Myosin VI powers self-organization of branched contractile actin network.

The actomyosin cytoskeletal network is responsible for a variety of fundamental cellular processes. Assembly and maintenance of actin networks involve an array of associated regulatory proteins for polymerization, branching, crosslinking and contractility-driven self-organization. In this study, we make the unexpected discovery in vitro that myosin VI and myosin X, motor proteins specialized in vesicle transport and filopodia formation, are capable of crosslinking and self-organizing actin into higher-order contractile structures in the absence of other actin-associated proteins. Moreover, myosin VI alone can initiate actin elongation and branching, and assemble branched force-generating networks from crosslinked actin polymers. Additional architectural control is provided by the actin crosslinking proteins α-actinin and fascin. Our data identify critical stages of tension-mediated connectivity in network development and provide a model system for further exploration of the nonequilibrium mechanics of actomyosin self-organization.

How myosin VI traps its off-state, is activated and dimerizes.

Myosin VI (Myo6) is the only minus-end directed nanomotor on actin, allowing it to uniquely contribute to numerous cellular functions. As for other nanomotors, the proper functioning of Myo6 relies on precise spatiotemporal control of motor activity via a poorly defined off-state and interactions with partners. Our structural, functional, and cellular studies reveal key features of myosin regulation and indicate that not all partners can activate Myo6. TOM1 and Dab2 cannot bind the off-state, while GIPC1 binds Myo6, releases its auto-inhibition and triggers proximal dimerization. Myo6 partners thus differentially recruit Myo6. We solved a crystal structure of the proximal dimerization domain, and show that its disruption compromises endocytosis in HeLa cells, emphasizing the importance of Myo6 dimerization. Finally, we show that the L926Q deafness mutation disrupts Myo6 auto-inhibition and indirectly impairs proximal dimerization. Our study thus demonstrates the importance of partners in the control of Myo6 auto-inhibition, localization, and activation.

The mechanical components of the dynein motor.

Unlike our understanding of the other two classes of cytoskeletal motor proteins, the myosins and kinesins, we have only recently begun to comprehend the molecular mechanism for how dynein produces force and movement. The slow progress has been attributed, in part, to the enormous size of the dynein force-producing head, but also to the complex interplay between its structural components, each of which has a unique role in regulating dynein motor activity. The integrated and highly coordinated mechanism by which these structures work together in powering the dynein machinery is discussed in this review.

The dynein stalk contains an antiparallel coiled coil with region-specific stability.

The dynein motor proteins interact with microtubules at the distal end of an unusual 12-15 nm stalk, which communicates with the sites for nucleotide hydrolysis and microtubule binding in a cyclical, bidirectional manner. Here, we report that the stalk shaft of rat cytoplasmic dynein is an antiparallel alpha-helical coiled coil, the stability of which is markedly altered by changes at its proximal and distal ends, consistent with a structure capable of rapid, cyclical rearrangement during the dynein cross-bridge cycle.

Whole blood clot optical clearing for nondestructive 3D imaging and quantitative analysis.

A technological revolution in both light and electron microscopy imaging now allows unprecedented views of clotting, especially in animal models of hemostasis and thrombosis. However, our understanding of three-dimensional high-resolution clot structure remains incomplete since most of our recent knowledge has come from studies of relatively small clots or thrombi, due to the optical impenetrability of clots beyond a few cell layers in depth. Here, we developed an optimized optical clearing method termed cCLOT that renders large whole blood clots transparent and allows confocal imaging as deep as one millimeter inside the clot. We have tested this method by investigating the 3D structure of clots made from reconstituted pre-labeled blood components yielding new information about the effects of clot contraction on erythrocytes. Although it has been shown recently that erythrocytes are compressed to form polyhedrocytes during clot contraction, observations of this phenomenon have been impeded by the inability to easily image inside clots. As an efficient and non-destructive method, cCLOT represents a powerful research tool in studying blood clot structure and mechanisms controlling clot morphology. Additionally, cCLOT optical clearing has the potential to facilitate imaging of ex vivo clots and thrombi derived from healthy or pathological conditions.

Strong Binding of Platelet Integrin αIIbß3 to Fibrin Clots: Potential Target to Destabilize Thrombi.

The formation of platelet thrombi is determined by the integrin αIIbß3-mediated interactions of platelets with fibrinogen and fibrin. Blood clotting in vivo is catalyzed by thrombin, which simultaneously induces fibrinogen binding to αIIbß3 and converts fibrinogen to fibrin. Thus, after a short time, thrombus formation is governed by αIIbß3 binding to fibrin fibers. Surprisingly, there is little understanding of αIIbß3 interaction with fibrin polymers. Here we used an optical trap-based system to measure the binding of single αIIbß3 molecules to polymeric fibrin and compare it to αIIbß3 binding to monomeric fibrin and fibrinogen. Like αIIbß3 binding to fibrinogen and monomeric fibrin, we found that αIIbß3 binding to polymeric fibrin can be segregated into two binding regimes, one with weaker rupture forces of 30-60 pN and a second with stronger rupture forces >60 pN that peaked at 70-80 pN. However, we found that the mechanical stability of the bimolecular αIIbß3-ligand complexes had the following order: fibrin polymer > fibrin monomer > fibrinogen. These quantitative differences reflect the distinct specificity and underlying molecular mechanisms of αIIbß3-mediated reactions, implying that targeting platelet interactions with fibrin could increase the therapeutic indices of antithrombotic agents by focusing on the destabilization of thrombi rather than the prevention of platelet aggregation.

Synthesis and biological evaluation of purealin and analogues as cytoplasmic dynein heavy chain inhibitors.

Cytoplasmic dynein plays important roles in membrane transport, mitosis, and other cellular processes. A few small-molecule inhibitors of cytoplasmic dynein have been identified. We report here the first synthesis of purealin, a natural product isolated from the sea sponge Psammaplysilla purea, which is known to inhibit axonemal dynein. Also described are the first syntheses, by modular amide coupling reactions, of the natural product purealidin A (a component of purealin) and a small library of analogues. The library was examined for inhibition of cytoplasmic dynein heavy chain and cell growth. The compounds showed effective antiproliferative activity against a mouse leukemia cell line but selective activities against human carcinoma cell lines. Purealin and some of the analogues inhibited the microtubule-stimulated ATPase activity of recombinant cytoplasmic dynein heavy chain motor domain. The inhibitory effect of purealin was concentration dependent and uncompetitive, supporting the hypothesis that it does not compete with the binding of ATP.

Control of cytoplasmic dynein force production and processivity by its C-terminal domain.

Cytoplasmic dynein is a microtubule motor involved in cargo transport, nuclear migration and cell division. Despite structural conservation of the dynein motor domain from yeast to higher eukaryotes, the extensively studied S. cerevisiae dynein behaves distinctly from mammalian dyneins, which produce far less force and travel over shorter distances. However, isolated reports of yeast-like force production by mammalian dynein have called interspecies differences into question. We report that functional differences between yeast and mammalian dynein are real and attributable to a C-terminal motor element absent in yeast, which resembles a 'cap' over the central pore of the mammalian dynein motor domain. Removal of this cap increases the force generation of rat dynein from 1 pN to a yeast-like 6 pN and greatly increases its travel distance. Our findings identify the CT-cap as a novel regulator of dynein function.

Dynein dynamics.

Dyneins are the largest of the cytoskeletal motor proteins, and their mechanochemical behavior is complex. Recent high-resolution crystallographic structures have revealed new surprises regarding motor domain organization and new insights into how force and movement are achieved.

Adaptation by alternative RNA splicing of slow troponin T isoforms in type 1 but not type 2 Charcot-Marie-Tooth disease.

Slow troponin T (TnT) plays an indispensable role in skeletal muscle function. Alternative RNA splicing in the NH(2)-terminal region produces high-molecular-weight (HMW) and low-molecular-weight (LMW) isoforms of slow TnT. Normal adult slow muscle fibers express mainly HMW slow TnT. Charcot-Marie-Tooth disease (CMT) is a group of inherited peripheral polyneuropathies caused by various neuronal defects. We found in the present study that LMW slow TnT was significantly upregulated in demyelination form type 1 CMT (CMT1) but not axonal form type 2 CMT (CMT2) muscles. Contractility analysis showed an increased specific force in single fibers isolated from CMT1 but not CMT2 muscles compared with control muscles. However, an in vitro motility assay showed normal velocity of the myosin motor isolated from CMT1 and CMT2 muscle biopsies, consistent with their unchanged myosin isoform contents. Supporting a role of slow TnT isoform regulation in contractility change, LMW and HMW slow TnT isoforms showed differences in the molecular conformation in conserved central and COOH-terminal regions with changed binding affinity for troponin I and tropomyosin. In addition to providing a biochemical marker for the differential diagnosis of CMT, the upregulation of LMW slow TnT isoforms under the distinct pathophysiology of CMT1 demonstrates an adaptation of muscle function to neurological disorders by alternative splicing modification of myofilament proteins.

Autoinhibitory and other autoregulatory elements within the dynein motor domain.

The dyneins are a family of microtubule motor proteins. The motor domain, which represents the C-terminal 2/3 of the dynein heavy chain, exhibits homology to the AAA family of ATPases. It consists of a ring of six related but divergent AAA+ units, with two substantial sized protruding projections, the stem, or tail, which anchors the protein to diverse subcellular sites, and the stalk, which binds microtubules. This article reviews recent efforts to probe the mechanism by which the dyneins produce force, and work from the authors' lab regarding long-range conformational regulation of dynein enzymatic activity.

Long range allosteric control of cytoplasmic dynein ATPase activity by the stalk and C-terminal domains.

The dynein motor domain consists of a ring of six AAA domains with a protruding microtubule-binding stalk and a C-terminal domain of unknown function. To understand how conformational information is communicated within this complex structure, we produced a series of recombinant and proteolytic rat motor domain fragments, which we analyzed enzymatically. A recombinant 210-kDa half-motor domain fragment surprisingly exhibited a 6-fold higher steady state ATPase activity than a 380-kDa complete motor domain fragment. The increased ATPase activity was associated with a complete loss of sensitivity to inhibition by vanadate and an approximately 100-fold increase in the rate of ADP release. The time course of product release was discovered to be biphasic, and each phase was stimulated approximately 1000-fold by microtubule binding to the 380-kDa motor domain. Both the half-motor and full motor domain fragments were remarkably resistant to tryptic proteolysis, exhibiting either two or three major cleavage sites. Cleavage near the C terminus of the 380-kDa motor domain released a 32-kDa fragment and abolished sensitivity to vanadate. Cleavage at this site was insensitive to ATP or 5'-adenylyl-beta,gamma-imidodiphosphate but was blocked by ADP-AlF3 or ADP-vanadate. Based on these data, we proposed a model for long range allosteric control of product release at AAA1 and AAA3 through the microtubule-binding stalk and the C-terminal domain, the latter of which may interact with AAA1 to close the motor domain ring in a cross-bridge cycle-dependent manner.