Microtubules or their subunits, tubulin dimers, interact with multiple components that contribute to intracellular metabolic pathways. Microtubules are required for insulin-dependent transport of glucose transporters (GLUT4) to the plasma membrane, they bind most glycolytic enzymes and are required for translation of the mRNA encoding hypoxia inducible factor -1α. Tubulin dimers bind the voltage dependent anion channel of the mitochondrial outer membrane; this channel functions in metabolite transport in and out of mitochondria. We hypothesize that tubulin partitioning between dimer and polymer pools regulates multiple steps in metabolism, where metabolic output is greatest when both tubulin dimers and microtubule polymers are present and reduced by drug treatments that disrupt this normal balance. Experimental evidence from these drug-induced changes in tubulin dimer/polymer partitioning supports our model for several metabolic steps. Signal transduction pathways that stabilize or destabilize microtubules can shift the normal ratio between unpolymerized and polymerized tubulin dimers, and one downstream consequence of this shift in tubulin partitioning could be a change in metabolic output. © 2012 Wiley Periodicals, Inc.

Characterization of the intestinal epithelium of the Snell's waltzer (sv/sv) mouse revealed that myosin VI (Myo6) is required for proper brush border (BB) ultrastructure, composition and membrane traffic. The defects observed were distinct from that observed in the myosin Ia KO, even though Myo6 is lost from the BB in this KO. Myo6 is expressed throughout the length of the small and large intestine; it is localized to the subapical inter-microvillar (MV) domain and basolateral membrane. Defects in the BB include apparent lifting of the plasma membrane off of the actin cytoskeleton in the inter-MV region, fusion of MV, and disorganized morphology of the terminal web. The molecular composition of the sv /sv BB is altered. This includes increased expression of myosin Va, myosin Ie and the MV actin binding proteins espin and phosphorylated-ezrin; myosin Id is reduced. Changes in endocytic components include reduced clathrin and adaptinβ, and increased disabled-2. Endocytic uptake of lumenal lactoferrin is inhibited in adult, but not neonatal intestinal epithelial cells. There is increased BB membrane-associated expression of both the Na⁺/H⁺ exchanger, NHE3 and the Na⁺/phosphate transporter, NaPi2b. These results suggest that Myo6 is involved in the regulated trafficking of NHE3 and NaPi2b between the BB membrane and endosome. © 2012 Wiley Periodicals, Inc.

Axonal growth cones turn away from repulsive guidance cues. This may start with reduced protrusive motility in the region the growth cone leading margin that is closer to the source of repulsive cue. Using explants of E7 chick temporal retina, we examine the effects of two repulsive guidance cues, ephrin-A2 and slit3, on retinal ganglion cell growth cone protrusive activity, total F-actin, free F-actin barbed ends, and the activities (phosphorylation states) of actin regulatory proteins, ADF/cofilin and ERM proteins. Ephrin-A2 rapidly stops protrusive activity simultaneously with reducing F-actin, free barbed ends and the activities of ADF/cofilin and ERM proteins. Slit3 also stops protrusion and reduces the activities of ADF/cofilin and ERM proteins. We interpret these results as indicating that repulsive guidance cues inhibit actin polymerization and actin-membrane linkage to stop protrusive activity. Retrograde F-actin flow withdraws actin to the C-domain, where F-actin bundles interact with myosin II to generate contractile forces that can collapse and retract the growth cone. Our results suggest that common mechanisms are used by repulsive guidance cue to disable growth cone motility and remodel growing axon terminals. © 2012 Wiley Periodicals, Inc.

Primary cilia are organelles that extend from the cell surface. More than 600 proteins have been identified in cilia, but ciliary targeting mechanisms are poorly understood. Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease with 11 responsible genes (NPHP1-11) thus far being identified. The mouse Nphp3 gene product is localized in the cilia and contains coiled-coil (CC) domains and tetratricopeptide repeats, but the ciliary targeting sequences are unknown. In the present study, we generated a series of GFP-tagged deletion constructs of Nphp3 and tried to find the ciliary targeting sequences of Nphp3. We found that the N-terminal 201 amino acid fragment (Nphp3 (1–201)), which contains two CC domains, is necessary and sufficient for cilia localization. Further analysis revealed that an N-terminal glycine (G2), which is a conserved myristoylation site among vertebrates, is also essential for trafficking of Nphp3 to the ciliary shaft. Interestingly, the N-terminal fragments, Nphp3 (8–201), Nphp3 (52–201) and Nphp3 (96–201), that contain the CC domains, targeted the basal body, but could not enter into the ciliary shaft. Our results showed the importance of myristoylation in ciliary trafficking, and suggest that Nphp3 trafficking to the ciliary shaft occurs in a two-step process. © 2012 Wiley Periodicals, Inc.

Very little is known about the function of the F-actin cytoskeleton in the regeneration and pathology of peripheral nerve fibers. The actin cytoskeleton has been associated with maintenance of tissue structure, transmission of traction and contraction forces, and an involvement in cell motility. Therefore, the state of the actin cytoskeleton strongly influences the mechanical properties of cells and intracellular transport therein. In this work, we analyze the distribution of F-actin at Schmidt-Lanterman Incisures (SLI) and nodes of Ranvier (NR) domains in normal, regenerating and pathologic Trembler J (TrJ/+) sciatic nerve fibers, of rats and mice. F-actin was quantified and it was found increased in TrJ/+, both in SLI and NR. However, SLI and NR of regenerating rat sciatic nerve did not show significant differences in F-actin, as compared with normal nerves. Cytochalasin-D and Latrunculin-A were used to disrupt the F-actin network in normal and regenerating rat sciatic nerve fibers. Both drugs disrupt F-actin, but in different ways. Cytochalasin-D did not disrupt Schwann cell F-actin at the NR. Latrunculin-A did not disrupt F-actin at the boundary region between SC and axon at the NR domain. We think that the rearrangement of F-actin in neurological disorders, as presented here, is an important feature of TrJ/+ pathology as a CMT model. © 2012 Wiley Periodicals, Inc.

Dendritic spines are the sites of most excitatory synapses in the central nervous system (CNS). Recent studies have shown that spines function independently of each other, and they are currently the smallest known processing units in the brain. Spines exist in an array of morphologies, and spine structure helps dictate synaptic function. Dendritic spines are rich in actin, and actin rearrangements are critical regulators of spine morphology and density. In this review we discuss the importance of actin in regulating dendritic spine morphogenesis, and discuss the upstream signal transduction pathways that either foster or inhibit actin polymerization. The understanding of actin regulatory pathways is best conceptualized as a hierarchical network in which molecules function in discrete levels defined by their molecular distance to actin. To this end, we focus on several classes of molecules, including guanine nucleotide exchange factors (GEFs), small GTPases, small GTPase effectors, and actin binding proteins. We discuss how individual proteins in these molecular classes impact spine morphogenesis, and reveal the biochemical interactions in these networks that are responsible for shaping actin polymerization. Finally, we discuss the importance of these actin regulatory pathways in neuropsychiatric disorders. © 2012 Wiley-Liss, Inc.

Many motile processes are regulated such that movement occurs only upon activation of a signaling cascade. Sperm from a variety of species are initially quiescent and must be activated prior to beating. The signaling events leading to the activation and regulation of sperm motility are not well characterized. Mature seminal vesicle sperm from the water strider Aquarius remigis are immotile in vitro, but vigorous motility is activated by trypsin. Trypsin-activated motility was blocked by pretreatment of the sperm with BAPTA-AM to chelate intracellular Ca²⁺ and was partially rescued by subsequent addition of A23187 and Ca²⁺. Thapsigargin stimulated motility in the absence of trypsin, suggesting that intracellular Ca²⁺ stores are available. In addition, motility could be fully activated by the phosphatase inhibitor calyculin A, suggesting that the immotile state is maintained by an endogenous phosphatase and that kinase activity is required for motility. The MEK1/2 inhibitor U0126 significantly reduced trypsin activated motility, and MPM-2, an antibody which recognizes proline-directed phosphorylation by kinases such as MAPK, recognized components of the water strider sperm flagellum. Antibodies specific for the mouse protease activated receptor PAR2 recognized an antigen on the sperm flagellum. These results suggest that trypsin stimulates a Ca²⁺ and MAPK mediated signaling pathway and potentially implicate a PAR2-like protein in regulating motility. © 2012 Wiley Periodicals, Inc.

Short microtubules move within the axon in both directions. In the past, it had been assumed that all of the short moving microtubules are oriented with their plus-ends distal to the cell body, regardless of their direction of movement. The anterogradely moving microtubules were posited to play critical roles in the establishment, expansion, and maintenance of the axonal microtubule array. There was no known function for the retrogradely moving microtubules. In considering the mechanism of their transport, we had assumed that all of the short microtubules have a plus-end-distal polarity orientation, as is characteristic of the long microtubules that dominate the axon. Here we discuss an alternative hypothesis, namely that the short microtubules moving retrogradely have the opposite polarity orientation of those moving anterogradely. Those that move anterogradely have their plus-ends distal to the cell body while those that move retrogradely have their minus ends distal to the cell body. In this view, retrograde transport is a means for clearing the axon of incorrectly oriented microtubules. This new model, if correct, has profound implications for the manner by which healthy axons preserve their characteristic pattern of microtubule polarity orientation. We speculate that pathological flaws in this mechanism may be a critical factor in the degeneration of axons during disease and injury, as well as in neuropathy caused by microtubule-active drugs. © 2012 Wiley Periodicals, Inc.

There is rising interest in modeling the noncentrosomal cortical microtubule cytoskeleton of plant cells, particularly its organization into ordered arrays and the mechanisms that facilitate this organization. In this review, we discuss quantitative models of this highly complex and dynamic structure both at a cellular and molecular level. We report differences in methodologies and assumptions of different models as well as their controversial results. Our review provides insights for future studies to resolve these controversies, in addition to underlining the common results between various models. We also highlight the need to compare the results from simulation and mathematical models with quantitative data from biological experiments in order to test the validity of the models and to further improve them. It is our hope that this review will serve to provide guidelines for how to combine quantitative and experimental techniques to develop higher-level models of the plant cytoskeleton in the future. © 2012 Wiley Periodicals, Inc

Numerous studies have indicated that each of the seven projections associated with the central pair of microtubules plays a distinct role in regulating eukaryotic ciliary/flagellar motility. Mutants which lack specific projections have distinct motility phenotypes. For example, Chlamydomonas pf6 mutants lack the C1a projection and have twitchy, non-beating flagella. The C1a projection is a complex of proteins including PF6, C1a-86, C1a-34, C1a-32, C1a-18, and calmodulin. To define functional domains within PF6 and to potentially assign functions to specific C1a components, we generated deletion constructs of the PF6 gene and tested for their ability to assemble and rescue motility upon transformation of mutant pf6 cells. Our results demonstrate that domains near the carboxyl-terminus of PF6 are essential for motility and/or assembly of the projection. The amino terminal half of PF6 is not required for C1a assembly; however, this region is important for stability of the C1a-34, C1a-32, and C1a-18 sub-complex and wild-type beat frequency. Analysis of double mutants lacking the amino terminus of PF6 and outer dynein arms reveal that C1a may play a role in modulating both inner and outer dynein arm activity. © 2012 Wiley Periodicals, Inc

Cytoplasmic linker associated proteins (CLASPs) comprise a class of microtubule (MT) plus end-binding proteins (+TIPs) that contribute to the dynamics and organization of MTs during many cellular processes, among them mitosis. Human CLASP proteins contain multiple MT-binding domains, including tumor over-expressed gene (TOG) domains, and a Ser-x-Ile-Pro (SxIP) motif known to target some +TIPs though interaction with end-binding protein 1 (EB1). However, how individual domains contribute to CLASP function is poorly understood. We generated full-length recombinant human CLASP1 and a series of truncation mutants and found that two N-terminal TOG domains make the strongest contribution to MT polymerization and bundling, but also identified a third TOG domain that further contributes to CLASP activity. Plus end tracking by CLASP requires the SxIP motif and interaction with EB1. The C-terminal coiled-coil domain mediates dimerization and association with many other factors, including the kinetochore motor centromere protein E (CENP-E), and the chromokinesin Xkid. Only the full-length protein was able to rescue spindle assembly in Xenopus egg extracts depleted of endogenous CLASP. Deletion of the C-terminal domain caused aberrant MT polymerization and dramatic spindle phenotypes, even with small amounts of added protein, indicating that proper localization of CLASP activity is essential to control MT polymerization during mitosis. © 2012 Wiley Periodicals, Inc.

The most common neurodegenerative disorder afflicting the aging human population is Alzheimer's disease (AD). A major hallmark of AD is dementia from a loss of neuronal function, attributed to the presence and accumulation of β-amyloid (Aβ) peptide into senile plaques. Preceding senile plaque formation, abnormalities in axons can be observed as changes in morphologies and intracellular trafficking. Recently, it has been recognized that Aβ also accumulates within neurons and this intraneuronal Aβ accumulation has been reported to be critical in the disruption of synapses and cognitive function. Here, we report on the internalization of a fluorescently labeled Aβ peptide into cultured chick retinal neurons. The pattern of Aβ distribution during the time course of incubation is reminiscent of the endocytic pathway. Furthermore, the distribution of the internalized Aβ peptide converges with that of myosin Vb and both relocalize from the axon to cell body. These observations are consistent with the hypothesis that AD proceeds as a result of an imbalance between Aβ production and Aβ clearance, suggesting a role for myosin Vb in this process. © 2012 Wiley Periodicals, Inc

All cells, from simple bacteria to complex human tissues, rely on extensive networks of protein fibers to help maintain their proper form and function. These filament systems usually do not operate as single filaments, but form complex suprastructures, which are essential for specific cellular functions. Here, we describe the progress in determining the architectures of molecular filamentous suprastructures, the principles leading to their formation, and the mechanisms by which they may facilitate function. The complex eukaryotic cytoskeleton is tightly regulated by a large number of actin- or microtubule-associated proteins. In contrast, recently discovered bacterial actins and tubulins have few associated regulatory proteins. Hence, the quest to find basic principles that govern the formation of filamentous suprastructures is simplified in bacteria. Three common principles, which have been probed extensively during evolution, can be identified that lead to suprastructures formation: cationic counterion fluctuations; self-association into liquid crystals; and molecular crowding. The underlying physics of these processes will be discussed with respect to physiological circumstance. © 2012 Wiley Periodicals, Inc.

In starfish oocytes, microtubules (MTs) form a spindle, which plays an important role in contributing to the selective loss of chromosomes and centrosomes to the polar bodies (PBs) during meiosis. When Taxol was locally injected near the germinal vesicle (GV) or the mitotic apparatus during meiosis I, PB formation was inhibited as mentioned below. In the oocytes, which were injected with Taxol after spindle formation, the spindle became large, and then the volume of the first PB also increased more than that of the control. In contrast, in the oocytes injected with Taxol before the spindle formation, chromosome capture and alignment were inhibited. These oocytes did not form PB, but only a bulge at the cell cortex was occasionally observed. Moreover, in the oocytes injected with Taxol before GV breakdown, the chromosomes did not gather in one place, and then two asters were observed at distant positions from the cell cortex. These results suggested that MTs lost not only the ability to obtain the bipolar attachment of chromosomes by Taxol injection but also the aster closer to the cell cortex lost its interaction with the cell cortex of the animal pole. © 2012 Wiley Periodicals, Inc.

Radial spokes (RSs) are ubiquitous components of motile cilia and flagella and play an essential role in transmitting signals that regulate the activity of the dynein motors, and thus ciliary and flagellar motility. In some organisms, the 96 nm axonemal repeat unit contains only a pair of spokes, RS1 and RS2, while most organisms have spoke triplets with an additional spoke RS3. The spoke pairs in Chlamydomonas flagella have been well characterized, while spoke triplets have received less attention. Here, we used cryoelectron tomography and subtomogram averaging to visualize the three-dimensional structure of spoke triplets in Strongylocentrotus purpuratus (sea urchin) sperm flagella in unprecedented detail. Only small differences were observed between RS1 and RS2, but the structure of RS3 was surprisingly unique and structurally different from the other two spokes. We observed novel doublet specific features that connect RS2, RS3, and the nexin-dynein regulatory complex, three key ciliary and flagellar structures. The distribution of these doublet specific structures suggests that they could be important for establishing the asymmetry of dynein activity required for the oscillatory movement of cilia and flagella. Surprisingly, a comparison with other organisms demonstrated both that this considerable RS heterogeneity is conserved and that organisms with RS pairs contain the basal part of RS3. This conserved RS heterogeneity may also reflect functional differences between the spokes and their involvement in regulating ciliary and flagellar motility. © 2011 Wiley Periodicals Inc.

The adhesive disc is a highly complex apparatus that allows mobilid ciliates to attach to the tissues of a variety of aquatic invertebrates and vertebrates. The disc comprises concentric rings of rigid skeletal pieces interconnected by filamentous material. This study explored the biochemical properties of the filamentous disc material in the trichodinid Trichodina pediculus. Calcium sensitivity of this material was suggested in vitro by the appearance of transverse cross-striation along bundles of filaments following calcium shock, and complete solubilization of the filamentous material in the presence of EGTA. A 23-kDa immunoanalog of centrins was immunoprecipitated from the EGTA extract. The protein binds calcium as indicated by ⁴⁵Ca²⁺ blot overlay and Ca²⁺-induced shifts in electrophoretic mobility. Using Ca²⁺/EGTA buffers, we demonstrated a direct relationship between extraction of the filaments and solubilization of the protein. Immunofluorescence and immunoelectron microscopy confirmed that the protein localized to the filamentous disc material and revealed cross-reactivity with the spasmoneme, which is the prototype of ion-sensitive, centrin-like contractile systems in ciliates. The possibility that the filamentous disc material may be a novel example of Ca²⁺-sensitive, centrin-based systems found in ciliates is discussed. © 2011 Wiley Periodicals Inc.

Force-extension curves obtained on intact human red blood cells (RBC) were compared with those of delipidated RBCs to assess the contribution of cytoskeletal flexibility to the extensibility of the intact membrane skeleton. The RBCs were first delipidated by treatment with phospholipase A₂; tensile properties of the exposed cytoskeletal structures were measured using an atomic force microscope (AFM). The AFM probes were modified either with the Band 3 specific lectin, concanavalin A, (Con A) or anti-F-actin antibody, to localize the point of interaction between the probe and the cytoskeleton. Extension of the spectrin-based cytoskeleton reached up to 2–3 μm with a force less than 70 pN without showing any force peaks before the final rupture of the adhesive bonds. Our interpretation of the result is that the spectrin-based network was slack enough to allow the observed degree of extension without unfolding the tetrameric spectrin molecules. The force-extension curves obtained either on Band 3-ankyrin loci or on junction nodes of the cytoskeleton were not significantly different. Experimental results were verified by computer simulation of pulling mechanics of a network model of the RBC cytoskeleton. Our experimental results are also in agreement with the theoretical prediction of Mirijanian and Voth [2008; Proc Natl Acad Sci USA 105:1204–1208]. © 2011 Wiley Periodicals Inc.

In this study, the effects of disturbance of the reactive oxygen species (ROS) homeostasis on the organization of tubulin cytoskeleton in interphase and mitotic root-tip cells of Triticum turgidum and Arabidopsis thaliana were investigated. Reduced ROS levels were obtained by treatment with diphenylene iodonium (DPI) and N-acetyl-cysteine, whereas menadione was applied to achieve ROS overproduction. Both increased and low ROS levels induced: (a) Macrotubule formation in cells with low ROS levels and tubulin paracrystals under oxidative stress. The protein MAP65-1 was detected in treated cells, exhibiting a conformation comparable to that of the atypical tubulin polymers. (b) Disappearance of microtubules (MTs). (c) Inhibition of preprophase band formation. (d) Delay of the nuclear envelope breakdown at prometaphase. (e) Prevention of perinuclear tubulin polymer assembly in prophase cells. (f) Loss of bipolarity of prophase, metaphase and anaphase spindles. Interestingly, examination of the A. thaliana rhd2/At respiratory burst oxidase homolog C (rbohc) NADPH oxidase mutant, lacking RHD2/AtRBOHC, gave comparable results. Similarly to DPI, the decreased ROS levels in rhd2 root-tip cells, interfered with MT organization and induced macrotubule assembly. These data indicate, for first time in plants, that ROS are definitely implicated in: (a) mechanisms controlling the assembly/disassembly of interphase, preprophase and mitotic MT systems and (b) mitotic spindle function. The probable mechanisms, by which ROS affect these processes, are discussed. © 2011 Wiley Periodicals, Inc.

Protein kinase A (PKA) signaling is targeted by interactions with A-kinase anchoring proteins (AKAPs) via a dimerization/docking domain on the regulatory (R) subunit of PKA. Four other mammalian proteins [AKAP-associated sperm protein (ASP), ropporin (ROPN1), sperm protein 17 (SP17) and calcium binding tyrosine-(Y)-phosphorylation regulated protein (CABYR)] share this highly conserved RII dimerization/docking (R2D2) domain. ASP and ROPN1 are 41% identical in sequence, interact with a variety of AKAPs in a manner similar to PKA, and are expressed in ciliated and flagellated human cells. To test the hypothesis that these proteins regulate motility, we developed mutant mouse lines lacking ASP or ROPN1. Both mutant lines produced normal numbers of cilia with intact ciliary ultrastructure. Lack of ROPN1 had no effect on ciliary motility. However, the beat frequency of cilia from mice lacking ASP is significantly slower than wild type, indicating that ASP signaling may regulate ciliary motility. This is the first demonstration of in vivo function for ASP. Similar localization of ASP in mice and humans indicates that these findings may translate to human physiology, and that these mice will be an excellent model for future studies related to the pathogenesis of human disease. © 2011 Wiley Periodicals, Inc.

Cilia and flagella, sensory and motile structures protruding from the cell body, rely on the continuous bidirectional traffic of intraflagellar transport (IFT) particles to ferry flagellar precursors into flagella for assembly. Cells synthesize a large pool of IFT particle proteins in the cell body, but only a small portion engages in active transport within the flagella at any given time. The atypical small G protein Rab-like 5 (RABL5) has been shown to move in an IFT-like manner in the flagella, but its function in ciliogenesis is controversial. In this report, we demonstrate that IFT22, the Chlamydomonas reinhardtii homolog of RABL5, is a bona fide IFT particle complex B subunit. Although the amount of IFT22 remains unaffected by depletion of either complex A or B, depletion of IFT22 leads to a smaller pool of both complex A and B. Strikingly, the smaller cellular pool of IFT particles does not lead to a reduced distribution of IFT particles to flagella. Instead, the amount of IFT particle proteins, including IFT22 itself, increase in the flagella. Moreover, cells over-expressing IFT22 also accumulate IFT particles in their flagella. Taken together, these data indicate that, in C. reinhardtii, IFT22 controls the cellular levels of both complex A and B, thus plays a critical role in determining the cellular availability of IFT particles. In addition, although IFT22 may not directly carry any precursors for flagellar assembly, it controls how many IFT particles participate in ferrying precursors into flagella. © 2011 Wiley Periodicals, Inc.

The cellular actin cytoskeleton plays a central role in the ability of cells to properly sense, propagate, and respond to external stresses and other mechanical stimuli. Calponin, an actin-binding protein found both in muscle and non-muscle cells, has been implicated in actin cytoskeletal organization and regulation. In this work, we studied the mechanical and structural interaction of actin with basic calponin, a differentiation marker in smooth muscle cells, on a single filament level. We imaged fluorescently labeled thermally fluctuating actin filaments and found that at moderate calponin binding densities, actin filaments were more flexible, evident as a reduction in persistence length from 8.0 to 5.8 μm. When calponin-decorated actin filaments were subjected to shear, we observed a marked reduction of filament lengths after decoration with calponin, which we argue was due to shear-induced filament rupture rather than depolymerization. This increased shear susceptibility was exacerbated with calponin concentration. Cryo-electron microscopy results confirmed previously published negative stain electron microscopy results and suggested alterations in actin involving actin subdomain 2. A weakening of F-actin intermolecular association is discussed as the underlying cause of the observed mechanical perturbations. © 2011 Wiley Periodicals, Inc

Myosin VI (Myo6) is unique among myosins in that it moves toward the minus (pointed) end of the actin filament. Thus to exert tension on, or move cargo along an actin filament, Myo6 is working against potentially multiple plus (barbed)-end myosins. To test the effect of plus-end motors on Myo6, the gliding actin filament assay was used to assess the motility of single-headed Myo6 in the absence and presence of cardiac myosin II (Myo2) and myosin Va (Myo5a). Myo6 alone exhibited a filament gliding velocities of 60.34 ± 13.68 nm/s. Addition of either Myo2 or Myo5a, at densities below that required to promote plus-end movement resulted in an increase in Myo6 velocity (∼100–150% increase). Movement in the presence of these plus-end myosins was minus-end directed as determined using polarity tagged filaments. High densities of Myo2 or Myo5a were required to convert to plus-end directed motility indicating that Myo6 is a potent inhibitor of Myo2 and Myo5a. Previous studies have shown that two-headed Myo6 slows and then stalls in an anchored state under load. Consistent with these studies, velocity of a two headed heavy mero myosin form of Myo6 was unaffected by Myo5a at low densities, and was inhibited at high Myo5a densities. © 2011 Wiley Periodicals Inc.