Dynein is required for spindle assembly in cytoplasmic extracts of Spisula solidissima oocytes.

Meiosis I spindle assembly is induced in lysate-extract mixtures prepared from clam (Spisula solidissima) oocytes. Unactivated lysate prepared from unactivated oocytes contain nuclei (germinal vesicles, GVs) which house condensed chromosomes. Treatment of unactivated lysate with clarified activated extract prepared from oocytes induced to complete meiosis by treatment with KCl induces GV breakdown (GVBD) and assembly of monopolar, bipolar, and multipolar aster-chromosome complexes. The process of in vitro meiosis I spindle assembly involves the assembly of microtubule asters and the association of these asters with the surfaces of the GVs, followed by GVBD and spindle assembly. Monoclonal antibody m74-1, known to react specifically with the N terminus of the intermediate chain of cytoplasmic dynein, recognizes Spisula oocyte dynein and inhibits in vitro meiosis I spindle assembly. Control antibody has no affect on spindle assembly. A similar inhibitory effect on spindle assembly was observed in the presence of orthovanadate, a known inhibitor of dynein ATPase activity. Neither m74-1 nor orthovanadate has any obvious affect on GVBD or aster formation. We propose that dynein function is required for the association of chromosomes with astral microtubules during in vitro meiosis I spindle assembly in these lysate-extract mixtures. However, we conclude that dynein function is not required for centrosome assembly and maturation or for centrosome-dependent aster formation.

Mammalian cells express three distinct dynein heavy chains that are localized to different cytoplasmic organelles.

We describe two dynein heavy chain (DHC)-like polypeptides (DHCs 2 and 3) that are distinct from the heavy chain of conventional cytoplasmic dynein (DHC1) but are expressed in a variety of mammalian cells that lack axonemes. DHC2 is a distant member of the "cytoplasmic" branch of the dynein phylogenetic tree, while DHC3 shares more sequence similarity with dynein-like polypeptides that have been thought to be axonemal. Each cytoplasmic dynein is associated with distinct cellular organelles. DHC2 is localized predominantly to the Golgi apparatus. Moreover, the Golgi disperses upon microinjection of antibodies to DHC2, suggesting that this motor is involved in establishing proper Golgi organization. DCH3 is associated with as yet unidentified structures that may represent transport intermediates between two or more cytoplasmic compartments. Apparently, specific cytoplasmic dyneins, like individual members of the kinesin superfamily, play unique roles in the traffic of cytomembranes.

Cytoplasmic dynein plays a role in mammalian mitotic spindle formation.

The formation and functioning of a mitotic spindle depends not only on the assembly/disassembly of microtubules but also on the action of motor enzymes. Cytoplasmic dynein has been localized to spindles, but whether or how it functions in mitotic processes is not yet known. We have cloned and expressed DNA fragments that encode the putative ATP-hydrolytic sites of the cytoplasmic dynein heavy chain from HeLa cells and from Dictyostelium. Monospecific antibodies have been raised to the resulting polypeptides, and these inhibit dynein motor activity in vitro. Their injection into mitotic mammalian cells blocks the formation of spindles in prophase or during recovery from nocodazole treatment at later stages of mitosis. Cells become arrested with unseparated centrosomes and form monopolar spindles. The injected antibodies have no detectable effect on chromosome attachment to a bipolar spindle or on motions during anaphase. These data suggest that cytoplasmic dynein plays a unique and important role in the initial events of bipolar spindle formation, while any later roles that it may play are redundant. Possible mechanisms of dynein's involvement in mitosis are discussed.

Pseudopodial activity at the active edge of migrating fibroblast is decreased after drug-induced microtubule depolymerization.

It is known that depolymerization of microtubules by colcemid or other similar drugs abolishes polarization of pseudopodial activity in migrating fibroblasts. In this work the effect of colcemid on the intensity of protrusion and retraction of lamellipodia at the active edges of human fibroblasts migrating into the wound was investigated with video-enhanced contrast microscopy. To characterize the pseudopodial activity quantitatively the outlines of the active edges in the pairs of frames taken at adjacent 20-sec intervals were compared and mean areas of protrusions and retractions per unit length of the perimeter of the edge were measured. The mean rates of protrusions and retractions were 4-6 times less in colcemid-treated cells than in controls. Thus, microtubules depolymerized by colcemid, and/or intermediate filaments undergoing perinuclear collapse in the presence of this drug, are essential not only for the restriction of pseudopodial activity to one particular zone of the cell edge but also for the development of maximal activity in this zone.

[45 kDa fragment of the kinesin molecule possesses high ATPase activity and binds to microtubules]. / 45 kDa-fragment molekuly kinezina obladaet vysokoi ATP-aznoi aktivnost'iu i sviazyvaetsia s mikrotrubochkami.

Kinesin is a mechano-chemical ATPase capable to move particles along microtubules and microtubules along the solid substrate. Molecule of bovine brain kinesin is a heterotetrameric unit consisting of two heavy (120 kDa) and two light (62 kDa) chains. We used limited proteolysis to study the location of the functional sites on the kinesin molecule. Chymotrypsin cleavage produced a stable 45 kDa fragment of the heavy chain which was purified from the digest using FPLC chromatography on a Superose 12 column. 45 kDa fragment contained both a microtubule-binding site and a ATPase site of the kinesin molecule. Cleavage of the 45 kDa fragment from the rest of the heavy chain significantly activated its ATPase activity. However, this activity remained fully dependent on microtubules. We suggest that the chymotrypsin cleavage uncouple ATPase activity of kinesin (found in the 45 kDa fragment) from its translocator activity (which, probably, required the presence of other parts of the molecule).

The quaternary structure of bovine brain kinesin.

In the present work we have studied the subunit composition of kinesin, the microtubule-activated, mechanochemical ATPase, isolated from bovine brain. Polypeptides with mol. wts of 120 and 62 kd are the major components of the kinesin preparation. These polypeptides could not be separated by electrophoresis under nondenaturing conditions or by FPLC on a MonoQ column, and are therefore assumed to form a tight complex. As shown by immunoblotting with polyclonal and monoclonal antibodies to the 120-kd polypeptide and by one-dimensional peptide mapping, the 62-kd polypeptide does not appear to be a proteolytic product of the 120-kd component. Densitometric scanning of polyacrylamide-SDS gels shows that these polypeptides are present in a complex in a 1:1 molar ratio. The mol. wt of native kinesin was studied by sedimentation equilibrium and was found to be 386 +/- 14 kd. A comparison of the mol. wts of individual polypeptides with the mol. wt of the intact molecule indicates that the native molecule contains two 120-kd subunits and two 62-kd subunits.

[Reorganization of the system of intermediate filaments and detection of the organization centers after centrifugation of cells attached to a substrate]. / Reorganizatsiia sistemy promezhutochnykh filamentov i vyiavlenie tsentrov ikh organizatsii posle tsentrifugirovaniia prikreplennykh k substratu kletok.

Cultured pig kidney epithelial cells were centrifuged at 20,000 gav so that the centrifugation force was oriented parallel to the substrate, fixed and processed for indirect immunofluorescent staining with tubulin and vimentin antibodies. After a 2 hour centrifugation vimentin filaments aggregated in the centripetal parts of the cells (probably, because of their association with floating lipid vesicles). Microtubule-organizing centers were found near the centripetal poles of the nuclei, which migrated in the direction of the centrifugal force. The distribution of the cytoplasmic microtubules did not change during centrifugation. The staining of the cultures one hour after centrifugation revealed vimentin-containing spots with radiating intermediate filaments in most of the cells. These spots were localized near the cell nuclei; double immunofluorescent staining with tubulin and vimentin antibodies showed that their position was identical to that of the microtubule-organizing centers. Similar foci of vimentin filaments were seen in the cells after a 3-4 hour centrifugation. Probably, these structures participate in organizing the intermediate filament cytoskeleton in cells.

Identification of a 100 kD protein associated with microtubules, intermediate filaments and coated vesicles in cultured cells.

We have obtained several hybridoma clones producing antibodies to microtubule-associated proteins (MAPs) from bovine brain. Interaction of one of these antibodies, named RN 17, with cultured cells was studied by indirect immunofluorescence and immunoelectron microscopy. RN 17 antibody recognized both high molecular weight (HMW) MAPs, MAP 1 and MAP 2, in immunoblotting reaction with brain microtubules. In lysates of cultured cells, it bound to a protein doublet with a molecular weight of 100 kD. By immunofluorescence microscopy we showed that RN 17 antibody stained cytoplasmic fibrils, mitotic spindles and small particles in the cytoplasm of various cultured cells. The cytoplasmic fibrils were identified as both microtubules and intermediate filaments by double fluorescence microscopy and by their response to colcemid and 0.6 M KCl. This identification was confirmed by immunoelectron microscopy which also showed that the particles stained by RN 17 antibody are coated vesicles. Thus, cultured non-neural cells may contain a novel protein that binds to microtubules, intermediate filaments, and coated vesicles.