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V I Gelfand

Publications and source records attributed to V I Gelfand.

At least 37 records · Page 2Linked to original sources
PubMed1993

Kinesin-like molecules involved in spindle formation.

To study the possible involvement of kinesin-like molecules in mitosis a polyclonal antibody against the head domain of Drosophila kinesin heavy chain (HD antibody) was microinjected into PtK1 cells at the prophase-prometaphase transition. Progress of the cell through mitosis was recorded for subsequent detailed analysis. Cells injected with pre-immune IgG progressed through mitosis at rates similar to those for noninjected cells. After HD antibody injections, chromosomes failed to congress to an equatorial plane and cells failed to form a bipolar spindle. Rather, the spindle poles came together, resulting in a monopolar-like configuration with chromosomes arranged about the poles in a rosette. Sometimes the monopolar array moved to the margin of the cell in a way similar to anaphase B movement in normal cells. Antibody-injected cells progressed into the next cell cycle as evidenced by chromosome decondensation and nuclear envelope reformation. Anti-tubulin immunofluorescence confirmed the presence of a radial monopolar array of microtubules in injected cells. HD antibody stained in a punctate pattern in interphase and the spindle region in mitotic PtK1 cells. The antibody also reacted with spindle fibers of isolated mitotic CHO spindles and with kinetochores of isolated CHO chromosomes. Immunoblotting indicated that the major component recognized by the antibody is the 120 kDa kinesin heavy chain. At higher protein loads the antibody recognized also a 34 kDa polypeptide in PtK1 cell extracts, a 135 kDa polypeptide in a preparation of CHO spindles and a 300 kDa polypeptide in a preparation of CHO mitotic chromosomes. We conclude that a kinesin-like molecule is important for the formation and/or maintenance of the structure of mitotic spindle.

Animals↗
PubMed1992

HSP70-related 65 kDa protein of beet yellows closterovirus is a microtubule-binding protein.

Beet yellows virus (BYV) genome encodes a 65 kDa protein homologous to the HSP70 family of cellular heat-shock proteins (Agranovsky, A.A., Boyko, V.P., Karasev, A.V., Koonin, E.V. and Dolja, V.V. (1991) J. Mol. Biol. 217, 603-610). The respective gene was cloned and expressed in vitro yielding a product of the expected size (p65). This product was found to bind to the purified microtubules with a binding constant of 4 x 10(-7) M. The binding of p65 was stimulated if ATP presented in the translation mixture was hydrolyzed by apyrase. Removal of the short C-terminal domains of alpha- and beta-tubulin by subtilisin digestion abolished the binding, demonstrating its specificity. The possible role of p65 association with microtubules in the movement of virus within and/or between plant cells is proposed.

Animals↗
PubMed1991

Coalignment of vimentin intermediate filaments with microtubules depends on kinesin.

Intermediate filaments in most types of cultured cells coalign with microtubules. Depolymerization of microtubules results in collapse of vimentin and desmin intermediate filaments to the nucleus where they form a perinuclear cap. Collapse can also be induced by microinjection of antibodies against intermediate filament or microtubule proteins. Thus, two filament systems interact with each other. But the molecules mediating this interaction are unknown. One of the candidates for this role is a microtubule motor kinesin. Recent data showed that kinesin is involved in the plus end-directed movement of the membranous organelles along microtubules such as radial extension of lysosomes in macrophages and centrifugal movement of pigment in melanophores. Here we report that injection of the anti-kinesin antibody into human fibroblasts results in the redistribution of intermediate filaments to a tight perinuclear aggregate but had no effect on the distribution of microtubules. Thus, kinesin is involved not only in organelle movement but also in interaction of the two major cytoskeletal systems, intermediate filaments and microtubules.

Adenosine Triphosphatases↗
PubMed1991

Kinesin is responsible for centrifugal movement of pigment granules in melanophores.

Kinesin is a mechanochemical ATPase that induces translocation of latex beads along microtubules and microtubule gliding on a glass surface. This protein is thought to be a motor for the movement of membranous organelles in cells. Recently Hollenbeck and Swanson [Hollenbeck, P. J. & Swanson, J. A. (1990) Nature (London) 346, 864-866] showed that kinesin is involved in the positioning of tubular lysosomes in macrophages. However, the role of this protein in the movement of organelles was not yet clear. We used a polyclonal antibody against the kinesin heavy chain that inhibited kinesin-dependent microtubule gliding in vitro to study the role of kinesin in the movement of pigment granules in melanophores of the teleost black tetra (Gymnocorymbus ternetzi). Microinjection of the antibody into cultured melanophores did not produce any specific effect on the aggregation of pigment granules in melanophores, but it did result in a strong dose-dependent inhibition of the dispersion. Immunoblotting of melanophore extracts showed that the kinesin antibody reacted in these cells with a single protein component with a molecular mass of 135 kDa. Thus, kinesin is responsible for the movement of pigment granules from the center to the periphery of the melanophore.

Adenosine Triphosphatases↗
PubMed1991

MAP2-mediated binding of chromaffin granules to microtubules.

We have examined the interaction of chromaffin granules from bovine adrenal medulla with microtubules. Chromaffin granules were mixed with microtubules made of phosphocellulose-purified tubulin, and pelleted through a 1.6 M sucrose cushion at 12,000 x g for 10 min. Both components (granules and microtubules) were pelleted when added together but not separately. This result indicates that granules form a heavy complex with the microtubules. Such a complex was visualized by an electron microscopy of the granule/microtubule mixture. Treatment of the granules with trypsin abolished their ability to interact with the microtubules. The binding of the granules to the microtubules; (i) was not sensitive to ATP; and (ii) was completely inhibited by the cleavage of C-terminal peptides of alpha- and beta-subunits of tubulin with subtilisin. These relationships suggest that the granule binding is mediated by one of the structural microtubule-associated proteins rather than by microtubule-dependent translocators. For identification of protein(s) mediating the binding, the granules were solubilized with Triton X-100, soluble proteins were mixed with the microtubules, and microtubules with bound proteins were pelleted through a glycerol cushion. At least one granule protein interacting with the microtubules was found in the pellet. This protein was identified as MAP2 according to its electrophoretic mobility and reactivity with a MAP2 antibody. Affinity chromatography of solubilized proteins on a column containing taxol-stabilized microtubules also revealed MAP2 as a protein of chromaffin granules interacting with the microtubules.

Adenosine Triphosphate↗
PubMed1991

Some of eukaryotic elongation factor 2 is colocalized with actin microfilament bundles in mouse embryo fibroblasts.

Indirect immunofluorescent microscopy was used to study the distribution of eukaryotic elongation factor 2 (EF-2) in cultured mouse embryo fibroblasts. The perinuclear area (endoplasm) of all the cells and many straight cables running along the whole cytoplasm were stained with monospecific goat or rabbit antibodies to rat liver EF-2. Double staining of the cells with antibodies to EF-2 and rhodaminyl-phalloidin (used for actin microfilament detection) showed that EF-2 containing cables coincided with bundles of actin microfilaments. Not all actin microfilament bundles contained EF-2: sometimes EF-2 was not observed in bundles running along the cell edges or in actin microfilament junctions. Triton X-100 extracted most of EF-2 from the cells and no actin microfilament bundles were stained with the EF-2 antibodies in the Triton-extracted cells. Thus, in mouse embryo fibroblasts EF-2 can be found along actin microfilament bundles, but it is unlikely to be their integral protein.

Actin Cytoskeleton↗
PubMed1990

Stimulation of actin synthesis in phalloidin-treated cells. Evidence for autoregulatory control.

We used the technique of scrape loading to introduce phalloidin into mouse embryo fibroblasts in mass culture. Phalloidin almost completely destroyed actin microfilament bundles, but the amount of polymerized cytoskeleton-associated actin was increased approximately two-fold and the amount of monomeric (Triton X-100 extractable) actin was significantly reduced. The major result of the present study is that the rate of actin synthesis in the phalloidin-treated cells was 2-3 times higher than in the control cells. Northern blot and translation in a cell-free system from rabbit reticulocytes showed that the actin mRNA level significantly increased as a result of phalloidin treatment.

Actin Cytoskeleton↗
PubMed1990

Microtubule-associated proteins and microtubule-based translocators have different binding sites on tubulin molecule.

It has been previously shown that a class of microtubule proteins, the so-called microtubule-associated proteins (MAPs), binds to the C-terminal part of tubulin subunits. We show here that microtubules composed of tubulin whose 4-kDa C-terminal domain was cleaved by subtilisin (S-microtubules) are unable to bind MAPs but can still bind the anterograde translocator protein kinesin and the retrograde translocator dynein. Binding of both motors to S-microtubules, like their binding to normal microtubules, was ATP-dependent. In addition, direct competition experiments showed that binding sites for kiensin and MAPs on the microtubule surface lattice do not overlap. Furthermore, S-microtubules stimulated the ATPase activity of kinesin at least 8-fold, and the affinities of kinesin for control and S-microtubules were identical. S-microtubules were able to glide along kinesin-coated coverslips at a rate of 0.2 microns/s, the same rate as control microtubules. We conclude, that unlike MAPs, kinesin and cytoplasmic dynein bind to the tubulin molecule outside the C-terminal region.

Adenosine Triphosphatases↗
PubMed1990

Effect of MAP 1, MAP 2, and tau-proteins on structural parameters of tubulin assemblies.

When temperature-dependent recycling procedures for purification were used, tubulin is usually accompanied by a mixture of microtubule-associated proteins (MAPs) primarily comprising MAP 1, MAP 2, and the tau-proteins. Formerly we reported that microtubules formed from tubulin in the presence of these MAPs have more protofilaments than those formed without MAPs. Furthermore, these MAPs suppress the formation of aberrant assemblies (Böhm et al., BBA 800, 119, 1984). Now, we report that each single MAP fraction is able to influence the protofilament number of microtubules and the ratio between aberrant assemblies and microtubules in the same manner as the MAP mixture. This implies that all the MAPs investigated play a certain role in lateral association of tubulin dimers.

Animals↗
PubMed1989

Isolation of a 45-kDa fragment from the kinesin heavy chain with enhanced ATPase and microtubule-binding activities.

Kinesin is a microtubule-activated, mechanochemical ATPase capable of moving particles along microtubules and making microtubules glide along a solid substrate. In this study we used limited proteolysis to study the structure of bovine brain kinesin, a heterotetramer composed of two heavy (120-kDa) and two light (62-kDa) chains. alpha-chymotrypsin, trypsin, and subtilisin all produced a protease-resistant 45-kDa fragment from the kinesin heavy chain. As isolated by gel-filtration chromatography, this fragment contains both the microtubule-binding site and the ATP catalytic site of the molecule. Proteolytic cleavage stimulated microtubule-dependent Mg2+-ATPase activity 4- to 5-fold up to 75-120 mumol ATP/min/mg. Cleavage also increased the affinity of the fragment for microtubules at least 10-fold. Since the purified fragment does not support the gliding of flagellar axonemes, we propose that cleavage of the heavy chain uncouples ATPase activity from its translocator activity, which may require other parts of the molecule.

Adenosine Triphosphatases↗
PubMed1989

Direct visualization of bipolar myosin filaments in stress fibers of cultured fibroblasts.

The authors examined the molecular organization of myosin in stress fibers (microfilament bundles) of cultured mouse embryo fibroblasts. To visualize the organization of myosin filaments in these cells, fibroblast cytoskeletons were treated with gelsolin-like protein from bovine brain (hereafter called brain gelsolin), which selectively disrupts actin filaments. As shown earlier [Verkhovsky et al., 1987], this treatment did not remove myosin from the stress fibers. The actin-free cytoskeletons then were lightly sonicated to loosen the packing of the remaining stress fiber components and fixed with glutaraldehyde. Electron microscopy of platinum replicas of these preparations revealed dumbbell-shaped structures of approximately 0.28 micron in length, which were identified as bipolar myosin filaments by using antibodies to fragments of myosin molecule (subfragment 1 and light meromyosin) and colloidal gold label. Bipolar filaments of myosin in actin-free cytoskeletons were often organized in chains and lattices formed by end-to-end contacts of individual filaments at their head-containing regions. Therefore, after extraction of actin, it was possible for the first time to display bipolar myosin filaments in the stress fibers of cultured cells.

Actin Cytoskeleton↗
PubMed1988

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.

Animals↗
PubMed1987

18 kDa microtubule-associated protein: identification as a new light chain (LC-3) of microtubule-associated protein 1 (MAP-1).

SDS gel electrophoresis of microtubule proteins obtained from bovine brain by polymerization cycles revealed a new protein of 18 kDa. This protein was copolymerized with tubulin and its stoichiometry to tubulin remained constant for at least 5 cycles of assembly. Moreover, this protein remained bound to microtubules stabilized with 10 microM taxol and pelleted through a 4 M glycerol cushion. The same 18 kDa protein was found in a purified preparation of the high molecular mass microtubule-associated protein 1 (MAP-1). The 18 kDa protein copurified with the MAP-1 heavy chains during column chromatography on phosphocellulose, DEAE-cellulose, hydroxyapatite and Bio-Gel A-15m. Incubation of the MAP-1 preparation with a mouse monoclonal antibody to the light chain 1 (LC-1) of MAP-1 and with a second precipitating antibody (a rabbit antibody to mouse IgG) immunoprecipitated from the solution all the known components of MAP-1 (heavy chains, LC-1, LC-2), as well as the 18 kDa protein. Immunoblotting showed, however, that this antibody does not interact directly with the 18 kDa protein. These results indicate that the 18 kDa protein forms a complex with all other components of MAP-1. This polypeptide, therefore, is a new light chain (LC-3) of MAP-1.

Animals↗
PubMed1987

Organization of stress fibers in cultured fibroblasts after extraction of actin with bovine brain gelsolin-like protein.

Mouse and quail embryo fibroblasts were extracted with Triton X-100 and the resulting cytoskeletons were treated with gelsolin-like actin-capping protein (the 90-kDa protein-actin complex isolated from bovine brain). Staining of cells with rhodamine-conjugated phalloin or an antibody to actin did not reveal any actin-containing structures after treatment with the 90-kDa protein-actin complex. Extraction of actin was confirmed by SDS-gel electrophoresis. Immunofluorescence microscopy showed that vinculin and alpha-actinin were released from the cytoskeletons together with actin. However, myosin remained associated with the cytoskeleton after treatment with the 90-kDa protein-actin complex. The distribution of myosin in treated cells showed no significant difference from that in control cells: in both cases myosin was localized mainly in the stress fibers. Double-fluorescence staining showed the absence of actin in myosin-containing stress fibers of treated cells. The ultrastructural organization of actin-depleted stress fibers was studied by transmission electron microscopy of platinum replicas. On electron micrographs these fibers appeared as bundles of filaments containing clusters of globular material. It is concluded that myosin localization in stress fibers does not depend on actin.

Actin Cytoskeleton↗
PubMed1987

Immunofluorescent localization of protein synthesis components in mouse embryo fibroblasts.

The distribution of initiation factor 2(eIF-2) and elongation factor 2(EF-2) in cultured mouse embryo fibroblasts was studied and compared with the distribution of ribosomes. We used immunofluorescence microscopy with monospecific antibodies to eIF-2, EF-2, and proteins S3a and S7 of the small ribosomal subunit. Ribosomes and factors eIF-2 and EF-2 were found mainly in the vicinity of the cell nucleus. This perinuclear zone coincides with the endoplasm - the central part of the cell containing numerous membraneous organelles and inclusions. Besides the perinuclear zone, small stained regions could be seen at the periphery of some cells. After treatment of the cells with Triton X-100 in a buffer conditions, that stabilizes the major cytoskeletal structures, some of the ribosomes, eIF-2, and EF-2 remained bound to the insoluble material. These components were found near the nucleus and some were located along the microfilament bundles.

Animals↗