No abstract

Photoreceptor outer segment (POS) phagosomes associate with the kinesin light chain 1 (KLC1) and move bidirectionally along microtubules in retinal pigment epithelium cells; lack of KLC1 results in impaired POS phagosome motility and degradation and, in aged mice, pathogenesis resembling age-related macular degeneration.

Dictyostelium myosin-5b is the gene product of myoJ and one of two closely related myosin-5 isoenzymes produced in Dictyostelium discoideum. Here we report a detailed investigation of the kinetic and functional properties of the protein. In standard assay buffer conditions, Dictyostelium myosin-5b displays high actin affinity in the presence of ADP, fast ATP hydrolysis, and a high steady-state ATPase activity in the presence of actin that is rate limited by ADP release. These properties are typical for a processive motor that can move over long distances along actin filaments without dissociating. Our results show that a physiological decrease in the concentration of free Mg 2؉ -ions leads to an increased rate of ADP release and shortening of the fraction of time the motor spends in the strong actin binding states. Consistently, the ability of the motor to efficiently translocate actin filaments at very low surface densities decreases with decreasing concentrations of free Mg 2؉ -ions. In addition, we provide evidence that the observed changes in Dd myosin-5b motor activity are of physiological relevance and propose a mechanism by which this molecular motor can switch between processive and non-processive movement.Class 5 myosins are dimeric actin-based motors that are involved in various forms of intracellular trafficking (1). Dependent on the isoform and cell type, class 5 myosins have been implicated in the movement of membranes and organelles (2-4), the transport of synaptic and secretory vesicles (5, 6), and the active delivery of receptors and mRNA-protein complexes to their place of action (7,8). The unique modular structure of class 5 myosins is essential for these specialized transport functions (9, 10). Each heavy chain of a dimeric myosin-5 molecule consists of a motor domain that binds actin and hydrolyzes ATP (11), followed by a long neck region to which up to six light chains can bind (12, 13). Parts of the adjacent tail region form a coiled-coil and the C terminus consists of a globular domain that mediates the binding to cargo and regulates activity of the motor (14, 15).Despite the high sequence similarity between myosin-5 isoforms, the individual members display differences in their mechanoenzymatic properties, which characterize them either as processive or non-processive motors. Processive myosins, like vertebrate myosin-5a, are capable of taking successive steps along actin as single molecules before detaching (16). The overall movement has been described as a coordinated stepping process of both heads in a hand-over-hand mechanism that is driven by intramolecular strain (17, 18). In contrast, non-processive myosins bind to the actin filament perform just one step and then dissociate rapidly. A notable difference between processive and non-processive myosins is displayed in the duty ratio, i.e. the fraction of the total ATPase cycle time a motor spends in the strong actin binding states. Processive myosins have a high duty ratio (Ͼ0.5), whereas non-processive myosins display a low duty ratio...

SummaryThe mitotic spindle in eukaryotic cells is composed of a bipolar array of microtubules (MTs) and associated proteins that are required during mitosis for the correct partitioning of the two sets of chromosomes to the daughter cells. In addition to the well-established functions of MT-associated proteins (MAPs) and MT-based motors in cell division, there is increasing evidence that the F-actin-based myosin motors are important mediators of F-actin-MT interactions during mitosis. Here, we report the functional characterization of the long-tailed class-1 myosin myosin-1C from Dictyostelium discoideum during mitosis. Our data reveal that myosin-1C binds to MTs and has a role in maintenance of spindle stability for accurate chromosome separation. Both myosin-1C motor function and taildomain-mediated MT-F-actin interactions are required for the cell-cycle-dependent relocalization of the protein from the cell periphery to the spindle. We show that the association of myosin-1C with MTs is mediated through the tail domain. The myosin-1C tail can inhibit kinesin motor activity, increase the stability of MTs, and form crosslinks between MTs and F-actin. These data illustrate that myosin-1C is involved in the regulation of MT function during mitosis in D. discoideum.