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.

Activation of the MKK/ERK pathway during somatic cell mitosis: direct interactions of active ERK with kinetochores and regulation of the mitotic 3F3/2 phosphoantigen.

The mitogen-activated protein (MAP) kinase pathway, which includes extracellular signal-regulated protein kinases 1 and 2 (ERK1, ERK2) and MAP kinase kinases 1 and 2 (MKK1, MKK2), is well-known to be required for cell cycle progression from G1 to S phase, but its role in somatic cell mitosis has not been clearly established. We have examined the regulation of ERK and MKK in mammalian cells during mitosis using antibodies selective for active phosphorylated forms of these enzymes. In NIH 3T3 cells, both ERK and MKK are activated within the nucleus during early prophase; they localize to spindle poles between prophase and anaphase, and to the midbody during cytokinesis. During metaphase, active ERK is localized in the chromosome periphery, in contrast to active MKK, which shows clear chromosome exclusion. Prophase activation and spindle pole localization of active ERK and MKK are also observed in PtK1 cells. Discrete localization of active ERK at kinetochores is apparent by early prophase and during prometaphase with decreased staining on chromosomes aligned at the metaphase plate. The kinetochores of chromosomes displaced from the metaphase plate, or in microtubule-disrupted cells, still react strongly with the active ERK antibody. This pattern resembles that reported for the 3F3/2 monoclonal antibody, which recognizes a phosphoepitope that disappears with kinetochore attachment to the spindles, and has been implicated in the mitotic checkpoint for anaphase onset (Gorbsky and Ricketts, 1993. J. Cell Biol. 122:1311-1321). The 3F3/2 reactivity of kinetochores on isolated chromosomes decreases after dephosphorylation with protein phosphatase, and then increases after subsequent phosphorylation by purified active ERK or active MKK. These results suggest that the MAP kinase pathway has multiple functions during mitosis, helping to promote mitotic entry as well as targeting proteins that mediate mitotic progression in response to kinetochore attachment.

Centriole duplication in lysates of Spisula solidissima oocytes.

A cell-free system has been developed that executes centriole duplication. Surf clam (Spisula solidissima) oocytes, arrested at late prophase of meiosis I, do not contain centrioles, centrosomes, or asters. Serial section high-voltage electron microscopy (HVEM) of asters and spindles isolated from potassium chloride-activated oocytes indicates that within 4 minutes oocytes assemble a single centriole that is duplicated by 15 minutes when assembly of the first meiotic spindle is complete. A mixture of lysates from unactivated oocytes and potassium chloride-activated oocytes induces centriole formation and duplication. Astral microtubule content in these lysate mixtures increases with time.

cut11(+): A gene required for cell cycle-dependent spindle pole body anchoring in the nuclear envelope and bipolar spindle formation in Schizosaccharomyces pombe.

The "cut" mutants of Schizosaccharomyces pombe are defective in spindle formation and/or chromosome segregation, but they proceed through the cell cycle, resulting in lethality. Analysis of temperature-sensitive alleles of cut11(+) suggests that this gene is required for the formation of a functional bipolar spindle. Defective spindle structure was revealed with fluorescent probes for tubulin and DNA. Three-dimensional reconstruction of mutant spindles by serial sectioning and electron microscopy showed that the spindle pole bodies (SPBs) either failed to complete normal duplication or were free floating in the nucleoplasm. Localization of Cut11p tagged with the green fluorescent protein showed punctate nuclear envelope staining throughout the cell cycle and SPBs staining from early prophase to mid anaphase. This SPB localization correlates with the time in the cell cycle when SPBs are inserted into the nuclear envelope. Immunoelectron microscopy confirmed the localization of Cut11p to mitotic SPBs and nuclear pore complexes. Cloning and sequencing showed that cut11(+) encodes a novel protein with seven putative membrane-spanning domains and homology to the Saccharomyces cerevisiae gene NDC1. These data suggest that Cut11p associates with nuclear pore complexes and mitotic SPBs as an anchor in the nuclear envelope; this role is essential for mitosis.

[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).

[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.

[A number of sorbents useful for the sorption of cephalosporin C]. / Izuchenie riada sorbentov primenitel'no k sorbtsii tsefalosporina C.

The physicochemical and sorption properties of a number of sorbents made in the USSR and abroad were studied with respect to the recovery of cephalosporin C from a model solution. It was shown that the sorption capacity of a sorbent depended on its specific surface. The macroporous styrene divinylbenzene sorbents with a specific surface of more than 500 m2/g had the best sorption properties.

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.

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.

[Sorption of aminoglycoside antibiotics by weakly acidic cation exchangers]. / Sorbtsiia antibiotikov aminoglikozidov slabokislotnymi kationitami.

Sorption of aminoglycoside antibiotics close by their chemical structures such as gentamicin, sisomicin and kanamycin by polyacrylic and polymetaacrylic cation exchange resins was studied. The generality of the sorption process (kinetics and statics) on the carboxylic cation exchange resins with participation of the ions of the aminoglycoside antibiotics was shown.

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.

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.

Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12.

A novel sequence discovered in a computational screen appears distantly related to the p35 subunit of IL-12. This factor, which we term p19, shows no biological activity by itself; instead, it combines with the p40 subunit of IL-12 to form a novel, biologically active, composite cytokine, which we term IL-23. Activated dendritic cells secrete detectable levels of this complex. IL-23 binds to IL-12R beta 1 but fails to engage IL-12R beta 2; nonetheless, IL-23 activates Stat4 in PHA blast T cells. IL-23 induces strong proliferation of mouse memory (CD4(+)CD45Rb(low)) T cells, a unique activity of IL-23 as IL-12 has no effect on this cell population. Similar to IL-12, human IL-23 stimulates IFN-gamma production and proliferation in PHA blast T cells, as well as in CD45RO (memory) T cells.