The Yeast Dynactin Complex Is Involved in Partitioning the Mitotic Spindle between Mother and Daughter Cells during Anaphase B

Although vertebrate cytoplasmic dynein can move to the minus ends of microtubules in vitro, its ability to translocate purified vesicles on microtubules depends on the presence of an accessory complex known as dynactin. We have cloned and characterized a novel gene, NIP100, which encodes the yeast homologue of the vertebrate dynactin complex protein p150glued. Like strains lacking the cytoplasmic dynein heavy chain Dyn1p or the centractin homologue Act5p, nip100Δ strains are viable but undergo a significant number of failed mitoses in which the mitotic spindle does not properly partition into the daughter cell. Analysis of spindle dynamics by time-lapse digital microscopy indicates that the precise role of Nip100p during anaphase is to promote the translocation of the partially elongated mitotic spindle through the bud neck. Consistent with the presence of a true dynactin complex in yeast, Nip100p exists in a stable complex with Act5p as well as Jnm1p, another protein required for proper spindle partitioning during anaphase. Moreover, genetic depletion experiments indicate that the binding of Nip100p to Act5p is dependent on the presence of Jnm1p. Finally, we find that a fusion of Nip100p to the green fluorescent protein localizes to the spindle poles throughout the cell cycle. Taken together, these results suggest that the yeast dynactin complex and cytoplasmic dynein together define a physiological pathway that is responsible for spindle translocation late in anaphase.

Cytoplasmic Dynein, the Dynactin Complex, and Kinesin Are Interdependent and Essential for Fast Axonal Transport

In axons, organelles move away from (anterograde) and toward (retrograde) the cell body along microtubules. Previous studies have provided compelling evidence that conventional kinesin is a major motor for anterograde fast axonal transport. It is reasonable to expect that cytoplasmic dynein is a fast retrograde motor, but relatively few tests of dynein function have been reported with neurons of intact organisms. In extruded axoplasm, antibody disruption of kinesin or the dynactin complex (a dynein activator) inhibits both retrograde and anterograde transport. We have tested the functions of the cytoplasmic dynein heavy chain (cDhc64C) and the p150Glued (Glued) component of the dynactin complex with the use of genetic techniques in Drosophila. cDhc64C and Glued mutations disrupt fast organelle transport in both directions. The mutant phenotypes, larval posterior paralysis and axonal swellings filled with retrograde and anterograde cargoes, were similar to those caused by kinesin mutations. Why do specific disruptions of unidirectional motor systems cause bidirectional defects? Direct protein interactions of kinesin with dynein heavy chain and p150Glued were not detected. However, strong dominant genetic interactions between kinesin, dynein, and dynactin complex mutations in axonal transport were observed. The genetic interactions between kinesin and either Glued or cDhc64C mutations were stronger than those between Glued and cDhc64C mutations themselves. The shared bidirectional disruption phenotypes and the dominant genetic interactions demonstrate that cytoplasmic dynein, the dynactin complex, and conventional kinesin are interdependent in fast axonal transport.

The interaction between cytoplasmic dynein and dynactin is required for fast axonal transport

Fast axonal transport is characterized by the bidirectional, microtubule-based movement of membranous organelles. Cytoplasmic dynein is necessary but not sufficient for retrograde transport directed from the synapse to the cell body. Dynactin is a heteromultimeric protein complex, enriched in neurons, that binds to both microtubules and cytoplasmic dynein. To determine whether dynactin is required for retrograde axonal transport, we examined the effects of anti-dynactin antibodies on organelle transport in extruded axoplasm. Treatment of axoplasm with antibodies to the p150Glued subunit of dynactin resulted in a significant decrease in the velocity of microtubule-based organelle transport, with many organelles bound along microtubules. We examined the molecular mechanism of the observed inhibition of motility, and we demonstrated that antibodies to p150Glued disrupted the binding of cytoplasmic dynein to dynactin and also inhibited the association of cytoplasmic dynein with organelles. In contrast, the anti-p150Glued antibodies had no effect on the binding of dynactin to microtubules nor on cytoplasmic dynein-driven microtubule gliding. These results indicate that the interaction between cytoplasmic dynein and the dynactin complex is required for the axonal transport of membrane-bound vesicles and support the hypothesis that dynactin may function as a link between the organelle, the microtubule, and cytoplasmic dynein during vesicle transport.

Na,K-ATPase transport from endoplasmic reticulum to Golgi requires the Golgi spectrin–ankyrin G119 skeleton in Madin Darby canine kidney cells

Spectrin (βIΣ∗) and ankyrin (AnkG119) associate with Golgi membranes and the dynactin complex, but their role in vesicle trafficking remains uncertain. We find that the actin-binding domain and membrane-association domain 1 (MAD1) of βI spectrin together form a constitutive Golgi targeting signal in transfected MDCK cells. Expression of this signal in transfected cells disrupts the endogenous Golgi spectrin skeleton and blocks transport of α- and β-Na,K-ATPase and vesicular stomatitis virus-G protein from the endoplasmic reticulum (ER) but does not disrupt the formation of Golgi stacks, the distribution of β-COP, or the transport and surface display of E-cadherin. The Golgi spectrin skeleton is thus required for the transport of a subset of membrane proteins from the ER to the Golgi. We postulate that together with polyfunctional adapter proteins such as AnkG119, Golgi spectrin forms a docking complex that acts prior to the cis-Golgi, presumably with vesicular–tubular clusters (VTCs or ERGIC), to sequester specific membrane proteins into vesicles transiting between the ER and Golgi, and subsequently (probably involving other isoforms of spectrin and ankyrin) to mediate cargo transport within the Golgi and to other membrane compartments. We hypothesize that this vesicular spectrin–ankyrin adapter-protein trafficking (or tethering) system (SAATS) mediates the capture and transport of many membrane proteins and acts in conjunction with vesicle-targeting molecules to effect the efficient transport of cargo proteins.

Genetic interactions among cytoplasmic dynein, dynactin, and nuclear distribution mutants of Neurospora crassa.

Cytoplasmic dynein is a multisubunit, microtubule-associated, mechanochemical enzyme that has been identified as a retrograde transporter of various membranous organelles. Dynactin, an additional multisubunit complex, is required for efficient dynein-mediated transport of vesicles in vitro. Recently, we showed that three genes defined by a group of phenotypically identical mutants of the filamentous fungus Neurospora crassa encode proteins that are apparent subunits of either cytoplasmic dynein or dynactin. These mutants, designated ropy (ro), display abnormal hyphal growth and are defective in nuclear distribution. We propose that mutations in other genes encoding dynein/dynactin subunits are likely to result in a ropy phenotype and have devised a genetic screen for the isolation of additional ro mutants. Cytoplasmic dynein/dynactin is the largest and most complex of the cytoplasmic motor proteins, and the genetic system described here is unique in its potentiality for identifying mutations in undefined genes encoding dynein/dynactin subunits or regulators. We used this screen to isolate > 1000 ro mutants, which were found to define 23 complementation groups. Unexpectedly, interallelic complementation was observed with some allele pairs of ro-1 and ro-3, which are predicted to encode the largest subunits of cytoplasmic dynein and dynactin, respectively. The results suggest that the Ro1 and Ro3 polypeptides may consist of multiple, functionally independent domains. In addition, approximately 10% of all newly isolated ro mutantsdisplay unlinked noncomplementation with two or more of the mutants that define the 23 complementation groups. The frequent appearance of ro mutants showing noncomplementation with multiple ro mutants having unlinked mutations suggests that nuclear distribution in filamentous fungi is a process that is easily disrupted by affecting either dosage or activity of cytoplasmic dynein, dynactin, and perhaps other cytoskeletal proteins or regulators.