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R L Margolis

Publications and source records attributed to R L Margolis.

At least 127 records · Page 7Linked to original sources
PubMed1987

Model of anaphase chromosome movement based on polymer-guided diffusion.

We propose a motility mechanism that may result in the displacement of objects within the cell. The mechanism, which we call polymer-guided diffusion, involves a microscopic cycle of polymer association and dissociation from a lateral binding site. Reassociation occurs at the polymer subunit adjacent to that which has just dissociated, thus generating an apparent sliding movement. The displacement involves only free diffusion and the spontaneous fluctuations of the polymer; the movement thus requires no other energy sources than thermal energy and the energy originally required for the formation of the polymer. In this manner polymer-associated organelles can be guided (inevitably) by diffusional processes toward a final destination. The specific example of the anaphase movement of chromosomes poleward is detailed.

Anaphase↗
PubMed1987

A 17-kD centromere protein (CENP-A) copurifies with nucleosome core particles and with histones.

We have detected and begun to characterize a 17-kD centromere-specific protein, CENP-A (Earnshaw, W. C., and N. Rothfield, 1985, Chromosoma., 91:313-321). Sera from several humans with CREST scleroderma autoimmune disease (CREST: calcinosis, Raynaud's phenomenon, esophageal dsymotility, sclerodactyly, and telangiectasia) bind this protein in immunoblot assays of HeLa whole cell or nuclear extracts. We have affinity purified the anti-17-kD centromere protein (anti-CENP-A) specific antibodies from immunoblots of HeLa nuclear protein. The antibodies react with epitopes present on CENP-A derived from humans but apparently do not recognize specific epitopes in either rat or chicken nuclei. Only human nuclear protein is CENP-A positive by immunoblot. Furthermore, human cells show localization of anti-CENP-A antibody to centromeres by immunofluorescence microscopy, whereas rat cells do not. On extraction from the nucleus, CENP-A copurifies with core histones and with nucleosome core particles. We conclude that this centromere-specific protein is a histone-like component of chromatin. The data suggest that CENP-A functions as a centromere-specific core histone.

Animals↗
PubMed1986

Identification of a distinct class of vinblastine binding sites on microtubules.

Vinblastine, at concentrations above approximately 1 to 2 microM, causes depolymerization of steady-state bovine brain microtubules in vitro by a fraying of microtubule ends into protofilament-like spirals. Microtubule depolymerization is associated with the binding of vinblastine in approximately molar stoichiometry to tubulin in microtubules with apparent low affinity, as determined by binding experiments with radiolabeled vinblastine and by the ability of vinblastine to inhibit DEAE-dextran decoration of microtubule surfaces. Our data suggest that depolymerization occurs by a propagated mechanism, initially involving binding of vinblastine to a limited number of available sites on microtubule surfaces. This appears to cause loosening of protofilament associations which results in the exposure of new vinblastine-binding sites. Additional vinblastine binding in turn results in further loosening of protofilament associations. Such loosening, when it occurs at microtubule ends, results in protofilament-like splaying and end-wise depolymerization. Microtubule depolymerization appears mechanistically distinct from inhibition of microtubule polymerization by the drug, which is associated with the binding of vinblastine to small numbers of high-affinity binding sites on tubulin at one or both microtubule ends.

Animals↗
PubMed1986

Purification and assay of a 145-kDa protein (STOP145) with microtubule-stabilizing and motility behavior.

The capacity of microtubules to disassemble in vitro is profoundly affected by a protein factor designated STOP (stable tubule only polypeptide). Here we report the isolation of STOP protein and confirm that its activity is, as predicted, highly substoichiometric to the tubulin in microtubules. The isolation of the 145-kDa STOP (STOP145) protein has been effected from isolated cold-stable microtubules by two column steps: DEAE ion-exchange and a calmodulin affinity column. To confirm the protein's activity we have produced an antibody against STOP145 and have used the antibody to specifically remove the protein and the activity using an antibody-linked affinity column. We conclude that the STOP145 protein accounts for the observed in vitro stabilization of microtubules.

Animals↗
PubMed1986

Sliding of STOP proteins on microtubules: a model system for diffusion-dependent microtubule motility.

STOP proteins, of 145 kD, act substoichiometrically to block end-wise disassembly of microtubules. STOPs bind to microtubules either during microtubule assembly or when added at steady state, and when binding to the polymers is apparently irreversible. They are not measurably lost from polymers under competition conditions, and there is no measurable exchange between polymers. Nonetheless, STOP proteins exhibit an extraordinary behavior: they "slide" laterally on the surface of the microtubule. Displacement is assayed by forming hybrid microtubules in which cold stable or cold labile region subunits are labeled. Displacement of STOPs on the polymer with time will cause labeled subunits of cold-stable regions to become increasingly cold labile in a manner reciprocal to cold stabilization of previously cold-labile subunits. Because equilibrium exchange of STOP proteins onto and off the polymers can be ruled out, the displacement of STOPs relative to subunits can only be explained by lateral diffusion or "sliding." Axonal transport and mitotic mechanisms were discussed as implications of such a lateral translocation mechanism for microtubule-dependent motility.

Animals↗
PubMed1985

Right and left cortical lesions asymmetrically alter cerebrovascular permeability in the rat.

Vascular permeability and intravascular space in the rat cerebral cortex and hypothalamus were assessed 5 and 14 days after the production of either right or left hemisphere frontal cortical suction lesions. Rats were intravenously injected with large (dextran, 50,000 kdalton) and small (inulin, 5000 kdalton) molecular tracers and tissue samples were removed for assay after 7 min. Five days following lesions of the right but not left hemisphere, inulin levels in the cortical region posterior and contralateral to the lesion site were significantly higher than control values. Dextran levels were not increased in this region by lesions of either hemisphere. However, both right and left hemisphere lesions led to a significant elevation of inulin and dextran levels (inulin higher than dextran) in the cortex surrounding the lesion site. Fourteen days after lesion of either hemisphere, tracer levels were not elevated over control in any region examined. These results suggest that both right and left hemisphere cortical lesions transiently increase local vasopermeability (and perhaps intravascular space), but that only right hemisphere lesions selectively increase inulin in cortex distant from the lesion site, probably from lesion-induced alterations in cerebrovascular permeability.

Animals↗
PubMed1985

The effects of methyl mercury binding to microtubules.

The effects of methyl mercury hydroxide (MeHg) on the in vitro polymerization and depolymerization of microtubules were studied. Polymerization was totally inhibited at 3.0 X 10(-5) M MeHg and depolymerization occurred at concentrations above 1.0 X 10(-5) M MeHg, reaching a maximal rate of -0.33%/min at 5.0 X 10(-5) M MeHg. At or above 1.0 X 10(-4) M MeHg, a mercury-protein aggregate formed in both the polymerization and depolymerization systems. Fifteen free sulfhydryl groups per tubulin dimer were determined, and MeHg bound to all 15. When MeHg bound to only 2 free sulfhydryl groups per dimer, it inhibited polymerization. MeHg bound to free sulfhydryl groups exposed uniquely on the surface of microtubules, as well as those free sulfhydryl groups exposed on the ends. These results show MeHg in vitro to be a potent microtubule assembly inhibitor at ratios stoichiometric with the tubulin dimer. The effects of MeHg on microtubules are presumably mediated through MeHg binding to free sulfhydryl groups both on the ends and on the surface of microtubules. The presence of binding sites (free sulfhydryl groups) on the microtubule surface suggests multiple classes of binding sites for MeHg.

Animals↗
PubMed1985

Generation of microtubule stability subclasses by microtubule-associated proteins: implications for the microtubule "dynamic instability" model.

We have developed a method to distinguish microtubule associated protein (MAP)-containing regions from MAP-free regions within a microtubule, or within microtubule sub-populations. In this method, we measure the MAP-dependent stabilization of microtubule regions to dilution-induced disassembly of the polymer. The appropriate microtubule regions are identified by assembly in the presence of [3H]GTP, and assayed by filter trapping and quantitation of microtubule regions that contain label. We find that MAPs bind very rapidly to polymer binding sites and that they do not exchange from these sites measurably once bound. Also, very low concentrations of MAPs yield measurable stabilization of local microtubule regions. Unlike the stable tubule only polypeptide (STOP) proteins, MAPs do not exhibit any sliding behavior under our assay conditions. These results predict the presence of different stability subclasses of microtubules when MAPs are present in less than saturating amounts. The data can readily account for the observed "dynamic instability" of microtubules through unequal MAP distributions. Further, we report that MAP dependent stabilization is quantitatively reversed by MAP phosphorylation, but that calmodulin, in large excess, has no specific influence on MAP protein activity when MAPs are on microtubules.

Animals↗
PubMed1985

Kinetochore components recognized by human autoantibodies are present on mononucleosomes.

We have developed a competitive enzyme-linked immunosorbent assay for solubilized kinetochore components, using human CREST (calcinosis, Raynaud's phenomenon, esophageal dysfunction, sclerodactyly, telangiectasia) scleroderma autoimmune antibodies specific for these kinetochore elements. Using this quantitative assay, we found interphase persistent or "pre-kinetochore" components in low- and moderately high-salt (375 mM salt) extracts of micrococcal nuclease-digested rat liver and chicken erythrocyte nuclei. The release of antigen activity from nuclei under these conditions has been correlated with loss of pre-kinetochore foci as determined by immunofluorescence microscopy. Combined biochemical and competition assay analysis of chicken erythrocyte nuclear extracts indicates that pre-kinetochore components are tightly bound to chromatin of mononucleosome size. The conclusions based on competition assay data are supported by a direct binding assay, which confirms that antigens recognized by CREST sera are present on chromatin. These results raise the possibility that the kinetochore-specific chromosomal antigen(s) we have detected substitutes for "standard" mononucleosome components, such as histone H1. Furthermore, they suggest approaches to the isolation of kinetochore-specific DNA sequences from higher eucaryotes.

Animals↗
PubMed1984

Sliding of STOP proteins on microtubules.

Microtubules are stabilized against cold temperature disassembly by 145-kilodalton proteins [stable tubule only polypeptides (STOPs)] that block the end-wise dissociation of subunits from the polymers. We describe here several kinetic parameters of the interaction of STOPs with microtubules. STOPs will bind to microtubules either during assembly of the polymer or at steady state. The addition appears random on the polymers and does not require the mediation of tubulin subunits. Tubulin subunits compete with microtubules for STOP binding, but binding to the polymers is apparently irreversible. We demonstrate that STOPs do not exchange measurably between polymers at steady state. Nonetheless, a displacement of STOPs within a single polymer is readily demonstrable. We have determined that the displacement is apparently due to a surface translocation, or "sliding", of STOPs on microtubules.

Animals↗
PubMed1984

Asymmetrical cerebrovascular response to right and left hemisphere cortical suction lesions in the rat.

We have previously demonstrated that focal lesions of the right but not left cerebral cortex in the rat produce hyperactivity and depletions of norepinephrine. To assess the cerebrovascular response to suction lesions, postoperative rats were infused intravenously with [14C]inulin. The brain inulin concentration in the right lesion group was significantly higher than control (no surgery) or left lesion groups in all cortical quandrants, but most prominently in the quadrant posterior and contralateral to the lesion site.

Animals↗
PubMed1984

Isolation from bovine brain of a superstable microtubule subpopulation with microtubule seeding activity.

Cold-stable microtubule protein isolated from beef brain is capable of seeding microtubule assembly under conditions that prevent the initiation of self-assembly of cold-labile microtubules. We have developed a quantitative assay for the determination of seeding activity. Using this assay, we find that seeding activity is apparently due to microtubule fragments that resist -80 degrees C, a condition that causes the depolymerization of cold-stable microtubules ("cold stability" is defined as resistance to 0 degree C disassembly), but rapidly depolymerize when exposed to 3.0 mM free calcium, to micromolar Ca2+-calmodulin, or to 0.2 M NaCl at 4 degrees C. After salt treatment, seeding activity is permanently lost although microtubule cold stability is retained through further assembly cycles. Similarly, after sedimentation of microtubule seeds the supernatant protein assembles into cold-stable microtubules, which are permanently devoid of seeding activity. By contrast, seeding activity can be recovered by recycling of supernatant protein from preparations exposed to 3.0 mM calcium or to Ca2+-calmodulin prior to centrifugation, indicating the solubilization of an active component (designated "preseeds") under these conditions. Polyacrylamide gels show some differences in polypeptides between seeding and non-seeding cold-stable microtubule preparations. Approximately 35% of the microtubule population assembled from beef brain crude extract is cold stable, while approximately 2% constitutes -80 degrees C resistant seeds. The formation of seeds from seed-forming subunits (preseeds) occurs rapidly, is apparently a cooperative phenomenon, and occurs on preexisting microtubules under either assembly initiating or steady-state conditions.

Animals↗
PubMed1984

Taxol-induced bundling of brain-derived microtubules.

Taxol has two obvious effects in cells. It stabilizes microtubules and it induces microtubule bundling. We have duplicated the microtubule-bundling effect of taxol in vitro and report preliminary characterization of this bundling using electron microscopy, sedimentation, and electrophoretic analyses. Taxol-bundled microtubules from rat brain crude extracts were seen as massive bundles by electron microscopy. Bundled microtubules sedimented through sucrose five times faster than control microtubules. Electrophoretic analysis of control and taxol-bundled microtubules pelleted through sucrose revealed no striking differences between the two samples except for a protein doublet of approximately 100,000 daltons. Taxol-induced microtubule bundling was not produced by using pure tubulin or recycled microtubule protein; this suggested that taxol-induced microtubule bundling was mediated by a factor present in rat brain crude extracts. Taxol cross-linked rat brain crude extract microtubules were entirely labile to ATP in the millimolar range. This ATP-dependent relaxation was also demonstrated in a more purified system, using taxol-bundled microtubules pelleted through sucrose and gently resuspended. Although the bundling factor did not recycle with microtubule protein, it was apparently retained on isolated taxol-stabilized microtubules. The bundling factor was salt extracted from taxol-stabilized microtubules and its retained activity was demonstrated in an add-back experiment with assembled phosphocellulose-purified tubulin.

Adenosine Triphosphate↗
PubMed1983

Regulation of microtubule cold stability by calmodulin-dependent and -independent phosphorylation.

Cold-labile microtubule protein can be rendered cold-stable by addition of a fraction containing a small number of polypeptides that are derived from cold-stable microtubules. These polypeptides can be obtained from purified cold-stable microtubules by passage through a DEAE-cellulose (DE-52) ion exchange column from which they emerge in the first eluate fraction. The stabilizing activity of these proteins is abolished by phosphorylation catalyzed by two types of protein kinases, one dependent on calmodulin and the other independent of that regulatory protein. The calmodulin-dependent reaction appears to phosphorylate mainly two polypeptides, 56 and 72 kilodaltons; the reaction is blocked by trifluoperazine. The calmodulin-independent reaction appears to phosphorylate different cold-stable microtubule-associated proteins. That reaction is observed only in purified material obtained from vigorously homogenized brain tissue. Gently homogenization yields cold-stable microtubules that are responsive only to the calmodulin-dependent protein kinase. A distinguishing feature of the calmodulin-independent reaction is that it does not occur on polypeptides while they are bound to the microtubules.

Animals↗
PubMed1983

Purification and characterization of sheep brain cold-stable microtubules.

The isolation of cold-stable microtubules in high yields, described previously only from rodents, was extended to the brain of higher animals. Under optimal conditions, yields of 30 mg of cold-stable microtubles per 100 g of sheep brain could be obtained routinely. Material purified by two polymerization cycles displayed the same stability to cold temperature or to millimolar concentrations of calcium and the same lability to calmodulin and to ATP as did the purified material obtained from the rat [Job, D., Rauch, C.T., Fischer, E.H. & Margolis, R.L. (1982) Biochemistry 21, 509]. Furthermore, DE-52 chromatography of this material yielded a fraction that restored cold stability when added to cold-labile microtubules. Known to bind to calmodulin and to enhance microtubule assembly, tau proteins had no cold-stabilizing activity. Protein profiles of the cold-stabilizing fraction from sheep and rat brain were similar to one another but showed no protein bands corresponding to the tau proteins.

Animals↗