Calcium-labile mitotic spindles isolated from sea urchin eggs (Lytechinus variegatus)

We isolated calcium-labile mitotic spindles from eggs of the sea urchin Lytechinus variegatus, using a low ionic strength, EGTA lysis buffer that contined 5.0 mM EGTA, 0.5 mM MgCl2, 10-50 mM PIPES, pH 6.8, with 1% Nonidet P-40 (detergent) and 20-25% glycerol. Isolated spindles were stored in EGTA buffer with 50% glycerol for 5-6 wk without deterioration. The isolated spindles were composed primarily of microtubules with the chromosomes attached. No membranes were seen. Isolated spindles, perfused with EGTA buffer to remove the detergent and glycerol, had essentially the same birefringent retardation (BR) as spindles in vivo at the same mitotic stage. Even in the absence of glycerol and exogenous tubulin, the isolated spindles were relatively stable in the EGTA buffer: BR decayed slowly to about half the initial value within 30-45 min. However, both the rate and extent of BR decay increased with concentrations of Ca2+ above 0.2-0.5 muM as assayed using Ca-EGTA buffers (0.2 mM EGTA, 0.5 mM MgCl2, 50 mM PIPES, pH 6.8, plus various amounts of CaCl2). Microtubules depolymerized almost completely in < 6 min at Ca2+ concentrations of 2 muM and within several seconds at 10 muM Ca2+. Of several divalent cations tested, only Sr2+ caused comparable changes in BR. The absence of membranes in the isolated spindles appeared to be associated with a lack of calcium- sequestering ability. Our results suggest that calcium ions play an important role in the depolymerization of spindle microtubules and that membrane components may function within the mitotic apparatus of living cells to sequester and release calcium ions during mitosis.     

The stabilization of microtubules in isolated spindles by tubulin-colchicine complex

We have analyzed the effect of colchicine and tubulin dimer-colchicine complex (T-C) on microtubule assembly in mitotic spindles. Cold- and calcium-labile mitotic spindles were isolated from embryos of the sea urchin Lytechinus variegatus employing EGTA/glycerol stabilization buffers. Polarization microscopy and measurements of spindle birefringent retardation (BR) were used to record the kinetics of microtubule assembly-disassembly in single spindles. When isolated spindles were perfused out of glycerol stabilizing buffer into a standard in vitro microtubule reassembly buffer (0.1 M Pipes, pH 6.8, 1 mM EGTA, 0.5 mM MgCl2, and 0.5 mM GTP) lacking glycerol, spindle BR decreased with a half-time of 120 s. Colchicine at 1 mM in this buffer had no effect on the rate of spindle microtubule disassembly. Inclusion of 20 microM tubulin or microtubule protein, purified from porcine brain, in this buffer resulted in an augmentation of spindle BR. Interestingly, in the presence of 20 microM T-C, spindle BR did not increase, but was reversibly stabilized; subsequent perfusion with reassembly buffer without T-C resulted in depolymerization. This behavior is striking in contrast to the rapid depolymerization of spindle microtubules induced by colchicine and T-C in vivo. These results support the current view that colchicine does not directly promote microtubule depolymerization. Rather, it is T-C complex that alters microtubule assembly, by reversibly binding to microtubules and inhibiting elongation. In vivo, colchicine can induce depolymerization of nonkinetochore spindle microtubules within 20 s. In vitro, colchicine blocks further microtubule assembly, but does not induce rapid disassembly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynein-like Mg2+-ATPase in mitotic spindles isolated from sea urchin embryos (Strongylocentrotus droebachiensis)

Two distinctly different ATPases have been reported to be endogenous to the mitotic apparatus: a Mg2+-ATPase resembling axonemal dynein, and a Ca2+-ATPase postulated to be bound in membranes. To examine the nature of the Mg2+-ATPase, we isolated membrane-free mitotic spindles from Stronglylocentrotus droebachiensis embryos by rapidly lysing these in a calcium-chelating, low-ionic-strength buffer (5 mM EGTA, 0.5 mM MgCl2, 10 mM PIPES, pH 6.8) that contained 1% Nonidet P-40. The fibrous isolated mitotic spindles closely resembled spindles in living cells, both in general morphology and in birefringence. In electron micrographs, the spindles were composed primarily of microtubules, free from membranes and highly extracted of intermicrotubular cytoplasmic ground substance. As analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the pelleted spindles contain 18% tubulin, variable amounts of actin (2-8%), and an unidentified protein of 55 kdaltons in a constant weight ratio to tubulin (1:2.5). The isolated spindles also contained two polypeptides, larger than 300 kdaltons, that comigrated with egg dynein polypeptides, and ATPase activity (0.02 mumol Pi/mg . min) that closely resembled both flagellar and egg dynein. The spindle Mg2+-ATPase showed a ratio of Ca2+-/Mg2+-ATPase = 0.85, had minimal activity in KCl and EDTA, and cleaved GTP at 35% of the rate of ATP. The Mg2+-ATPase was insensitive to ouabain or oligomycin. The spindle Mg2+-ATPase was inhibited by sodium vanadate but, like egg dynein, was less sensitive to vanadate than flagellar dynein. The spindle Mg2+- ATPase does not resemble the mitotic Ca2+-ATPase described by others. We propose that the spindle Mg2+-ATPase is egg dynein. Bound carbohydrate on the two high-molecular-weight polypeptides of both egg dynein and the spindle enzyme suggest that these proteins may normally associate with membranes in the living cell.     

THE GLYCEROL-ISOLATED MITOTIC APPARATUS: A RESPONSE TO PORCINE BRAIN TUBULIN AND INDUCTION OF CHROMOSOME MOTION

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 M 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, Mg²⁺ 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 to half the way to the poles accompanied by an increase in spindle length by 10 to 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 .     

In vitro polymerization of microtubules into asters and spindles in homogenates of surf clam eggs

The eggs of the surf clam Spisula solidissima were artificially activated, homogenized at various times in cold 0.5 M MES buffer, 1mM EGTA at pH 6.5, and microtubule polymerization was induced by raising the temperature to 28 degrees C. In homogenates of unactivated eggs few microtubules form and no asters are observed. By 2.5 min after activation microtubules polymerize in association with a dense central cylinder, resulting in the formation of small asterlike structures. By 4.5 min after activation the asters formed in vitro contain a distinct centriole, and microtubules now radiate from a larger volume of granular material which surrounds the centriole. By 15 min (metaphase I) the granular material is more disperse and only loosely associated with the centriole. Microtubules are occasionally observed which appear to radiate directly from one end of the centriole. The organizing center can be partially isolated by centrifugation of homogenates of metaphase eggs and will induce aster formation if mixed with tubulin from either activated or unactivated eggs. Pretreatment of the eggs with colchicine does not prevent the formation of a functional organizing center. Complete spindles can also be obtained under polymerizing conditions by either homogenizing the eggs directly into warm buffer or by adding a warm high-speed supernate to spindles which have been isolated in a microtubule stabilizing medium. Extensive addition of new tubulin occurs onto the isolated spindles, resulting primarily in growth of astral fibers, although there occasionally appears to be growth of chromosomal fibers and of pole-to-pole fibers. Negatively stained aster microtubules have a strong tendency to associate side by side, and under some conditions distinct cross bridges can be observed. However, under other conditions large numbers of 300-400-A particles surround the microtubules; the presence of stain between particles can give the appearance of cross bridges.     

Myosin localization during meiosis I of crane-fly spermatocytes gives indications about its role in division

We showed previously that in crane-fly spermatocytes myosin is required for tubulin flux [Silverman-Gavrila and Forer, 2000a: J Cell Sci 113:597-609], and for normal anaphase chromosome movement and contractile ring contraction [Silverman-Gavrila and Forer, 2001: Cell Motil Cytoskeleton 50:180-197]. Neither the identity nor the distribution of myosin(s) were known. In the present work, we used immunofluorescence and confocal microscopy to study myosin during meiosis-I of crane-fly spermatocytes compared to tubulin, actin, and skeletor, a spindle matrix protein, in order to further understand how myosin might function during cell division. Antibodies to myosin II regulatory light chain and myosin II heavy chain gave similar staining patterns, both dependent on stage: myosin is associated with nuclei, asters, centrosomes, chromosomes, spindle microtubules, midbody microtubules, and contractile rings. Myosin and actin colocalization along kinetochore fibers from prometaphase to anaphase are consistent with suggestions that acto-myosin forces in these stages propel kinetochore fibres poleward and trigger tubulin flux in kinetochore fibres, contributing in this way to poleward chromosome movement. Myosin and actin colocalization at the cell equator in cytokinesis, similar to studies in other cells [e.g., Fujiwara and Pollard, 1978: J Cell Biol 77:182-195], supports a role of actin-myosin interactions in contractile ring function. Myosin and skeletor colocalization in prometaphase spindles is consistent with a role of these proteins in spindle formation. After microtubules or actin were disrupted, myosin remained in spindles and contractile rings, suggesting that the presence of myosin in these structures does not require the continued presence of microtubules or actin. BDM (2,3 butanedione, 2 monoxime) treatment that inhibits chromosome movement and cytokinesis also altered myosin distributions in anaphase spindles and contractile rings, consistent with the physiological effects, suggesting also that myosin needs to be active in order to be properly distributed.     

Evidence for microtubule subunit addition to the distal end of mitotic structures in vitro

HeLa cells blocked in metaphase with 0.04 micrograms/ml of the microtubule poison nocodazole were shown to contain large numbers of microtubules with typical mitotic organization but no cenriole. Lysis of nocodazole-poisoned cells in a microtubule reassembly buffer containing 0.5 M PIPES, 2.5% dimethyl sulfoxide, 1 mM EDTA, 1 mM MgCl2, 1 mM GTP, 1% Triton X-165, 0.5% sodium deoxycholate, 0.2% SDS, pH 6.9, preserved metaphase aster structures 5 micrograms in diameter surrounded only by a thin, fibrous cell remnant. Inclusion of 2 mg/ml porcine brain microtubule protein in the lysis buffer produced asters up to 20 micrometers in diameter with a birefringent retardation of 5-6 nm. In these large asters the central microtubules had normal morphology, but peripheral microtubules were clearly abnormal. Our interpretation is that in high PIPES lysis buffer, exogenous brain tubulin adds to the distal ends of preexisting aster microtubules to form abnormal microtubules. This observation supports the assumptions made by Borisy and by Summers and Kirschner in their interpretation of growth experiments to determine the microtubule polarity in mitotic structures.     

Identification of a new protein localized at sites of cell-substrate adhesion

Tipulid spermatocytes form normally functioning bipolar spindles after one of the centrosomes is experimentally dislocated from the nucleus in late diakinesis (Dietz, R., 1959, Z. Naturforsch., 14b:749-752; Dietz, R., 1963, Zool. Anz. Suppl., 23:131-138; Dietz, R., 1966, Heredity, 19:161-166). The possibility that dissociated pericentriolar material (PCM) is nevertheless responsible for the formation of the spindle in these cells cannot be ruled out based on live observation. In studying serial sections of complete cells and of lysed cells, it was found that centrosome-free spindle poles in the crane fly show neither pericentriolar-like material nor aster microtubules, whereas the displaced centrosomes appear complete, i.e., consist of a centriole pair, aster microtubules, and PCM. Exposure to a lysis buffer containing tubulin resulted in an increase of centrosomal asters due to aster microtubule polymerization. Aster-free spindle poles did not show any reaction, also indicating the absence of PCM at these poles. The results favor the hypothesis of chromosome-induced spindle pole formation at the onset of prometaphase and the dispensability of PCM in Pales.     

Protein 4.1R, a microtubule-associated protein involved in microtubule aster assembly in mammalian mitotic extract

Non-erythroid protein 4.1R (4.1R) consists of a complex family of isoforms. We have shown that 4.1R isoforms localize at the mitotic spindle/spindle poles and associate in a complex with the mitotic-spindle organization proteins Nuclear Mitotic Apparatus protein (NuMA), dynein, and dynactin. We addressed the mitotic function of 4.1R by investigating its association with microtubules, the main component of the mitotic spindles, and its role in mitotic aster assembly in vitro. 4.1R appears to partially co-localize with microtubules throughout the mitotic stages of the cell cycle. In vitro sedimentation assays showed that 4.1R isoforms directly interact with microtubules. Glutathione S-transferase (GST) pull-down assays using GST-4.1R fusions and mitotic cell extracts further showed that the association of 4.1R with tubulin results from both the membrane-binding domain and C-terminal domain of 4.1R. Moreover, 4.1R, but not actin, is a mitotic microtubule-associated protein; 4.1R associates with microtubules in the microtubule pellet of the mitotic asters assembled in mammalian cell-free mitotic extract. The organization of microtubules into asters depends on 4.1R in that immunodepletion of 4.1R from the extract resulted in randomly dispersed microtubules. Furthermore, adding a 135-kDa recombinant 4.1R reconstituted the mitotic asters. Finally, we demonstrated that a mitotic 4.1R isoform appears to form a complex in vivo with tubulin and NuMA in highly synchronized mitotic HeLa extracts. Our results suggest that a 135-kDa non-erythroid 4.1R is important to cell division, because it participates in the formation of mitotic spindles and spindle poles through its interaction with mitotic microtubules.     

Contribution of noncentrosomal microtubules to spindle assembly in Drosophila spermatocytes

Previous data suggested that anastral spindles, morphologically similar to those found in oocytes, can assemble in a centrosome-independent manner in cells that contain centrosomes. It is assumed that the microtubules that build these acentrosomal spindles originate over the chromatin. However, the actual processes of centrosome-independent microtubule nucleation, polymerisation, and sorting have not been documented in centrosome-containing cells. We have identified two experimental conditions in which centrosomes are kept close to the plasma membrane, away from the nuclear region, throughout meiosis I in Drosophila spermatocytes. Time-lapse confocal microscopy of these cells labelled with fluorescent chimeras reveals centrosome-independent microtubule nucleation, growth, and sorting into a bipolar spindle array over the nuclear region, away from the asters. The onset of noncentrosomal microtubule nucleation is significantly delayed with respect to nuclear envelope breakdown and coincides with the end of chromosome condensation. It takes place in foci that are close to the membranes that ensheath the nuclear region, not over the condensed chromosomes. Metaphase plates are formed in these spindles, and, in a fraction of them, some degree of polewards chromosome segregation takes place. In these cells that contain both membrane-bound asters and an anastral spindle, the orientation of the cytokinesis furrow correlates with the position of the asters and is independent of the orientation of the spindle. We conclude that the fenestrated nuclear envelope may significantly contribute to the normal process of spindle assembly in Drosophila spermatocytes. We also conclude that the anastral spindles that we have observed are not likely to provide a robust back-up able to ensure successful cell division. We propose that these anastral microtubule arrays could be a constitutive component of wild-type spindles, normally masked by the abundance of centrosome-derived microtubules and revealed when asters are kept away. These observations are consistent with a model in which centrosomal and noncentrosomal microtubules contribute to the assembly and are required for the robustness of the cell division spindle in cells that contain centrosomes.     

Spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching

The rate of exchange of tubulin that is incorporated into spindle microtubules with dimeric tubulin in the cytoplasm has been measured in sea urchin eggs by studying fluorescence redistribution after photobleaching (FRAP). Dichlorotriazinyl amino fluorescein (DTAF) has been used to label bovine brain tubulin. DTAF-tubulin has been injected into fertilized eggs of Lytechinus variegatus and allowed to equilibrate with the endogenous tubulin pool. Fluorescent spindles formed at the same time that spindles were seen in control eggs, and the injected embryos proceeded through many cycles of division on schedule, suggesting that DTAF-tubulin is a good analogue of tubulin in vivo. A microbeam of argon laser light has been used to bleach parts of the fluorescent spindles, and FRAP has been recorded with a sensitive video camera. Laser bleaching did not affect spindle structure, as seen with polarization optics, nor spindle function, as seen by rate of progress through mitosis, even when one spindle was bleached several times in a single cell cycle. Video image analysis has been used to measure the rate of FRAP and to obtain a low resolution view of the fluorescence redistribution process. The half-time for spindle FRAP is approximately 19 s, even when an entire half-spindle is bleached. Complete exchange of tubulin in nonkinetochore spindle and astral microtubules appeared to occur within 60-80 s at steady state. This rate is too fast to be explained by a simple microtubule end-dependent exchange of tubulin. Efficient microtubule treadmilling would be fast enough, but with current techniques we saw no evidence for movement of the bleached spot during recovery, which we would expect on the basis of Margolis and Wilson's model (Nature (Lond.)., 1981, 293:705)-- fluorescence recovers uniformly. Microtubules may be depolymerizing and repolymerizing rapidly and asynchronously throughout the spindle and asters, but the FRAP data are most compatible with a rapid exchange of tubulin subunits all along the entire lengths of nonkinetochore spindle and astral microtubules.     

Contribution of Noncentrosomal Microtubules to Spindle Assembly in Drosophila Spermatocytes

Previous data suggested that anastral spindles, morphologically similar to those found in oocytes, can assemble in a centrosome-independent manner in cells that contain centrosomes. It is assumed that the microtubules that build these acentrosomal spindles originate over the chromatin. However, the actual processes of centrosome-independent microtubule nucleation, polymerisation, and sorting have not been documented in centrosome-containing cells. We have identified two experimental conditions in which centrosomes are kept close to the plasma membrane, away from the nuclear region, throughout meiosis I in Drosophila spermatocytes. Time-lapse confocal microscopy of these cells labelled with fluorescent chimeras reveals centrosome-independent microtubule nucleation, growth, and sorting into a bipolar spindle array over the nuclear region, away from the asters. The onset of noncentrosomal microtubule nucleation is significantly delayed with respect to nuclear envelope breakdown and coincides with the end of chromosome condensation. It takes place in foci that are close to the membranes that ensheath the nuclear region, not over the condensed chromosomes. Metaphase plates are formed in these spindles, and, in a fraction of them, some degree of polewards chromosome segregation takes place. In these cells that contain both membrane-bound asters and an anastral spindle, the orientation of the cytokinesis furrow correlates with the position of the asters and is independent of the orientation of the spindle. We conclude that the fenestrated nuclear envelope may significantly contribute to the normal process of spindle assembly in Drosophila spermatocytes. We also conclude that the anastral spindles that we have observed are not likely to provide a robust back-up able to ensure successful cell division. We propose that these anastral microtubule arrays could be a constitutive component of wild-type spindles, normally masked by the abundance of centrosome-derived microtubules and revealed when asters are kept away. These observations are consistent with a model in which centrosomal and noncentrosomal microtubules contribute to the assembly and are required for the robustness of the cell division spindle in cells that contain centrosomes.     

Experimental manipulation of the amount of tubulin available for assembly into the spindle of dividing sea urchin eggs

Spindle assembly is studied in the eggs of the sea urchin Lytechinus variegatus by experimentally varying the amount of polymerizable tubulin within the egg. Aliquots of fertilized eggs from the same female are individually pulsed for 1-6 min with 1 X 10(-6) M Colcemid at least 20 min before first nuclear envelope breakdown. This treatment inactivates a portion of the cellular tubulin before the spindle is formed. Upon entering mitosis, treated eggs form functional spindles that are reduced in length and birefringent retardation but not width. With increased exposure to Colcemid, the length and retardation of the metaphase spindles are progressively reduced. Similar results are obtained by pulsing the eggs with Colcemid before fertilization, which demonstrates that the tubulin found in unfertilized sea urchin eggs is later used in spindle formation. Spindles, once assembled, are responsive to increases in the amount of polymerizable tubulin within the cell. Rapid increases in the amount of polymerizable tubulin within a Colcemid-treated cell can be experimentally effected by irradiating the cells with 366-nm light. This treatment photochemically inactivates the Colcemid, thereby freeing the tubulin to polymerize. Upon irradiation, the small prometaphase spindles of Colcemid-treated cells immediately increase in length and retardation. In these irradiated cells, spindle length and retardation increase as much as four times faster than they do during prometaphase for normal spindles. This suggests that the rate of the normal prometaphase increase in retardation and spindle size may be determined by factors other than the maximum rate of tubulin polymerization in the cell.     

The adenomatous polyposis coli protein is required for the formation of robust spindles formed in CSF Xenopus extracts

Mutations in the adenomatous polyposis coli (APC) protein occur early in colon cancer and correlate with chromosomal instability. Here, we show that depletion of APC from cystostatic factor (CSF) Xenopus extracts leads to a decrease in microtubule density and changes in tubulin distribution in spindles and asters formed in such extracts. Addition of full-length APC protein or a large, N-terminally truncated APC fragment to APC-depleted extracts restored normal spindle morphology and the intact microtubule-binding site of APC was necessary for this rescue. These data indicate that the APC protein plays a role in the formation of spindles that is dependent on its effect on microtubules. Spindles formed in cycled extracts were not sensitive to APC depletion. In CSF extracts, spindles predominantly formed from aster-like intermediates, whereas in cycled extracts chromatin was the major site of initial microtubule polymerization. These data suggest that APC is important for centrosomally driven spindle formation, which was confirmed by our finding that APC depletion reduced the size of asters nucleated from isolated centrosomes. We propose that lack of microtubule binding in cancer-associated mutations of APC may contribute to defects in the assembly of mitotic spindles and lead to missegregation of chromosomes.     

Rapid rate of tubulin dissociation from microtubules in the mitotic spindle in vivo measured by blocking polymerization with colchicine

At metaphase, the amount of tubulin assembled into spindle microtubules is relatively constant; the rate of tubulin association equals the rate of dissociation. To measure the intrinsic rate of dissociation, we microinjected high concentrations of colchicine, or its derivative colcemid, into sea urchin embryos at metaphase to bind the free tubulin, thereby rapidly blocking polymerization. The rate of microtubule disassembly was measured from a calibrated video signal by the change in birefringent retardation (BR). After an initial delay after injection of colchicine or colcemid at final intracellular concentrations of 0.1-3.0 mM, BR decreased rapidly and simultaneously throughout the central spindle and aster. Measured BR in the central half-spindle decreased exponentially to 10% of its initial value within a characteristic period of approximately 20 s; the rate constant, k = 0.11 +/- 0.023 s-1, and the corresponding half-time, t 1/2, of BR decay was approximately 6.5 +/- 1.1 s in this concentration range. Below 0.1 mM colchicine or colcemid, the rate at which BR decreased was concentration dependent. Electron micrographs showed that the rapid decrease in BR corresponded to the disappearance of nonkinetochore microtubules; kinetochore fiber microtubules were differentially stable. As a control, lumicolchicine, which does not bind to tubulin with high affinity, was shown to have no effect on spindle BR at intracellular concentrations of 0.5 mM. If colchicine and colcemid block only polymerization, then the initial rate of tubulin dissociation from nonkinetochore spindle microtubules is in the range of 180-992 dimers per second. This range of rates is based on k = 11% of the initial polymer per second and an estimate from electron micrographs that the average length of a half-spindle microtubule is 1- 5.5 micron. Much slower rates of tubulin association are predicted from the characteristics of end-dependent microtubule assembly measured previously in vitro when the association rate constant is corrected for the lower rate of tubulin diffusion in the embryo cytoplasm. Various possibilities for this discrepancy are discussed.     

Anastral Spindle Assembly: A Mathematical Model

Assembly of an anastral spindle was modeled as a two-stage process: first, the aggregation of microtubule foci or asters around the chromosomes, and second, the elongation of cross-linked microtubules and onset of bipolarity. Several possibilities involving diffusion and transport were investigated for the first stage, and the most feasible was found to be binding of the asters to cytoskeletal filaments and directed transport toward the chromosomes. For the second stage, a differential-equation model was formulated and solved numerically; it involves cross-linking of microtubules with those aligned with the spindle axis and between microtubules bound to different chromosomes, and sliding of microtubules along the spindle axis to elongate the spindle. Ncd was postulated to perform both functions. The model shows that spindle formation begins with rapid cross-linking of microtubules, followed by elongation, which continues until the population of microtubules aligned with the spindle axis is depleted and microtubules cross-linking different chromosomes dominate. It also shows that when sliding is inhibited, short bipolar spindles still form, and if clustering is enhanced, normal-length spindles can form, although requiring longer assembly time. These findings are consistent with spindle assembly in live wild-type and ncd mutant Drosophila oocytes.     

Mitosis-specific anchoring of gamma tubulin complexes by pericentrin controls spindle organization and mitotic entry

Microtubule nucleation is the best known function of centrosomes. Centrosomal microtubule nucleation is mediated primarily by gamma tubulin ring complexes (gamma TuRCs). However, little is known about the molecules that anchor these complexes to centrosomes. In this study, we show that the centrosomal coiled-coil protein pericentrin anchors gamma TuRCs at spindle poles through an interaction with gamma tubulin complex proteins 2 and 3 (GCP2/3). Pericentrin silencing by small interfering RNAs in somatic cells disrupted gamma tubulin localization and spindle organization in mitosis but had no effect on gamma tubulin localization or microtubule organization in interphase cells. Similarly, overexpression of the GCP2/3 binding domain of pericentrin disrupted the endogenous pericentrin-gamma TuRC interaction and perturbed astral microtubules and spindle bipolarity. When added to Xenopus mitotic extracts, this domain uncoupled gamma TuRCs from centrosomes, inhibited microtubule aster assembly, and induced rapid disassembly of preassembled asters. All phenotypes were significantly reduced in a pericentrin mutant with diminished GCP2/3 binding and were specific for mitotic centrosomal asters as we observed little effect on interphase asters or on asters assembled by the Ran-mediated centrosome-independent pathway. Additionally, pericentrin silencing or overexpression induced G2/antephase arrest followed by apoptosis in many but not all cell types. We conclude that pericentrin anchoring of gamma tubulin complexes at centrosomes in mitotic cells is required for proper spindle organization and that loss of this anchoring mechanism elicits a checkpoint response that prevents mitotic entry and triggers apoptotic cell death.     

Initiation and growth of microtubules from mitotic centers in lysed mammalian cells

Metaphase PtK1 cells, lysed into polymerization-competent microtubule protein, maintain a spindle which will gain or lose birefringence depending on the concentration of disassembled tubulin subunits used in the lysis medium. Concentrations of tubulin subunits greater than the equilibrium monomer value promote a rate and extent of birefringence increase that is proportional to the subunit concentration. Increase in spindle birefringence can be correlated with an increase in tubule number, though the relationship is not strictly linear. Increase in spindle tubule number is due to an vivo-like initiation of tubules at the mitotic centers, as well as tubulin addition onto pre-existing spindle fragments. Colcemid-treated prometaphase cells lysed into polymerization-competent tubulin develop large asters in the region of the centrioles and short tubules at kinetochores, making it unlikely that all microtubule formation in lysed cell preparations is dependent on tubulin addition to short tubule fragments. Asters can also form in colcemid-treated prometaphase cells lysed in tubulin that is incapable of spontaneous tubule initiation, suggesting that the centriolar region serves a tubule-initiator function in our lysed cell preparations. The ability of the centriole to initiate microtubule assembly is a time-dependent process-a ripening effect takes place between prophase and late prometaphase. Ripening is expressed by an increase in the number and length of tubules found associated with the centriolar region.     

A Highlights from MBoC Selection: Fast Microtubule Dynamics in Meiotic Spindles Measured by Single Molecule Imaging: Evidence That the Spindle Environment Does Not Stabilize Microtubules

Metaphase spindles are steady-state ensembles of microtubules that turn over rapidly and slide poleward in some systems. Since the discovery of dynamic instability in the mid-1980s, models for spindle morphogenesis have proposed that microtubules are stabilized by the spindle environment. We used single molecule imaging to measure tubulin turnover in spindles, and nonspindle assemblies, in Xenopus laevis egg extracts. We observed many events where tubulin molecules spend only a few seconds in polymer and thus are difficult to reconcile with standard models of polymerization dynamics. Our data can be quantitatively explained by a simple, phenomenological model—with only one adjustable parameter—in which the growing and shrinking of microtubule ends is approximated as a biased random walk. Microtubule turnover kinetics did not vary with position in the spindle and were the same in spindles and nonspindle ensembles nucleated by Tetrahymena pellicles. These results argue that the high density of microtubules in spindles compared with bulk cytoplasm is caused by local enhancement of nucleation and not by local stabilization. It follows that the key to understanding spindle morphogenesis will be to elucidate how nucleation is spatially controlled.     

Two protein 4.1 domains essential for mitotic spindle and aster microtubule dynamics and organization in vitro

Multifunctional structural proteins belonging to the 4.1 family are components of nuclei, spindles, and centrosomes in vertebrate cells. Here we report that 4.1 is critical for spindle assembly and the formation of centrosome-nucleated and motor-dependent self-organized microtubule asters in metaphase-arrested Xenopus egg extracts. Immunodepletion of 4.1 disrupted microtubule arrays and mislocalized the spindle pole protein NuMA. Remarkably, assembly was completely rescued by supplementation with a recombinant 4.1R isoform. We identified two 4.1 domains critical for its function in microtubule polymerization and organization utilizing dominant negative peptides. The 4.1 spectrin-actin binding domain or NuMA binding C-terminal domain peptides caused morphologically disorganized structures. Control peptides with low homology or variant spectrin-actin binding domain peptides that were incapable of binding actin had no deleterious effects. Unexpectedly, the addition of C-terminal domain peptides with reduced NuMA binding caused severe microtubule destabilization in extracts, dramatically inhibiting aster and spindle assembly and also depolymerizing preformed structures. However, the mutant C-terminal peptides did not directly inhibit or destabilize microtubule polymerization from pure tubulin in a microtubule pelleting assay. Our data showing that 4.1 is a crucial factor for assembly and maintenance of mitotic spindles and self-organized and centrosome-nucleated microtubule asters indicates that 4.1 is involved in regulating both microtubule dynamics and organization. These investigations underscore an important functional context for protein 4.1 in microtubule morphogenesis and highlight a previously unappreciated role for 4.1 in cell division.