Biomedical subjects

P Sherline

Publications and source records attributed to P Sherline.

32 records · Page 2Linked to original sources

PubMed1982

Localization of high molecular weight microtubule-associated proteins (MAP1 and MAP2) in a HeLa microtubule-organizing centre.

With a rabbit antiserum specific for high molecular weight microtubule-associated proteins (HMW/MAPs: MAP1 and MAP2), centrosomes in interphase and mitotic HeLa cells were visualized by indirect immunofluorescence. Pre-immune serum did not stain a similar organelle. Microtubules were observed to originate from the centrosomes, demonstrating their microtubule organizing (MTOC) function. The MTOC antigen(s) to which the anti-HMW/MAP antiserum reacted was resistant to Colcemid, Triton-X-100, Nonidet P-40 and Brij 35. Immunofluorescence localization of the MTOCs was blocked by pre-incubation of the antiserum with a MAP1--MAP2 preparation but not with tubulin, actin, or a 58 K intermediate filament protein.

Demecolcine↗

PubMed1981

Unpolymerized tubulin modulates the level of tubulin mRNAs.

Although numerous studies have suggested ways in which the assembly of cytoskeletal proteins can be regulated physiologically, less information has been generated on the regulation of the synthesis of these proteins. Ben-Ze'ev et al. recently suggested that the synthesis of tubulin in mouse 3T6 cells is affected by the state of assembly of microtubules. We have investigated the level at which this apparent modulation of tubulin synthesis takes place, using cloned cDNA probes for alpha- and beta-tubulin mRNAs to measure the amounts of tubulin mRNAs combined with immunoprecipitation of tubulin to monitor the rate of protein synthesis. We have found that in many, but not all, cell types tubulin synthesis decreases very rapidly in response to microtubule inhibitors that increase the monomer pool. This decline in synthesis is associated with decline in the amounts of both alpha- and beta-tubulin mRNAs. Kinetic studies of tubulin protein synthesis and RNA levels suggest that the tubulin monomer may regulate the rate of tubulin mRNA transcription. It is likely that tubulin synthesis can be shut off quickly in the cell as the result of short half-lives of the tubulin mRNAs, which may be as short as 1--2 hr. These data suggest that the cell exploits the instability of the tubulin mRNAs as a means to regulate precise levels of the monomer-tubulin pool.

Alkaloids↗

PubMed1979

Endogenous inhibitor of colchicine-tubulin binding in rat brain.

A competitive inhibitor of colchicine binding to tubulin has been found in rat brain. Most of the inhibitor is associated with microsomes but some inhibitor, with an apparent molecular weight of approximately 250,000, is found in the cytosol. Both the microsomal and cytosol inhibitors are heat- and trypsin-sensitive, indicating that a protein moiety is required for activity. The microsomes bind tubulin directly; the microsomal and cytosol fractions both inhibit microtubule assembly in vitro. The inhibitor may function in the living cell to bind and sequester non-polymerized tubulin. Regulation of tubulin attachment to microsomes could then control the concentration of cytosolic tubulin available for microtubule assembly.

Animals↗

PubMed1978

High molecular weight MAPs are part of the mitotic spindle.

We have found that the microtubule-associated proteins of high molecular weight are located in the mitotic spindle. Indirect immunofluorescence studies reveal that the pattern of distribution of these proteins is similar to that described for tubulin and corresponds to the known phases of mitosis.

Cell Line↗

PubMed1977

Immunofluorescence localization of proteins of high molecular weight along intracellular microtubules.

To help clarify the role, in cytoplasmic microtubule function, of the proteins of high molecular weight that coassemble with tubulin in vitro, a monospecific antibody against the proteins of high molecular weight was prepared from the serum of immunized rabbits by affinity chromatography. With indirect immunofluorescence we found that the protein in both cultured neuroblastoma cells and 3T3 cells in distributed in an extensive filamentous array that originates at sites near the nucleus and extends to the cell periphery or, in neuroblastoma cells, gathers into bundles which enter neurites. No filaments were seen with nonspecific antibodies from serum taken before immunization, and prior incubation of the specific antibody with purified protein of high molecular weight (but not tubulin) prevented filament visualization. The filamentous pattern in 3T3 cells was disrupted by colchicine. This evidence indicates that the proteins of high molecular weight are found in cells in association with cytoplasmic microtubules.

Cell Line↗

PubMed1977

Binding of microtubules to pituitary secretory granules and secretory granule membranes.

Microtubules assembled in vitro were bound to purified porcine pituitary secretory granules and to isolated granule membranes. The interaction between microtubules and whole secretory granules was demonstrated by alteration in the sedimentation properties of the microtubules. Incubation of secretory granules with microtubules resulted in pelleting of microtubules which increased as a function of the number of granules added. Binding was quantitated by measurement of the tubulin remaining in the supernate after centrifugation. The interaction of secretory granules and microtubules was inhibited by nucleoside triphosphates and augmented by adenosine 5'-monophosphate and adenosine. When depolymerized protein from microtubules was incubated with secretory granules, the granules did not appear to bind the soluble tubulin dimer present in these preparations. However, the high molecular weight protein associated with microtubules was adsorbed by secretory granules during the binding process. Incubation of isolated secretory granule membranes with microtubules followed by centrifugation to density equilibrium in a discontinuous sucrose density gradient caused pelleting of the membranes, which otherwise banded higher in the gradient. The visible alteration in membrane sedimentation was confirmed by measurements of the membrane-associated magnesium-ATPase activity and by a shift in radioactivity in iodinated membrane preparations. Our data suggest a role for microtubules in the intracellular movement of secretory granules; this movement is perhaps brought about by dynein-like cross bridges which link the tubulin backbone and granule surface.

Adenosine↗

PubMed1977

A sensitive method for measuring polymerized and depolymerized forms of tubulin in tissues.

A rapid method for measuring polymerized and depolymerized forms of tubulin in tissues has been developed. The procedure consists of homogenization and centrifugation of the tissue in a microtubule-stabilizing solution and depolymerization of the precipitated microtubules; polymerized and depolymerized forms of tubulin are quantitated by a colchicine-binding assay. The validity of the technique was assessed by electron microscopy and recovery studies with labeled and unlabeled preparations of polymerized and depolymerized forms of rat brain tubulin. The sensitivity of this technique allows quantitation of tubulin in 150 micrograms of tissue, wet weight. The method demonstrated that both the polymerized and depolymerized forms of tubulin were present in rat liver cells, and that in the fed state 31.3 +/-0.7% of the total tubulin pool was in the polymerized form.

Animals↗

PubMed1977

Role of the tubulin-microtubule system in lymphocyte activation.

The role of the tubulin-microtubule system was examined in human peripheral blood leukocytes after activation with phytohemagglutinin (PHA). Soluble tubulin and microtubules were measured with a [(3)H]colchicine-binding assay. It was found that the tubulin content of PHA-activated lymphocytes was consistently increased relative to total protein content after 36 h of culture. There was no increase in the proportion of total tubulin synthesis which was present as microtubules at 36 h. Nevertheless, as a result of increased tubulin synthesis, there was a two-to three-fold increase in total microtubular mass. Colchicine, which disrupts microtubles, was used to assess the role of microtubule assembly in the sequence of events which follow lymphocyte activation, namely lymphokine release, protein synthesis, RNA synthesis, and DNA synthesis. Colchicine consistently inhibited DNA synthesis but did not inhibit release of the lymphokine, osteoclast activating factor (OAF). Protein and RNA syntheses were inhibited much less than DNA synthesis. The fact that some effects of PHA on lymphocytes appear to require intact microtubules and at least one does not suggest that the microtubule dependent step in PHA-stimulated lymphocyte activation occurs at a stage after propagation of the signal from the membrane to the cell interior.

Blood Proteins↗

PubMed1975

Binding of colchicine to purified microtubule protein.

The binding of colchicine to tubulin, purified by two cycles of assembly-disassembly, has been studied. Equilibrium studies indicated a dissociation constant which declined during incubation approaching a minimum value of approximately 0.30 times 10- minus 6 M after 13 hours of incubation. Because tubulin is unstable during prolonged incubation (t1/2 of 5.2 hours for free tubulin, t1/2 of 12.5 hours for tubulin bound to colchicine), the equilibrium Kd was felt to be an overestimation of the true Kd. The rate constant of dissociation (k-1 equal to 0.009 hour- minus 1 hour- minus 1) and the rate constant of association (k1 equal to 0.37 times 10-6 M-minus 1) were measured under conditions designed to circumvent or correct for tubulin instability. The dissociation constant determined by the ratio k-1/k1 was 0.024 times -minus 6 M. To determine whether the discrepancy between the "equilibrium" and "kinetic" determined dissociation constants could be accounted for on the basis of tubulin instability, the binding reaction was computer-simulated using the measured association and dissociation rate constants and the rate constants for decay of bound and free tubulin. Computer simulation was in close agreement with the experimentally determined behavior of the reaction during a 13-hour incubation. It is concluded that the Kd determined by equilibrium methodology results in a considerable overestimation due to the instability of tubulin, and that the best estimate for the Kd of the colchicine-tubulin equilibrium is the value determined by the ratio of the rate constants.

Animals↗

PubMed1974