The polarisome is a cortical proteinaceous microcompartment that organizes the growth of actin filaments and the fusion of secretory vesicles in yeasts and filamentous fungi. Polarisomes are compact, spotlike structures at the growing tips of their respective cells. The molecular forces that control the form and size of this microcompartment are not known. Here we identify a complex between the polarisome subunit Pea2 and the type V Myosin Myo2 that anchors Myo2 at the cortex of yeast cells. We discovered a point mutation in the cargo-binding domain of Myo2 that impairs the interaction with Pea2 and consequently the formation and focused localization of the polarisome. Cells carrying this mutation grow round instead of elongated buds. Further experiments and biophysical modeling suggest that the interactions between polarisome-bound Myo2 motors and dynamic actin filaments spatially focus the polarisome and sustain its compact shape.

In the apex-directed RAB11 exocytic pathway of Aspergillus nidulans, kinesin-1/KinA conveys secretory vesicles (SVs) to the hyphal tip, where they are transferred to the type V myosin MyoE. MyoE concentrates SVs at an apical store located underneath the PM resembling the presynaptic active zone. A rod-shaped RAB11 effector, UDS1, and the intrinsically disordered and coiled-coil HMSV associate with MyoE in a stable HUM (HMSV-UDS1-MyoE) complex recruited by RAB11 to SVs through an interaction network involving RAB11 and HUM components, with the MyoE globular tail domain (GTD) binding both HMSV and RAB11-GTP and RAB11-GTP binding both the MyoE-GTD and UDS1. UDS1 bridges RAB11-GTP to HMSV, an avid interactor of the MyoE-GTD. The interaction between the UDS1-HMSV sub-complex and RAB11-GTP can be reconstituted in vitro. Ablating UDS1 or HMSV impairs actomyosin-mediated transport of SVs to the apex, resulting in spreading of RAB11 SVs across the apical dome as KinA/microtubule-dependent transport gains prominence.

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 myosin-dependent 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.

Class V myosin (myosin-V) is a cargo transporter that moves along an actin filament with large (approximately 36-nm) successive steps. It consists of two heads that each includes a motor domain and a long (23 nm) neck domain. One of the more popular models describing these steps, the hand-over-hand model, assumes the two-headed structure is imperative. However, we previously succeeded in observing successive large steps by one-headed myosin-V upon optimizing the angle of the acto-myosin interaction. In addition, it was reported that wild type myosin-VI and myosin-IX, both one-headed myosins, can also generate successive large steps. Here, we describe the mechanical properties (stepsize and stepping kinetics) of successive large steps by one-headed and two-headed myosin-Vs. This study shows that the stepsize and stepping kinetics of one-headed myosin-V are very similar to those of the two-headed one. However, there was a difference with regards to stability against load and the number of multisteps. One-headed myosin-V also showed unidirectional movement that like two-headed myosin-V required 3.5 k(B)T from ATP hydrolysis. This value is also similar to that of smooth muscle myosin-II, a non-processive motor, suggesting the myosin family uses a common mechanism for stepping regardless of the steps being processive or non-processive. In this present paper, we conclude that one-headed myosin-V can produce successive large steps without following the hand-over-hand mechanism.

Unlike wild-type mouse melanocytes, where melanosomes are concentrated in dendrites and dendritic tips, melanosomes in dilute (myosin Va-) melanocytes are concentrated in the cell center. Here we sought to define the role that myosin Va plays in melanosome transport and distribution. Actin filaments that comprise a cortical shell running the length of the dendrite were found to exhibit a random orientation, suggesting that myosin Va could drive the outward spreading of melanosomes by catalyzing random walks. In contrast to this mechanism, time lapse video microscopy revealed that melanosomes undergo rapid ( approximately 1.5 microm/s) microtubule-dependent movements to the periphery and back again. This bidirectional traffic occurs in both wild-type and dilute melanocytes, but it is more obvious in dilute melanocytes because the only melanosomes in their periphery are those undergoing this movement. While providing an efficient means to transport melanosomes to the periphery, this component does not by itself result in their net accumulation there. These observations, together with previous studies showing extensive colocalization of myosin Va and melanosomes in the actin-rich periphery, suggest a mechanism in which a myosin Va-dependent interaction of melanosomes with F-actin in the periphery prevents these organelles from returning on microtubules to the cell center, causing their distal accumulation. This "capture" model is supported by the demonstration that (a) expression of the myosin Va tail domain within wild-type cells creates a dilute-like phenotype via a process involving initial colocalization of tail domains with melanosomes in the periphery, followed by an approximately 120-min, microtubule-based redistribution of melanosomes to the cell center; (b) microtubule-dependent melanosome movement appears to be damped by myosin Va; (c) intermittent, microtubule-independent, approximately 0.14 microm/s melanosome movements are seen only in wild-type melanocytes; and (d) these movements do not drive obvious spreading of melanosomes over 90 min. We conclude that long-range, bidirectional, microtubule-dependent melanosome movements, coupled with actomyosin Va-dependent capture of melanosomes in the periphery, is the predominant mechanism responsible for the centrifugal transport and peripheral accumulation of melanosomes in mouse melanocytes. This mechanism represents an alternative to straightforward transport models when interpreting other myosin V mutant phenotypes.

Because of the industrial and medical importance of members of the fungal genus Aspergillus, there is considerable interest in the functions of cytoskeletal components in growth and secretion in these organisms. We have analyzed the genome of Aspergillus nidulans and found that there are two previously unstudied myosin genes, a myosin II homolog, myoB (product = MyoB) and a myosin V homolog, myoE (product = MyoE). Deletions of either cause significant growth defects. MyoB localizes in strings that coalesce into contractile rings at forming septa. It is critical for septation and normal deposition of chitin but not for hyphal extension. MyoE localizes to the Spitzenkörper and to moving puncta in the cytoplasm. Time-lapse imaging of SynA, a v-SNARE, reveals that in myoE deletion strains vesicles no longer localize to the Spitzenkörper. Tip morphology is slightly abnormal and branching occurs more frequently than in controls. Tip extension is slower than in controls, but because hyphal diameter is greater, growth (increase in volume/time) is only slightly reduced. Concentration of vesicles into the Spitzenkörper before incorporation into the plasma membrane is, thus, not required for hyphal growth but facilitates faster tip extension and a more normal hyphal shape.

Pentabromopseudilin (PBrP) is a marine antibiotic isolated from the marine bacteria Pseudomonas bromoutilis and Alteromonas luteoviolaceus. PBrP exhibits antimicrobial, anti-tumour, and phytotoxic activities. In mammalian cells, PBrP is known to act as a reversible and allosteric inhibitor of myosin Va (MyoVa). In this study, we report that PBrP is a potent inhibitor of transforming growth factor-β (TGF-β) activity. PBrP inhibits TGF-β-stimulated Smad2/3 phosphorylation, plasminogen activator inhibitor-1 (PAI-1) protein production and blocks TGF-β-induced epithelial-mesenchymal transition in epithelial cells. PBrP inhibits TGF-β signalling by reducing the cell-surface expression of type II TGF-β receptor (TβRII) and promotes receptor degradation. Gene silencing approaches suggest that MyoVa plays a crucial role in PBrP-induced TβRII turnover and the subsequent reduction of TGF-β signalling. Because, TGF-β signalling is crucial in the regulation of diverse pathophysiological processes such as tissue fibrosis and cancer development, PBrP should be further explored for its therapeutic role in treating fibrotic diseases and cancer.

Lgl family members play an important role in the regulation of cell polarity in eukaryotic cells. The yeast homologues Sro7 and Sro77 are thought to act downstream of the Rab GTPase Sec4 to promote soluble N-ethylmaleimide-sensitive factor adaptor protein receptor (SNARE) function in post-Golgi transport. In this article, we characterize the interaction between Sro7 and the type V myosin Myo2 and show that this interaction is important for two distinct aspects of Sro7 function. First, we show that this interaction plays a positive role in promoting the polarized localization of Sro7 to sites of active growth. Second, we find evidence that Myo2 negatively regulates Sro7 function in vesicle clustering. Mutants in either Myo2 or Sro7 that are defective for this interaction show hypersensitivity to Sro7 overexpression, which results in Sec4-dependent accumulation of large groups of vesicles in the cytoplasm. This suggests that Myo2 serves a dual function, to both recruit Sro7 to secretory vesicles and inhibit its Rab-dependent tethering activity until vesicles reach the plasma membrane. Thus Sro7 appears to coordinate the spatial and temporal nature of both Rab-dependent tethering and SNARE-dependent membrane fusion of exocytic vesicles with the plasma membrane.

Myosin V (myoV) is a two-headed myosin capable of taking many successive steps along actin per diffusional encounter, enabling it to transport vesicular and ribonucleoprotein cargos in the dense and complex environment within cells. To better understand how myoV navigates along actin, we used polarized total internal reflection fluorescence microscopy to examine angular changes of bifunctional rhodamine probes on the lever arms of single myoV molecules in vitro. With a newly developed analysis technique, the rotational motions of the lever arm and the local orientation of each probe relative to the lever arm were estimated from the probe's measured orientation. This type of analysis could be applied to similar studies on other motor proteins, as well as other proteins with domains that undergo significant rotational motions. The experiments were performed on recombinant constructs of myoV that had either the native-length (six IQ motifs and calmodulins [CaMs]) or truncated (four IQ motifs and CaMs) lever arms. Native-length myoV-6IQ mainly took straight steps along actin, with occasional small azimuthal tilts around the actin filament. Truncated myoV-4IQ showed an increased frequency of azimuthal steps, but the magnitudes of these steps were nearly identical to those of myoV-6IQ. The results show that the azimuthal deflections of myoV on actin are more common for the truncated lever arm, but the range of these deflections is relatively independent of its lever-arm length.

There is growing evidence for a coupling of actin assembly and myosin motor activity in cells. However, mechanisms for recruitment of actin nucleators and motors on specific membrane compartments remain unclear. Here we report how Spir actin nucleators and myosin V motors coordinate their specific membrane recruitment. The myosin V globular tail domain (MyoV-GTD) interacts directly with an evolutionarily conserved Spir sequence motif. We determined crystal structures of MyoVa-GTD bound either to the Spir-2 motif or to Rab11 and show that a Spir-2:MyoVa:Rab11 complex can form. The ternary complex architecture explains how Rab11 vesicles support coordinated F-actin nucleation and myosin force generation for vesicle transport and tethering. New insights are also provided into how myosin activation can be coupled with the generation of actin tracks. Since MyoV binds several Rab GTPases, synchronized nucleator and motor targeting could provide a common mechanism to control force generation and motility in different cellular processes.

Sensory neuron terminal differentiation tasks apical secretory transport with delivery of abundant biosynthetic traffic to the growing sensory membrane. We recently showed Drosophila Rab11 is essential for rhodopsin transport in developing photoreceptors and asked here if myosin V and the Drosophila Rab11 interacting protein, dRip11, also participate in secretory transport. Reduction of either protein impaired rhodopsin transport, stunting rhabdomere growth and promoting accumulation of cytoplasmic rhodopsin. MyoV-reduced photoreceptors also developed ectopic rhabdomeres inappropriately located in basolateral membrane, indicating a role for MyoV in photoreceptor polarity. Binary yeast two hybrids and in vitro protein-protein interaction predict a ternary complex assembled by independent dRip11 and MyoV binding to Rab11. We propose this complex delivers morphogenic secretory traffic along polarized actin filaments of the subcortical terminal web to the exocytic plasma membrane target, the rhabdomere base. A protein trio conserved across eukaryotes thus mediates normal, in vivo sensory neuron morphogenesis.

Synaptic vesicle fusion occurs at specialized release sites at the active zone. How refilling of release sites with new vesicles is regulated in central synapses remains poorly understood. Using nanoscale-resolution detection of individual release events in rat hippocampal synapses we found that inhibition of myosin V, the predominant vesicle-associated motor, strongly reduced refilling of the release sites during repetitive stimulation. Single-vesicle tracking revealed that recycling vesicles continuously shuttle between a plasma membrane pool and an inner pool. Vesicle retention at the membrane pool was regulated by neural activity in a myosin V dependent manner. Ultrastructural measurements of vesicle occupancy at the plasma membrane together with analyses of single-vesicle trajectories during vesicle shuttling between the pools suggest that myosin V acts as a vesicle tether at the plasma membrane, rather than a motor transporting vesicles to the release sites, or directly regulating vesicle exocytosis.

While vesicle transport is one of the principal functions of myosin motors in neurones, the role played by specific myosin subtypes in discrete vesicle trafficking is poorly understood. We conducted electrophysiological and morphological experiments to determine whether myosin isoforms II and V might be involved in the transport of small synaptic vesicles in presynaptic nerve terminals of a model cholinergic synapse. Electron microscopy revealed the presence of normal synaptic architecture and synaptic vesicle density in presynaptic terminals of cultured superior cervical ganglion neurones (SCGNs) from myosin Va null rats (dilute-opisthotonus, dop). Similarly, electrophysiological analyses of synaptic transmission and synaptic vesicle cycling at paired SCGN synapses failed to uncover any significant differences in synaptic development and function between normal and dop rats. Immunocytochemistry and in situ localization of green fluorescent protein (GFP)-fusion proteins in wild-type synapses revealed that myosins IIB and Va were distributed throughout the cell soma and processes of SCGNs, while myosins IIA and Vb were not detected in SCGNs. Myosin Va was conspicuously absent in presynaptic nerve terminals, but myosin IIB alone was found to be expressed. Furthermore, synaptic transmission was inhibited by introduction of myosin IIB heavy chain fragments into presynaptic terminals of SCGNs. Together these results suggest that only myosin IIB isoform participates in vesicle trafficking in presynaptic nerve terminals of cultured SCGNs.

Transforming Growth Factor Beta (TGF-β) is involved in regulating many biological processes and disease states. Cells secrete cytokine as a latent complex that must be activated for it to exert its biological functions. We previously discovered that the epithelial-restricted integrin α(v)β(6) activates TGF-β and that this process is important in a number of in vivo models of disease. Here, we show that agonists of G-protein coupled receptors (Sphingosine-1-Phosphate and Lysophosphatidic Acid) which are ligated under conditions of epithelial injury directly stimulate primary airway epithelial cells to activate latent TGF-β through a pathway that involves Rho Kinase, non-muscle myosin, the α(v)β(6) integrin, and the generation of mechanical tension. Interestingly, lung epithelial cells appear to exert force on latent TGF-β using sub-cortical actin/myosin rather than the stress fibers utilized by fibroblasts and other traditionally "contractile" cells. These findings extend recent evidence suggesting TGF-β can be activated by integrin-mediated mechanical force and suggest that this mechanism is important for an integrin (α(v)β(6)) and a cell type (epithelial cells) that have important roles in biologically relevant TGF-β activation in vivo.

This study aimed to identify the genetic defect in a multigenerational family presenting an autosomal dominant myopathy with histological features of congenital fiber type disproportion. Linkage analysis and genetic sequencing identified, in all affected members of the family, the c.5807A>G heterozygous mutation in MYH7, which encodes the slow/β-cardiac myosin heavy chain. This mutation causes skeletal but not cardiac involvement. Myosin heavy chain expression pattern was also characterized by immunohistochemistry, western blot and q-PCR in muscle biopsies from two patients aged 25 and 62, respectively. While only congenital fiber type disproportion was observed in the younger patient, older patient's biopsy presented aggregates of slow myosin heavy chains, in fiber sub-sarcolemmal region. These clinico-pathologic findings suggest a novel phenotype within the emerging group of hereditary myosin myopathies, which in this family presents typical characteristics of congenital fiber type disproportion in early stages and later evolves to myosin storage myopathy.

Skeletal muscle consists of different fiber types which adapt to exercise, aging, disease, or trauma. Here we present a protocol for fast staining, automatic acquisition, and quantification of fiber populations with ImageJ.

The vegetal polyphenol curcumin displays beneficial effects against skeletal muscle derangement induced by oxidative stress, disuse or aging. Since oxidative stress and inflammation are involved in the progression of muscle dystrophy, the effects of curcumin administration were investigated in the diaphragm of mdx mice injected intraperitoneally or subcutaneously with curcumin for 4-12-24 weeks. Curcumin treatment independently of the way and duration of administration (i) ameliorated myofiber maturation index without affecting myofiber necrosis, inflammation and degree of fibrosis; (ii) counteracted the decrease in type 2X and 2B fiber percentage; (iii) increased about 30% both twitch and tetanic tensions of diaphragm strips; (iv) reduced myosin nitrotyrosination and tropomyosin oxidation; (v) acted on two opposite nNOS regulators by decreasing active AMP-Kinase and increasing SERCA1 protein levels, the latter effect being detectable also in myotube cultures from mdx satellite cells. Interestingly, increased contractility, decreased myosin nitrotyrosination and SERCA1 upregulation were also detectable in the mdx diaphragm after a 4-week administration of the NOS inhibitor 7-Nitroindazole, and were not improved further by a combined treatment. In conclusion, curcumin has beneficial effects on the dystrophic muscle, mechanistically acting for the containment of a deregulated nNOS activity.

Nonmuscle myosin type II (Myo1p) is required for cytokinesis in the budding yeast Saccharomyces cerevisiae Loss of Myo1p activity has been associated with growth abnormalities and enhanced sensitivity to osmotic stress, making it an appealing antifungal therapeutic target. The Myo1p tail-only domain was previously reported to have functional activity equivalent to the full-length Myo1p whereas the head-only domain did not. Since Myo1p tail-only constructs are biologically active, the tail domain must have additional functions beyond its previously described role in myosin dimerization or trimerization. The identification of new Myo1p-interacting proteins may shed light on the other functions of the Myo1p tail domain. To identify novel Myo1p-interacting proteins, and determine if Myo1p can serve as a scaffold to recruit proteins to the bud neck during cytokinesis, we used the integrated split-ubiquitin membrane yeast two-hybrid (iMYTH) system. Myo1p was iMYTH-tagged at its C-terminus, and screened against both cDNA and genomic prey libraries to identify interacting proteins. Control experiments showed that the Myo1p-bait construct was appropriately expressed, and that the protein colocalized to the yeast bud neck. Thirty novel Myo1p-interacting proteins were identified by iMYTH. Eight proteins were confirmed by coprecipitation (Ape2, Bzz1, Fba1, Pdi1, Rpl5, Tah11, and Trx2) or mass spectrometry (AP-MS) (Abp1). The novel Myo1p-interacting proteins identified come from a range of different processes, including cellular organization and protein synthesis. Actin assembly/disassembly factors such as the SH3 domain protein Bzz1 and the actin-binding protein Abp1 represent likely Myo1p interactions during cytokinesis.

The β-myosin heavy chain expressed in ventricular myocardium and the myosin heavy chain (MyHC) in slow-twitch skeletal Musculus soleus (M. soleus) type-I fibers are both encoded by MYH7. Thus, these myosin molecules are deemed equivalent. However, some reports suggested variations in the light chain composition between M. soleus and ventricular myosin, which could influence functional parameters, such as maximum velocity of shortening. To test for functional differences of the actin gliding velocity on immobilized myosin molecules, we made use of in vitro motility assays. We found that ventricular myosin moved actin filaments with ∼0.9 µm/s significantly faster than M. soleus myosin (0.3 µm/s). Filaments prepared from isolated actin are not the native interaction partner of myosin and are believed to slow down movement. Yet, using native thin filaments purified from M. soleus or ventricular tissue, the gliding velocity of M. soleus and ventricular myosin remained significantly different. When comparing the light chain composition of ventricular and M. soleus β-myosin, a difference became evident. M. soleus myosin contains not only the "ventricular" essential light chain (ELC) MLC1sb/v, but also an additional longer and more positively charged MLC1sa. Moreover, we revealed that on a single muscle fiber level, a higher relative content of MLC1sa was associated with significantly slower actin gliding. We conclude that the ELC MLC1sa decelerates gliding velocity presumably by a decreased dissociation rate from actin associated with a higher actin affinity compared to MLC1sb/v. Such ELC/actin interactions might also be relevant in vivo as differences between M. soleus and ventricular myosin persisted when native thin filaments were used.

Cytokinesis in Saccharomyces cerevisiae involves coordination between actomyosin ring contraction and septum formation and/or targeted membrane deposition. We show that Mlc1p, a light chain for Myo2p (type V myosin) and Iqg1p (IQGAP), is the essential light chain for Myo1p, the only type II myosin in S. cerevisiae. However, disruption or reduction of Mlc1p-Myo1p interaction by deleting the Mlc1p binding site on Myo1p or by a point mutation in MLC1, mlc1-93, did not cause any obvious defect in cytokinesis. In contrast, a different point mutation, mlc1-11, displayed defects in cytokinesis and in interactions with Myo2p and Iqg1p. These data suggest that the major function of the Mlc1p-Myo1p interaction is not to regulate Myo1p activity but that Mlc1p may interact with Myo1p, Iqg1p, and Myo2p to coordinate actin ring formation and targeted membrane deposition during cytokinesis. We also identify Mlc2p as the regulatory light chain for Myo1p and demonstrate its role in Myo1p ring disassembly, a function likely conserved among eukaryotes.