Cytoskeletal molecular motors belonging to the kinesin and dynein families transport cargos (for example, messenger RNA, endosomes, virus) on polymerized linear structures called microtubules in the cell. These 'nanomachines' use energy obtained from ATP hydrolysis to generate force, and move in a step-like manner on microtubules. Dynein has a complex and fundamentally different structure from other motor families. Thus, understanding dynein's force generation can yield new insight into the architecture and function of nanomachines. Here, we use an optical trap to quantify motion of polystyrene beads driven along microtubules by single cytoplasmic dynein motors. Under no load, dynein moves predominantly with a mixture of 24-nm and 32-nm steps. When moving against load applied by an optical trap, dynein can decrease step size to 8 nm and produce force up to 1.1 pN. This correlation between step size and force production is consistent with a molecular gear mechanism. The ability to take smaller but more powerful strokes under load--that is, to shift gears--depends on the availability of ATP. We propose a model whereby the gear is downshifted through load-induced binding of ATP at secondary sites in the dynein head.

Microtubule-associated proteins (MAPs) use particular microtubule-binding domains that allow them to interact with microtubules in a manner specific to their individual cellular functions. Here, we have identified a highly basic microtubule-binding domain in the p150 subunit of dynactin that is only present in the dynactin members of the CAP-Gly family of proteins. Using single-particle microtubule-binding assays, we found that the basic domain of dynactin moves progressively along microtubules in the absence of molecular motors - a process we term 'skating'. In contrast, the previously described CAP-Gly domain of dynactin remains firmly attached to a single point on microtubules. Further analyses showed that microtubule skating is a form of one-dimensional diffusion along the microtubule. To determine the cellular function of the skating phenomenon, dynein and the dynactin microtubule-binding domains were examined in single-molecule motility assays. We found that the basic domain increased dynein processivity fourfold whereas the CAP-Gly domain inhibited dynein motility. Our data show that the ability of the basic domain of dynactin to skate along microtubules is used by dynein to maintain longer interactions for each encounter with microtubules.

Multiple dyneins working together suppress shortcomings of a single motor and generate robust motion under in vitro conditions. There appears to be no need for additional cofactors (e.g., dynactin) for this improvement. Because cargos are often driven by multiple dyneins in vivo, our results show that changing the number of dynein motors could allow modulation of dynein function from the mediocre single-dynein limit to robust in vivo-like dynein-driven motion.

The intermediate filament (IF) proteins paranemin and synemin are unique members of the IF protein superfamily. Paranemin and synemin were originally identified as IFassociated proteins because they colocalize and copurify with desmin in avian muscle cells.The IFs wrap around the periphery of myofibrils at their Z-lines in adult skeletal muscle cells, connect adjacent myofibrils to one another and to any nearby mitochondria and nuclei, and connect the peripheral layer of myofibrils to the sarcolemma at costameres. We have followed the co localization of paranemin, synemin, and desmin during skeletal muscle myogenesis at different stages of development by immunofluorescence and immunoelectron microscopy. Localization of paranemin precedes that of desmin in cultured avian skeletal muscle presumptive myoblasts. Paranemin, synemin and desmin then colocalize throughout muscle cell development but, in relation to desmin and synemin, paranemin immunofluorescence is increased at the growth tips of elongating myotubes and decreased in areas of the cell where the myofibrils are in a more mature pattern of alignment. The IF proteins colocalized within longitudinal filaments dispersed around/between myofibrils, but there was no IF colocalization with the myofibrillar Z-line protein a-actinin until after myofibril alignment had occurred. At the ultrastructural level, IFs were observed near the ends of early nascent myofibrils and at cytoskeletal filament junctions. A direct interaction of paranemin with both desmin and synemin was demonstrated by in vitro binding assays. Furthermore, expression of GFP-tagged paranemin rod domain in cultured myotubes resulted in colocalization with, and eventual aggregation of, the desmin-containing IFs, but did not noticeably affect myofibril assembly and alignment. Rather than playing a direct role in myofibrillogenesis, it is likely that IFs play a role at the next level of cytoskeletal organization by preserving the overall shape and integrity of developing muscle cells as they grow into elongated myotubes. myofibrils, especially within growth tips of elongating myotubes, between adjacent myofibrils, and between the peripheral layer of myofibrils and the sarcolemma.