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Vimentin intermediate filaments move bi-directionally along microtubules in the cell. Recent work has identified the microtubule motor cytoplasmic dynein as the missing inward-directed motor that drives this movement.
A crowd of proteins seems to have gathered around the plus-ends of microtubules. A rapidly expanding group of proteins known as plus-end tracking proteins (+TIPs) have been identified that seem to be able to 'surf' the dynamic ends of microtubules. Microtubule plus-ends exist in multiple conformational and chemical states. In principle, altering this plus-end microenvironment is an appealing way for regulators such as the +TIPS to control microtubule dynamics; however, specific mechanisms are poorly defined. Here, we focus on new findings addressing the underlying mechanisms of plus-end tracking and the mechanisms by which +TIPS control microtubule dynamics. We review the evidence that plus-end-binding and the control of microtubule dynamics are mechanistically linked. We also consider the possibility that, by studying +TIPs, we might learn more about the dynamic structural changes at the microtubule ends that are at the heart of dynamic instability.
We reconstituted dynein-driven, dynactin-dependent vesicle transport using protein-free liposomes and soluble components from squid axoplasm. Dynein and dynactin, while necessary, are not the only essential cytosolic factors; axonal spectrin is also required. Spectrin is resident on axonal vesicles, and rebinds from cytosol to liposomes or proteolysed vesicles, concomitant with their dynein-dynactin-dependent motility. Binding of purified axonal spectrin to liposomes requires acidic phospholipids, as does motility. Using dominant negative spectrin polypeptides and a drug that releases PH domains from membranes, we show that spectrin is required for linking dynactin, and thereby dynein, to acidic phospholipids in the membrane. We verify this model in the context of liposomes, isolated axonal vesicles, and whole axoplasm. We conclude that spectrin has an essential role in retrograde axonal transport.
Molecular motors are the long-haul carriers of eukaryotic cells, moving cargos bidirectionally along microtubule tracks. In the December 12th issue of Cell, report that HDAC6, a tubulin deacetylase, functions as an adaptor that links cargos of aggregated protein to the minus end-directed motor, cytoplasmic dynein.
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Here we show that p53 protein is physically associated with tubulin in vivo and in vitro, and that it localizes to cellular microtubules. Treatment with vincristine or paclitaxel before DNA-damage or before leptomycin B treatment reduces nuclear accumulation of p53 and expression of mdm2 and p21. Overexpression of dynamitin or microinjection of anti-dynein antibody before DNA damage abrogates nuclear accumulation of p53. Our results indicate that transport of p53 along microtubules is dynein-dependent. The first 25 amino acids of p53 contain the residues that are essential for binding to microtubules. We propose that functional microtubules and the dynein motor protein participate in transport of p53 and facilitate its accumulation in the nucleus after DNA damage.
Whereas total loss of Lis1 is lethal, disruption of one allele of the Lis1 gene results in brain abnormalities, indicating that developing neurons are particularly sensitive to a reduction in Lis1 dosage. Here we show that Lis1 is enriched in neurons relative to levels in other cell types, and that Lis1 interacts with the microtubule motor cytoplasmic dynein. Production of more Lis1 in non-neuronal cells increases retrograde movement of cytoplasmic dynein and leads to peripheral accumulation of microtubules. These changes may reflect neuron-like dynein behaviours induced by abundant Lis1. Lis1 deficiency produces the opposite phenotype. Our results indicate that abundance of Lis1 in neurons may stimulate specific dynein functions that function in neuronal migration and axon growth.
Mutations in the LIS1 gene cause gross histological disorganization of the developing human brain, resulting in a brain surface that is almost smooth. Here we show that LIS1 protein co-immunoprecipitates with cytoplasmic dynein and dynactin, and localizes to the cell cortex and to mitotic kinetochores, which are known sites for binding of cytoplasmic dynein. Overexpression of LIS1 in cultured mammalian cells interferes with mitotic progression and leads to spindle misorientation. Injection of anti-LIS1 antibody interferes with attachment of chromosomes to the metaphase plate, and leads to chromosome loss. We conclude that LIS1 participates in a subset of dynein functions, and may regulate the division of neuronal progenitor cells in the developing brain.
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The movement of chromosomes during mitosis occurs on a bipolar, microtubule-based protein machine, the mitotic spindle. It has long been proposed that poleward chromosome movements that occur during prometaphase and anaphase A are driven by the microtubule motor cytoplasmic dynein, which binds to kinetochores and transports them toward the minus ends of spindle microtubules. Here we evaluate this hypothesis using time-lapse confocal microscopy to visualize, in real time, kinetochore and chromatid movements in living Drosophila embryos in the presence and absence of specific inhibitors of cytoplasmic dynein. Our results show that dynein inhibitors disrupt the alignment of kinetochores on the metaphase spindle equator and also interfere with kinetochore- and chromatid-to-pole movements during anaphase A. Thus, dynein is essential for poleward chromosome motility throughout mitosis in Drosophila embryos.
Cytoplasmic dynein supports long-range intracellular movements of cargo in vivo but does not appear to be a processive motor protein by itself. We show here that the dynein activator, dynactin, binds microtubules and increases the average length of cytoplasmic-dynein-driven movements without affecting the velocity or microtubule-stimulated ATPase kinetics of cytoplasmic dynein. Enhancement of microtubule binding and motility by dynactin are both inhibited by an antibody to dynactin's microtubule-binding domain. These results indicate that dynactin acts as a processivity factor for cytoplasmic-dynein-based motility and provide the first evidence that cytoskeletal motor processivity can be affected by extrinsic factors.
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We describe the dynamics of kinetochore dynein-dynactin in living Drosophila embryos and examine the effect of mutant dynein on the metaphase checkpoint. A functional conjugate of dynamitin with green fluorescent protein accumulates rapidly at prometaphase kinetochores, and subsequently migrates off kinetochores towards the poles during late prometaphase and metaphase. This behaviour is seen for several metaphase checkpoint proteins, including Rough deal (Rod). In neuroblasts, hypomorphic dynein mutants accumulate in metaphase and block the normal redistribution of Rod from kinetochores to microtubules. By transporting checkpoint proteins away from correctly attached kinetochores, dynein might contribute to shutting off the metaphase checkpoint, allowing anaphase to ensue.
A microtubule network on the basal cortex of polarized epithelial cells consists of non-centrosomal microtubules of mixed polarity. Here, we investigate the proteins that are involved in organizing this network, and we show that end-binding protein 1 (EB1), adenomatous polyposis coli protein (APC) and p150Glued - although considered to be microtubule plus-end-binding proteins - are localized along the entire length of microtubules within the network, and at T-junctions between microtubules. The network shows microtubule behaviours that arise from physical interactions between microtubules, including microtubule plus-end stabilization on the sides of other microtubules, and sliding of microtubule ends along other microtubules. APC also localizes to the basal cortex. Microtubules grew over and paused at APC puncta; an in vitro reconstituted microtubule network overlaid APC puncta; and microtubule network reconstitution was inhibited by function-blocking APC antibodies. Thus, APC is a component of a cortical template that guides microtubule network formation.
Drosophila melanogaster cellularization is a dramatic form of cytokinesis in which a membrane furrow simultaneously encapsulates thousands of cortical nuclei of the syncytial embryo to generate a polarized cell layer. Formation of this cleavage furrow depends on Golgi-based secretion and microtubules. During cellularization, specific Golgi move along microtubules, first to sites of furrow formation and later to accumulate within the apical cytoplasm of the newly forming cells. Here we show that Golgi movements and furrow formation depend on cytoplasmic dynein. Furthermore, we demonstrate that Lava lamp (Lva), a golgin protein that is required for cellularization, specifically associates with dynein, dynactin, cytoplasmic linker protein-190 (CLIP-190) and Golgi spectrin, and is required for the dynein-dependent targeting of the secretory machinery. The Lva domains that bind these microtubule-dependent motility factors inhibit Golgi movement and cellularization in a live embryo injection assay. Our results provide new evidence that golgins promote dynein-based motility of Golgi membranes.
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.