inhibits the actin-activated ATPase activity of tailless DM7A at low [Ca.sup.2+] but not high [Ca.sup.2+]. By examining various deletion constructs, we found that deletion of the distal IQ domain, the C-terminal region of the tail, and the N-terminal region of the tail abolishes the tail-induced inhibition of ATPase activity. Single-particle EM analysis of full-length DM7A at low [Ca.sup.2+] suggests that the tail folds back on to the head, where it contacts both the motor core domain and the neck domain, forming an inhibited conformation. We concluded that unconventional myosin that may be present a monomer in the cell can be regulated by intramolecular interaction of the tail with the head. molecular motor | regulation | unconventional myosin | Usher syndrome type 1B | EM images

Estrogen ([E.sub.2]) is reported to regulate skeletal muscle mass and contractile function; whether [E.sub.2] exerts its effects through estrogen receptor-[alpha] (ER[alpha]) or -[beta] (ER[beta]) is unclear. We determined the effect of ER[alpha] or ER[beta] elimination on muscle mass and contractile function in multiple muscles of the lower limb, muscles with different locomotor tasks and proportions of fiber types I and II: soleus (Sol), plantaris (Plan), tibialis anterior (TA), and gastrocnemius (Gast) in mature female mice. To determine [E.sub.2] elimination effects on muscle, we also used aromatase (Ar) knockout (KO) and wild-type (WT) mice. ER[alpha] and ArKO body weights were ~10 and 20% higher than WT. Although muscle mass tended to show a commensurate increase in both groups, only the TA was significantly larger in ER[alpha] (P < 0.05). Ratios of muscle mass to body mass revealed significantly lower values for Gast and TA in ArKO mice (P < 0.05). Tetanic tension ([P.sub.o]) per calculated anatomical cross-sectional area (aCSA) in ER[alpha] KO was lower in TA and Gast than in WT. Lower [P.sub.o]/aCSA in ER[alpha] KO Gast and TA was also supported histologically by significantly less [P.sub.o]/fiber areas (P < 0.05). ArKO mice also had lower [P.sub.o]/aCSA in Gast and TA compared with WT. ER[beta] KO and WT mice were comparable in all measures. Our results support the hypothesis that [E.sub.2] effects on skeletal muscle are mediated in part via the ER[alpha] but that [E.sub.2] effects may be mediated via more than one mechanism or receptor. muscle mass; peak tetanic tension; fiber area; myosin protein

Full-length Drosophila myosin 7a (myosin 7a-FL) has a complex tail containing a short predicted coiled coil followed by a MyTH4-FERM domain, an SH3 domain, and a C-terminal MyTH4-FERM domain. Myosin 7a-FL expressed in Sf9 cells is monomeric despite the predicted coiled coil. We showed previously that Subfragment-1 (S1) from this myosin has MgATPase of [V.sub.max] [approximately equal to] [1S-.sup.-1] and [K.sub.ATPase] [approximately equal to] 1 [micro]M actin. We find that myosin 7a-FL has [V.sub.max] similar to S1 but [K.sub.ATPase] [approximately equal to] 30 [micro]M. Thus, at low actin concentrations (5 [micro]M), the MgATPase of S1 is fully activated, whereas that of myosin 7a-FL is low, suggesting that the tail regulates activity. Electron microscopy of myosin 7a-FL with ATP shows the tail is tightly bent back against the motor domain. Myosin 7a-FL extends at either high ionic strength or without ATP, revealing the motor domain, lever, and tail. A series of C-terminal truncations show that deletion of 99 aa (the MYTH7 subdomain of the C-terminal FERM domain) is sufficient to abolish bending, and the [K.sub.ATPase] is then similar to S1. This region is highly conserved in myosin 7a. We found that a double mutation in it, R2140A-K2143A, abolishes bending and reduces [K.sub.ATPase] to S1 levels. In addition, the expressed C-terminal FERM domain binds actin with [K.sub.d] [approximately equal to] 30 [micro]M regardless of ATP, similar to the [K.sub.ATPase] value for myosin 7a-FL. We propose that at low cellular actin concentrations, myosin 7a-FL is bent and inactive, but at high actin concentrations, it is unfolded and active because the C-terminal FERM domain binds to actin. regulation | electron microscopy | ATPase activity

Flowering plants have evolved multigene families of the class XI myosin motors, the functions of which remain poorly understood. Here, we investigated functional profiles of the Arabidopsis myosins that belong to two paralogous pairs, XI-K/XI-1 and XI-2/XI-B, using single and double gene-knockout mutants. It was found that the myosins XI-K, XI-2, and XI-B, but not XI-1 have overlapping and additive roles in the root hair elongation. A nonidentical set of the three myosins, XI-K, XI-1, and XI-2, exhibited partially redundant and additive roles in the transport of Golgi stacks, peroxisomes, and mitochondria. Conspicuously, the double xi-k/1 knockout plants that showed the largest cumulative reduction of the organelle velocities also exhibited a stunted plant growth and reduced fecundity phenotype. Collectively, these results suggest that the rapid, myosin-powered organelle trafficking is required for the optimal plant growth, whereas a distinct myosin function, presumably the vesicular transport, is involved in elongation of the root hairs. In addition, our data imply that the myosin gene duplication in plants has been followed by a gradual functional specialization of the resulting pairs of myosin paralogs. myosin | organelle transport | plant development

Ractopamine-HC1 (RAC) is a [beta]-adrenergic agonist with variable effects on cattle performance and carcass variables. Cull cows fed RAC (200 mg x [head.sup.-1] x [d.sup.-1]) demonstrate an increased size of type I and II muscle fibers that does not translate into a larger ribeye area. The objective of this study was to examine the dose-dependent effects of RAC on cull cow muscle morphometrics. Eighty-eight cull beef cows representing 2 breed types (n = 44 each) were fed 0, 100, 200, and 300 mg x [head.sup.-1] x [.sup.d-1] of RAC for the last 28 d of a 54-d feeding period. On d 54, cows were slaughtered, and samples of the LM and semimembranosus muscle (SM) from 16 randomly selected carcasses (n = 4 per treatment) were taken for measurement of [[beta].sub.2]-adrenergic receptors and type I, IIA, and IIX myosin heavy chain (MyHC) gene expression. Twenty-four hours postmortem, LM, SM, infraspinatus (INF), and vastus lateralis samples from 40 randomly selected carcasses (n = 10 per treatment) were obtained and frozen for immunohistochemical analysis. Muscle fiber cross-sectional area and diameter, MyHC isoform expression, and fiber-associated nuclei numbers were measured. Ractopamine dosage exhibited differential effects on muscle morphometrics and MyHC gene expression. Muscle fiber cross-sectional area and diameter were increased (P < 0.05) by RAC in INF type I and IIA fibers and SM type IIA fibers. Ractopamine increased (P < 0.05) MyHC type IIX mRNA and tended to increase (P < 0.10) [[beta].sub.2]-adrenergic receptors in the SM; a change in mRNA abundance was not detected for either gene in the LM. Treatment with RAC decreased (P < 0.05) fiber-associated nuclei numbers in the INF, vastus lateralis, and LM but did not affect (P > 0.05) MyHC or [beta]-adrenergic receptor expression. These results indicate that cull cow feeding programs may consider supplementing RAC as a means of adding value to cuts within the chuck, such as the INF. Key words: hypertrophy, myosin heavy chain, ractopamine, skeletal muscle, type I fiber

p21-activated kinases (Paks) have been identified in a variety of eukaryotic cells as key effectors of the Cdc42 family of guanosine triphosphatases. Pak kinases play important roles in regulating the filamentous actin cytoskeleton. In this study, we describe a function for the Schizosaccharomyces pombe Pak-related protein Pak1p/Orb2p in cytokinesis. Pak1p localizes to the actomyosin ring during mitosis and cytokinesis. Loss of Pak1 p function leads to accelerated cytokinesis. Pak1p mediates phosphorylation of myosin II regulatory light chain Rlc1p at serine residues 35 and 36 in vivo. Interestingly, loss of Pak1 p function or substitution of serine 35 and serine 36 of Rlc1p with alanines, thereby mimicking a dephosphorylated state of Rlc1p, leads to defective coordination of mitosis and cytokinesis. This study reveals a new mechanism involving Pak1p kinase that helps ensure the fidelity of cytokinesis.

Dendrite morphogenesis is regulated by neuronal activity or neurotrophins, which may function by activating intrinsic signaling proteins, including Rho family GTPases. Here we report that activity- and brain-derived neurotrophic factor (BDNF)--dependent dendritic morphogenesis requires activation of geranylgeranyltransferase I (GGT), a prenyltransferase that mediates lipid modification of Rho GTPases. Dendritic arborization in cultured hippocampal neurons was promoted by over-expression of GGT, and reduced by inhibition or down-regulation of GGT. Furthermore, GGT was activated by neuronal depolarization or BDNF, both of which promote dendritic arborization, in cultured hippocampal neurons. Moreover, exploration of a novel environment caused activation of GGT in the mice hippocampus, suggesting that neural activity activates GGT in vivo. Interestingly, GGT was physically associated with tropomyosin-related kinase B (TrkB), the receptor for BDNF, and this association was enhanced by depolarization. Disrupting the GGT-TrkB interaction or down-regulating GGT activity attenuated depolarization- or BDNF-induced dendrite development. Finally, the GGT effect on dendrite arborization was prevented by over-expressing Rac1 with the prenylation site deleted or mutated. Thus depolarization- or BDNF-dependent dendrite development may be mediated by GGT-induced prenylation of Rho GTPases. BDNF | dendrite | neuronal activity | Rac | prenylation