A Functional Mitotic Spindle Prepared from Mammalian Cells in Culture

Cande, W. Zacheus; Snyder, Judith A.; Smith, Dayle M. A.; Summers, Keith; McIntosh, J. Richard

doi:10.1073/pnas.71.4.1559

Cited by 87 publications

(56 citation statements)

References 24 publications

(18 reference statements)

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“…This has also been shown in recent studies in which brain tubulin was assembled onto the isolated, microtubule-depleted mitotic apparatuses of marine eggs (17,18) and HeLa cells (19) as well as onto the isolated flagellar basal bodies of Chlamydomonas (20).…”

Section: Discussionmentioning

confidence: 68%

Assembly of chick brain tubulin onto flagellar microtubules from Chlamydomonas and sea urchin sperm.

Binder¹,

Dentler²,

Rosenbaum³

1975

Proc. Natl. Acad. Sci. U.S.A.

105

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Flagellar microtubules from Chlamydomonas and sea urchin sperm were used as in vitro assembly sites for chick brain tubulin. Brain microtubules assembled onto the A-tubules, central tubules, and, to a limited extent, onto the B-tubules of flagellar axonemes. Assembly occurred onto the distal ends of the axonemes at low tubulin concentrations and onto distal and proximal ends at high tubulin concentrations; however, the rate of assembly onto the distal end was always greater. The rate of neurotubule assembly onto axonemes was shown to be dependent upon tubulin concentration and a forward rate constant for assembly was determined.It has been possible to study a variety of aspects of microtubule protein synthesis and assembly in vivo by the use of systems of flagellar regeneration in flagellated protozoans (1,2). Among these studies were those which demonstrated by use of light and electron microscopic autoradiography that the flagellum, and specifically the flagellar microtubules, assembled by the addition of subunits to their distal ends during flagellar elongation (tip growth) (1-3). Additional studies on the mechanisms controlling the directionality, rate, and initiation of flagellar microtubule assembly would be facilitated if means were available to assemble flagellar microtubules in vitro. As yet, this has not been possible. However, it has been possible to assemble brain microtubules (neurotubules) in vitro (4) and, because of the chemical similarity of tubulin isolated from a variety of organisms (5), studies were initiated on the assembly of brain tubulin subunits onto flagellar microtubules (axonemes) from Chlamydomonas and sea urchin sperm. MATERIALS AND METHODSAxoneme Isolation. Chlamydomonas reinhardi (strain 21 gr) was grown to a cell density of 1 X 106 cells per ml in Medium I of Sager and Granick (6) at 250. Growth was with continuous aeration on a cycle of 13 hr of light and 11 hr of dark. The cells were harvested by centrifugation and washed once in 10 mM Tris HCl buffer, pH 7.8, at 25°. All subsequent steps were carried out at 250. The cells were then resuspended in either 0.5% Triton X-100 or 0.5% Nonidet P-40 (Shell), 1 mM ATP, 2 mM MgSO4, 1 mM EDTA, 10 mM .Tris, pH 7.8, or in 0.04% Nonidet P40, 5 mM MgSO4, 0.35 mM dithiothreitol, 0.5 mM EDTA, and 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), pH 7.5. Treatment with either solution caused simultaneous flagellar detachment and demembranation. Five milliliters of the suspension of cells and axonemes were underlayed with 3 ml of 10% sucrose in 10 mM Tris, pH 7.8, and centrifuged on an Abbreviation: Pipes, piperazine-N-N'-bis(2-ethanesulfonic acid).IEC model HN centrifuge at 350 X g for 3 min, sedimenting the cells and leaving the axonemes above the sucrose. The layer above the sucrose was drawn off and centrifuged at 35,000 X g for 20 min to sediment the axonemes, which were resuspended in PM [1 mM GTP, 2 mM ethyleneglycol-bis-(P-aminoethylether)-N-N'-tetraacetic acid (EGTA), 1 mM MgSO4, 100 mM piperazine-N-N'-bis(2-et...

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Section: Discussionmentioning

confidence: 68%

Assembly of chick brain tubulin onto flagellar microtubules from Chlamydomonas and sea urchin sperm.

Binder¹,

Dentler²,

Rosenbaum³

1975

Proc. Natl. Acad. Sci. U.S.A.

105

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“…We refer to these bands collectively as high-molecular-weight components (HMW). Bands of high molecular weight have also been observed by others using a similar assembly procedure (11)(12)(13), an assembly procedure using glycerol (14), and in purified tubule preparations stabilized in hexylene glycol (15). Previous results showed that the HMW components copurified in constant stoichiometry with tubulin through repeated cycles of assembly-disassembly (9,10,14).…”

mentioning

confidence: 56%

Association of high-molecular-weight proteins with microtubules and their role in microtubule assembly in vitro.

Murphy¹,

Borisy²

1975

Proc. Natl. Acad. Sci. U.S.A.

491

232

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High-molecular-weight components (HMW) specifically associated with microtubule protein purified from porcine brain tissue were separated from tubulin by DEAE-Sephadex ion exchange chromatography. Analysis by viscometry, sedimentation, and electron microscopy of the unfractionated microtubule protein, separated HMW and tubulin fractions, and reconstituted mixtures showed that HMW promoted formation of ring structures at 50 and tubule polymerization at 370. The HMW reassociated with tubulin and was identified in thin sections as 18.9 X 5.6 nm projections attached to the microtubules with a longitudinal periodicity of 32.5 nm. These studies: (1) indicate that the HMW fraction stimulates microtubule assembly by facilitating the formation of ring structures which are apparently intermediates in polymerization, and (2) demonstrate that the HMW associates with microtubules as a structural component projecting from the surface of the microtubule wall.Cytoplasmic microtubules play important roles in cell elongation and motile processes such as mitosis and intracellular transport (1). For some of these functions microtubule-associated elements such as crossbridges have been postulated (2, 3), and structural components attached to microtubules have been observed in neurons (4), myoblasts (5), and the mitotic spindle (6, 7). Although a microtubule sidearm which produces the force for axonemal motility has been isolated from cilia and flagella (8), in no case have the structural components observed on cytoplasmic microtubules been characterized biochemically or had their exact functions defined.Microtubule-associated proteins have been observed in preparations of purified microtubule protein obtained from brain tissue by an in vitro assembly procedure (9, 10). These components account for 15-20% of the total purified material and are typically resolved on 5% acrylamide gels as a pair of closely spaced bands (286,000 and 271,000 MW) together with a minor band of higher molecular weight (345,000 MW). We refer to these bands collectively as high-molecular-weight components (HMW). Bands of high molecular weight have also been observed by others using a similar assembly procedure (11-13), an assembly procedure using glycerol (14), and in purified tubule preparations stabilized in hexylene glycol (15). Previous results showed that the HMW components copurified in constant stoichiometry with tubulin through repeated cycles of assembly-disassembly (9,10,14). Agents that inhibited microtubule assembly also inhibited the sedimentation of the HMW under conditions that sedimented microtubules but not the unpolymerized material (10). Therefore, we concluded that these components were not contaminants but rather were specifically associated with microtubules. The HMW components were therefore further investigated to determine the nature of their association with microtubules and their effect on tubule assembly in vitro. MATERIALS AND METHODSPreparation of HMW and Tubulin Fractions. Purified microtubule protein was obtained from p...

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“…Using 0.05% Triton models of mitotic Pt K, cell, CANDE et al (1974) have shown that anaphase chromosomes separate without addition of ATP. In another Triton model prepared in the presence of 2% Carbowax, 0.1-2.5 mM ATP or GTP, and none of exogenous tubulin, early anaphase chromosomes were kept on moving in a normal fashion.…”

Section: Discussionmentioning

confidence: 99%

The Glycerol-Isolated Mitotic Apparatus: A Response to Porcine Brain Tubulin and Induction of Chromosome Motion

1975

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The birefringence of the MAS or spindles isolated from sea urchin eggs with the 1 M glycerol-isolation medium was stabilized when more than 0.5 mg/ml tubulin was contained in the medium. The addition of glycerol up to a final concentration of of 4 w strongly stabilized the MAS even in the absence of GTP and tubulin. The birefringence of the spindle and asters was not reduced even for the periods of several hours. The incorporation of heterogeneous tubulin into the isolated anaphase MAS was demonstrated by augmentation of the birefringence at the interzonal region as well as half spindles accompanied by enlargement of spindle and asters. In the anaphase MAS isolated in the absence of brain tubulin, chromosomes moved a short distance toward the poles upon addition of ATP, Mga and 0.5 mg/ml tubulin. When the MAS were isolated in the presence of 0.5 mg/ml tubulin, the chromosomes moved in a more regular fashion t o half the way to the poles accompanied by an increase in spindle length by 10 t o 15%. GTP could not be substituted for ATP for inducing the motion. The chromosome motion of the isolated anaphase spindle was less significant than that of the isolated MA. Increasing tubulin concentration to 3 mg/ml, the chromosomes in the isolated MA separated at random by an unusual growth of the spindle. The stretch of the interzonal region by incorporating heterogeneous tubulin seemed to push the chromosomes apart abnormally. It was suggested that brain tubulin in a range of 0.5 mg/ml supports a tubulin-MA microtubule equilibrium favoring more regular motion of chromosomes in vitro.It has been recently demonstrated that glycerol stabilizes reconstituted microtubules ( SHELANSKI et al, 1973) and strikingly prolongs the decay time of tubulin polymerizability (KURIYAMA & SAKAI, 1974a). The stabilizing effect of glycerol was also demonstrated in the mitotic apparatus (MA). The MAS were stable in the isolation medium containing 4 M glycerol for a period of several hours. Furthermore, MAS in the glycerol-containing medium were shown to retain tubulin having high ability to bind colchicine. (SAKAI & KURIYAMA, 1974). These facts bring forth the possibility that the isolated MA or spindle bears as a model for investigating 265

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A Functional Mitotic Spindle Prepared from Mammalian Cells in Culture

Cited by 87 publications

References 24 publications

Assembly of chick brain tubulin onto flagellar microtubules from Chlamydomonas and sea urchin sperm.

Assembly of chick brain tubulin onto flagellar microtubules from Chlamydomonas and sea urchin sperm.

Association of high-molecular-weight proteins with microtubules and their role in microtubule assembly in vitro.

The Glycerol-Isolated Mitotic Apparatus: A Response to Porcine Brain Tubulin and Induction of Chromosome Motion

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