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A Roobol

Publications and source records attributed to A Roobol.

At least 19 recordsLinked to original sources
PubMed1999

Disassembly of the cytosolic chaperonin in mammalian cell extracts at intracellular levels of K+ and ATP.

The eukaryotic, cytoplasmic chaperonin, CCT, is essential for the biogenesis of actin- and tubulin-based cytoskeletal structures. CCT purifies as a doubly toroidal particle containing two eight-membered rings of approximately 60-kDa ATPase subunits, each encoded by an essential and highly conserved gene. However, immunofluorescence detection with subunit-specific antibodies has indicated that in cells CCT subunits do not always co-localize. We report here that CCT ATPase activity is highly dependent on K+ ion concentration and that in cell extracts, at physiological levels of K+ and ATP, there is considerable dissociation of CCT to a smaller oligomeric structure and free subunits. This dissociation is consequent to ATP hydrolysis and is readily reversed on removal of ATP. The ranking order for ease with which subunits can exit the chaperonin particle correlates well with the length of a loop structure, identified by homology modeling, in the intermediate domain of CCT subunits. K+-ATP-induced disassembly is not an intrinsic property of purified CCT over a 40-fold concentration range and requires the presence of additional factor(s) present in cell extracts.

Adenosine Triphosphate↗
PubMed1999

Selected subunits of the cytosolic chaperonin associate with microtubules assembled in vitro.

The molecular chaperone activities of the only known chaperonin in the eukaryotic cytosol (cytosolic chaperonin containing T-complex polypeptide 1 (CCT)) appear to be relatively specialized; the main folding substrates in vivo and in vitro are identified as tubulins and actins. CCT is unique among chaperonins in the complexity of its hetero-oligomeric structure, containing eight different, although related, gene products. In addition to their known ability to bind to and promote correct folding of newly synthesized and denatured tubulins, we show here that CCT subunits alpha, gamma, zeta, and theta also associated with in vitro assembled microtubules, i.e. behaved as microtubule-associated proteins. This nucleotide-dependent association between microtubules and CCT polypeptides (Kd approximately 0.1 microM CCT subunit) did not appear to involve whole oligomeric chaperonin particles, but rather free CCT subunits. Removal of the tubulin COOH termini by subtilisin digestion caused all eight CCT subunits to associate with the microtubule polymer, thus highlighting the non-chaperonin nature of the selective CCT subunit association with normal microtubules.

Animals↗
PubMed1999

Subunits of the eukaryotic cytosolic chaperonin CCT do not always behave as components of a uniform hetero-oligomeric particle.

The chaperonin CCT is an hetero-oligomeric molecular chaperone complex. Studies in yeast suggest each of its eight gene products are required for its major identified functions in producing native tubulins and actins. However, it is unclear whether these eight components always form a single particle, covering all functions, or else can also exist as heterogeneous mixtures and/or free subunits in cells. Using mouse P19 embryonal carcinoma cells, which divide rapidly, yet in retinoic acid adopt a neuronal phenotype, admixed with occasional (approximately 10%) fibroblast-like cells, together with a panel of peptide-specific antibodies raised to 7 of the 8 CCT subunits we show that; (1) adoption of a post mitotic phenotype is accompanied by reduced CCT protein expression, significantly more so for CCTbeta, CCTdelta, CCTepsilon, and CCTtheta than for CCTalpha (TCP-1), CCTgamma and CCTzeta; (2) CCTalpha is detected preferentially over other subunits in neurites of P19 neurons; (3) small amounts of CCTalpha and gamma are localised in nuclei (i.e. are not exclusively cytoplasmic), selectively so compared with other subunits; (4) numerous cytosolic foci exist in the cytoplasm which, when detected by double immunofluorescence can contain only one of the subunits probed for; (5) while a "core" chaperonin particle can be immunoprecipitated under native conditions, epitope access is modified both by nucleotides and by non-CCT co-precipitating proteins. Collectively, these findings indicate that CCT subunits are not only components of the hetero-oligomeric chaperonin particle but exist as significant populations of free subunits or smaller oligomers in cells.

Animals↗
PubMed1995

Cytoplasmic chaperonin complexes enter neurites developing in vitro and differ in subunit composition within single cells.

Chaperonins containing t-complex polypeptide-1 (CCT) are cytosolic molecular chaperone particles implicated especially in the biogenesis of cytoskeletal proteins by promoting the correct folding of the major ubiquitous cytoskeletal components, tubulin and actin. We have purified cytosolic chaperonins from the ND7/23 cell line, determined their subunit composition and examined changes in the intracellular locations of their components during differentiation of ND7/23 cells to a neuronal phenotype by using immunocytochemistry and immunoblots. Chaperonins containing the CCT alpha (TCP1) subunit enter neuritic processes and are particularly noticeable at the leading edge of growth cone-like structures where they co-localise with actin. Chaperonins containing three other components (CCT beta, epsilon and gamma), however, remain predominantly restricted to perikaryal cytoplasm. These findings suggest a heterogeneous population of chaperonin particles within single differentiated ND7/23 cells and this may reflect specialisation of chaperonin function in different cytoplasmic compartments of a neurone. Further, since ribosomes do not enter neurites while CCT alpha-containing chaperonins do, the latter may play roles, subsequent to translation, which influence cytoskeletal elaboration during neuritogenesis.

Actins↗
PubMed1993

Identification of chaperonin particles in mammalian brain cytosol and of T-complex polypeptide 1 as one of their components.

An approximately 950-kDa heteromeric particle was purified from guinea-pig and rat brain by sucrose gradient fractionation of post-mitochondrial supernatants. Further purification, by affinity chromatography on ATP-Sepharose and anion exchange FPLC on MonoQ, yielded a particle with typical chaperonin ultrastructure. One of the component polypeptides was recognized by a monoclonal antibody to murine T-complex polypeptide 1. Brain cytosolic chaperonin particles formed a binary complex with unfolded tubulin subunits. The polypeptide compositions of the cytosolic chaperonin particles appeared very similar between brain and testicular tissues of the same animal, but differed subtly between the guinea-pig and rat.

Animals↗
PubMed1987

Patterns of tubulin isotype synthesis and usage during mitotic spindle morphogenesis in Physarum.

Tubulin synthesis in the naturally synchronous plasmodium of Physarum polycephalum is a markedly periodic event restricted to the late G2 period of the cell cycle. Mitosis in the plasmodium is intranuclear, and there are no cytoplasmic microtubules at any stage of the cell cycle. We have combined a biochemical investigation of the synthesis of the plasmodial tubulin isotypes and their participation in the mitotic spindle with a microscopic study (immunofluorescence) of the development of spindle microtubules throughout the cell cycle. We have shown that all four tubulin isotypes identified in the plasmodium (alpha 1, alpha 2, beta 1 and beta 2) are present in the mitotic spindle. The stoichiometry of isotype usage in the mitotic spindle generally reflects the overall abundance of isotypes in the plasmodium as a whole: beta 2 greater than alpha 1 greater than alpha 2 greater than beta 1. We have also shown that tubulins synthesized in the G2 period of one cell cycle can be incorporated into the spindles of the immediately ensuing mitosis and have sufficient biological longevity to allow participation in the mitotic divisions of future cell cycles. Thus, the phenomenon of periodic tubulin synthesis does not reflect a restricted use of tubulin to the cell cycle in which it was synthesized. The major polymerization of tubulin in the nucleus occurred less than 30 min before metaphase. A novel tubulin-containing structure was, however, present in the nucleus approximately 60 min before metaphase. Polymerized tubulin is rapidly removed from the nucleus following nucleokinesis.

Cell Cycle↗
PubMed1984

Identification of tubulin isoforms in the plasmodium of Physarum polycephalum by in vitro microtubule assembly.

The tubulins of the plasmodium of Physarum polycephalum have been identified by in vitro microtubule assembly from partially purified extracts of asynchronous microplasmodia and late G2 macroplasmodia. The plasmodial tubulin group comprised of 2 alpha tubulins (app. m.w. 51000 daltons) and 2 beta tubulins (app. m.w. 58000 daltons and 55000 daltons) and appeared to be identical with a group of polypeptides which are synthesized periodically in late G2. Two of the plasmodial tubulin subunits (one alpha and one beta) were identical to the Physarum amoebal tubulin alpha and beta subunits as characterised by 2D gel positions.

Electrophoresis, Polyacrylamide Gel↗
PubMed1984

The isolation of nuclei containing mitotic spindles from the plasmodium of the slime mould Physarum polycephalum.

We have developed a protocol which facilitates the isolation of nuclei containing mitotic spindles from the plasmodium of Physarum polycephalum. The nuclei are isolated in a spindle stabilization buffer (SSB) consisting of -4 M glycerol, 5 mM EGTA, 10 mM MgCl2, 10 mM PIPES and 0.1% (v/v) Triton X-100 at pH 6.5. Isolation at 20 degrees C, chelation of Ca2+ and the inclusion of the 4 M glycerol were found to be essential for stabilization of the spindle microtubules. The method allows the isolation of clean nuclei in a reasonable yield. Nuclei were isolated at each of the main stages of mitosis (prophase, metaphase and anaphase) and transmission electron microscopy of the isolated nuclei revealed well preserved arrangements of spindle microtubules which were characteristic of the particular stage of mitosis.

Cell Nucleus↗
PubMed1982

Microtubule nucleation by the isolated microtubule-organizing centre of Physarum polycephalum myxamoebae.

The nucleus--centrosome complex from Physarum polycephalum myxamoebae has been purified. The complex contained the centriole pair and pericentriolar material in association with the nucleus. Apart from some unusually stable microtubules, which appeared to be involved in maintaining the nucleus-centrosome association, endogenous microtubule arrays had been stripped from the complex during isolation. When the nucleus--centrosome complex was incubated with purified brain or myxamoebal tubulins the growth of 45-70 microtubules was initiated onto the pericentriolar material. The number and length of the nucleated microtubules was proportional to the tubulin concentration. Pretreatment of the nucleus--centrosome complex with DNase 1, RNase A, antitubulin antibody and anticentriolar antibody did not affect pericentriolar nucleation capacity, although pretreatment with DNase 1 did expose perinuclear nucleation sites that had a much lower minimal tubulin concentration for assembly than the pericentriolar site. After pretreatment with trypsin pericentriolar material and nucleation were destroyed, and microtubule elongation occurred directly onto the centriole microtubules.

Cell Nucleus↗
PubMed1981

A correlation between in vivo and in vitro effects of the microtubule inhibitors colchicine, parbendazole and nocodazole on myxamoebae of Physarum polycephalum.

The effects of the microtubule inhibitors colchicine, parbendazole and nocodazole on the growth of myxamoebae of Physarum polycephalum were closely paralleled by the effects of these drugs on the assembly in vitro of purified amoebal microtubule protein. Colchicine at 100 microM did not inhibit amoebal growth and did not inhibit formation, or depolymerization, of amoebal microtubules. The benzimidazole carbamate derivatives nocodazole and parbendazole were very effective in both inhibiting growth and inhibiting the assembly in vitro of amoebal microtubule protein. Parbendazole was the most effective.

Benzimidazoles↗
PubMed1980

Identification and characterization of microtubule proteins from myxamoebae of Physarum polycephalum.

Cell extracts of myxamoebae of Physarum polycephalum have been prepared in such a way that they do not inhibit assembly of brain microtubule protein in vitro even at high extract-protein concentration. Co-polymers of these extracts and brain tubulin have been purified to constant stoichiometry and amoebal components identified by radiolabelling. Amoebal tubulin has been identified as having an alpha-subunit, mol.wt. 54 000, which co-migrates with brain alpha-tubulin and a beta-subunit, mol.wt. 50 000, which co-migrates with Tetrahymena ciliary beta-tubulin. Non-tubulin amoebal proteins that co-purify with tubulin during co-polymer formation have been shown to be essential for microtubule formation in the absence of glycerol and appear to be rather more effective than brain microtubule-associated proteins in stimulating assembly. The mitotic inhibitor griseofulvin (7-chloro-2',4,6-trimethoxy-6'-methylspiro[benzofuran-2(3H),1'-cyclohex-2'-ene] -3,4'-dione), which binds to brain microtubule-associated proteins and inhibits brain microtubule assembly in vitro, affected co-polymer microtubule protein in a similar way, but to a slightly greater extent.

Animals↗
PubMed1977

Griseofulvin-induced aggregation of microtubule protein.

Griseofulvin (7-chloro-2',4,6-trimethoxy-6'-methylspiro[benzofuran-2(3H),1'-[2]cyclohexene]-3,4'-dione) induces aggregation of microtubule protein at 0 degrees C. This aggregate contains approx. 90% of the microtubule-associated proteins originally present in the microtubule protein. The supernatant obtained after removal of the griseofulvin-induced aggregate does not form microtubules on warming at 37 degrees C. Addition of the griseofulvin-aggregated protein to this supernatant and warming to 37 degrees C gives rise to a limited amount of microtubule assembly. The possible involvement of griseofulvin-induced aggregation of microtubule protein at 0 degrees C in the inhibition by griseofulvin of microtubule assembly in vitro is discussed.

Animals↗