Microinjection of biotin-tubulin into anaphase cells induces transient elongation of kinetochore microtubules and reversal of chromosome-to- pole motion

During prometaphase and metaphase of mitosis, tubulin subunit incorporation into kinetochore microtubules occurs proximal to the kinetochore, at the plus-ends of kinetochore microtubules. During anaphase, subunit loss from kinetochore fiber microtubules is also thought to occur mainly from microtubule plus-ends, proximal to the kinetochore. Thus, the kinetochore can mediate both subunit addition and loss while maintaining an attachment to kinetochore microtubules. To examine the relationship between chromosome motion and tubulin subunit assembly in anaphase, we have injected anaphase cells with biotin-labeled tubulin subunits. The pattern of biotin-tubulin incorporation was revealed using immunoelectron and confocal fluorescence microscopy of cells fixed after injection; chromosome motion was analyzed using video records of living injected cells. When anaphase cells are examined approximately 30 s after injection with biotin-tubulin, bright "tufts" of fluorescence are detected proximal to the kinetochores. Electron microscopic immunocytochemistry further reveals that these tufts of biotin-tubulin-containing microtubules are continuous with unlabeled kinetochore fiber microtubules. Biotin-tubulin incorporation proximal to the kinetochore in anaphase cells is detected after injection of 3-30 mg/ml biotin-tubulin, but not in cells injected with 0.3 mg/ml biotin-tubulin. At intermediate concentrations of biotin-tubulin (3-5 mg/ml), incorporation at the kinetochore can be detected within 15 s after injection; by approximately 1 min after injection discrete tufts of fluorescence are no longer detected, although some incorporation throughout the kinetochore fiber and into nonkinetochore microtubules is observed. At higher concentrations of injected biotin-tubulin (13 mg/ml), incorporation at the kinetochore is more extensive and occurs for longer periods of time than at intermediate concentrations. Incorporation of biotin-tubulin proximal to the kinetochore can be detected in cells injected during anaphase A, but not during anaphase B. Analysis of video records of microinjection experiments reveals that kinetochore proximal incorporation of biotin-tubulin is accompanied by a transient reversal of chromosome-to-pole motion. Chromosome motion is not altered after injection of 0.3 mg/ml biotin-tubulin or 5 mg/ml BSA. These results demonstrate that kinetochore microtubules in anaphase cells can elongate in response to the elevation of the tubulin concentration and that kinetochores retain the ability to mediate plus-end-dependent assembly of KMTs and plus-end-directed chromosome motion after anaphase onset.     

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.     

Minor alteration of microtubule dynamics causes loss of tension across kinetochore pairs and activates the spindle checkpoint

We have previously identified the opium alkaloid noscapine as a microtubule interacting agent that binds stoichiometrically to tubulin and alters its conformation. Here we show that, unlike many other microtubule inhibitors, noscapine does not significantly promote or inhibit microtubule polymerization. Instead, it alters the steady-state dynamics of microtubule assembly, primarily by increasing the amount of time that the microtubules spend in an attenuated (pause) state. Further studies reveal that even at high concentrations, noscapine does not alter the tubulin polymer/monomer ratio in HeLa cells. Cells treated with noscapine arrest at mitosis with nearly normal bipolar spindles. Strikingly, although most of the chromosomes in these cells are aligned at the metaphase plate, the rest remain near the spindle poles, both of which exhibit loss of tension across kinetochore pairs. Furthermore, levels of the spindle checkpoint proteins Mad2, Bub1, and BubR1 decrease by 138-, 3.7-, and 3.9-fold, respectively, at the kinetochore region upon chromosome alignment. Our results thus suggest that an exquisite control of microtubule dynamics is required for kinetochore tension generation and chromosome alignment during mitosis. Our data also support the idea that Mad2 and Bub1/BubR1 respond to kinetochore-microtubule attachment and/or tension to different degrees.     

The Microtubule-dependent Motor Centromere–associated Protein E (CENP-E) Is an Integral Component of Kinetochore Corona Fibers That Link Centromeres to Spindle Microtubules

Centromere-associated protein E (CENP-E) is a kinesin-related microtubule motor protein that is essential for chromosome congression during mitosis. Using immunoelectron microscopy, CENP-E is shown to be an integral component of the kinetochore corona fibers that tether centromeres to the spindle. Immediately upon nuclear envelope fragmentation, an associated plus end motor trafficks cytoplasmic CENP-E toward chromosomes along astral microtubules that enter the nuclear volume. Before or concurrently with initial lateral attachment of spindle microtubules, CENP-E targets to the outermost region of the developing kinetochores. After stable attachment, throughout chromosome congression, at metaphase, and throughout anaphase A, CENP-E is a constituent of the corona fibers, extending at least 50 nm away from the kinetochore outer plate and intertwining with spindle microtubules. In congressing chromosomes, CENP-E is preferentially associated with (or accessible at) the stretched, leading kinetochore known to provide the primary power for chromosome movement. Taken together, this evidence strongly supports a model in which CENP-E functions in congression to tether kinetochores to the disassembling microtubule plus ends.     

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.     

Microtubules of the kinetochore fiber turn over in metaphase but not in anaphase

In previous work we injected mitotic cells with fluorescent tubulin and photobleached them to mark domains on the spindle microtubules. We concluded that chromosomes move poleward along kinetochore fiber microtubules that remain stationary with respect to the pole while depolymerizing at the kinetochore. In those experiments, bleached zones in anaphase spindles showed some recovery of fluorescence with time. We wished to determine the nature of this recovery. Was it due to turnover of kinetochore fiber microtubules or of nonkinetochore microtubules or both? We also wished to investigate the question of turnover of kinetochore microtubules in metaphase. We microinjected cells with x- rhodamine tubulin (x-rh tubulin) and photobleached spindles in anaphase and metaphase. At various times after photobleaching, cells were detergent lysed in a cold buffer containing 80 microM calcium, conditions that led to the disassembly of almost all nonkinetochore microtubules. Quantitative analysis with a charge coupled device image sensor revealed that the bleached zones in anaphase cells showed no fluorescence recovery, suggesting that these kinetochore fiber microtubules do not turn over. Thus, the partial fluorescence recovery seen in our earlier anaphase experiments was likely due to turnover of nonkinetochore microtubules. In contrast fluorescence in metaphase cells recovered to approximately 70% the control level within 7 min suggesting that many, but perhaps not all, kinetochore fiber microtubules of metaphase cells do turn over. Analysis of the movements of metaphase bleached zones suggested that a slow poleward translocation of kinetochore microtubules occurred. However, within the variation of the data (0.12 +/- 0.24 micron/min), it could not be determined whether the apparent movement was real or artifactual.     

A unique kinesin-8 surface loop provides specificity for chromosome alignment

Microtubule length control is essential for the assembly and function of the mitotic spindle. Kinesin-like motor proteins that directly attenuate microtubule dynamics make key contributions to this control, but the specificity of these motors for different subpopulations of spindle microtubules is not understood. Kif18A (kinesin-8) localizes to the plus ends of the relatively slowly growing kinetochore fibers (K-fibers) and attenuates their dynamics, whereas Kif4A (kinesin-4) localizes to mitotic chromatin and suppresses the growth of highly dynamic, nonkinetochore microtubules. Although Kif18A and Kif4A similarly suppress microtubule growth in vitro, it remains unclear whether microtubule-attenuating motors control the lengths of K-fibers and nonkinetochore microtubules through a common mechanism. To address this question, we engineered chimeric kinesins that contain the Kif4A, Kif18B (kinesin-8), or Kif5B (kinesin-1) motor domain fused to the C-terminal tail of Kif18A. Each of these chimeric kinesins localizes to K-fibers; however, K-fiber length control requires an activity specific to kinesin-8s. Mutational studies of Kif18A indicate that this control depends on both its C-terminus and a unique, positively charged surface loop, called loop2, within the motor domain. These data support a model in which microtubule-attenuating kinesins are molecularly “tuned” to control the dynamics of specific subsets of spindle microtubules.     

Survivin modulates microtubule dynamics and nucleation throughout the cell cycle

Survivin is a member of the chromosomal passenger complex implicated in kinetochore attachment, bipolar spindle formation, and cytokinesis. However, the mechanism by which survivin modulates these processes is unknown. Here, we show by time-lapse imaging of cells expressing either green fluorescent protein (GFP)-alpha-tubulin or the microtubule plus-end binding protein GFP-EB1 that depletion of survivin by small interfering RNAs (siRNAs) increased both the number of microtubules nucleated by centrosomes and the incidence of microtubule catastrophe, the transition from microtubule growth to shrinking. In contrast, survivin overexpression reduced centrosomal microtubule nucleation and suppressed both microtubule dynamics in mitotic spindles and bidirectional growth of microtubules in midbodies during cytokinesis. siRNA depletion or pharmacologic inhibition of another chromosomal passenger protein Aurora B, had no effect on microtubule dynamics or nucleation in interphase or mitotic cells even though mitosis was impaired. We propose a model in which survivin modulates several mitotic events, including spindle and interphase microtubule organization, the spindle assembly checkpoint and cytokinesis through its ability to modulate microtubule nucleation and dynamics. This pathway may affect the microtubule-dependent generation of aneuploidy and defects in cell polarity in cancer cells, where survivin is commonly up-regulated.     

MCAK-independent functions of ch-Tog/XMAP215 in microtubule plus-end dynamics

The formation of a functional bipolar mitotic spindle is essential for genetic integrity. In human cells, the microtubule polymerase XMAP215/ch-Tog ensures spindle bipolarity by counteracting the activity of the microtubule-depolymerizing kinesin XKCM1/MCAK. Their antagonistic effects on microtubule polymerization confer dynamic instability on microtubules assembled in cell-free systems. It is, however, unclear if a similar interplay governs microtubule behavior in mammalian cells in vivo. Using real-time analysis of spindle assembly, we found that ch-Tog is required to produce or maintain long centrosomal microtubules after nuclear-envelope breakdown. In the absence of ch-Tog, microtubule assembly at centrosomes was impaired and microtubules were nondynamic. Interkinetochore distances and the lengths of kinetochore fibers were also reduced in these cells. Codepleting MCAK with ch-Tog improved kinetochore fiber length and interkinetochore separation but, surprisingly, did not rescue centrosomal microtubule assembly and microtubule dynamics. Our data therefore suggest that ch-Tog has at least two distinct roles in spindle formation. First, it protects kinetochore microtubules from depolymerization by MCAK. Second, ch-Tog plays an essential role in centrosomal microtubule assembly, a function independent of MCAK activity. Thus, the notion that the antagonistic activities of MCAK and ch-Tog determine overall microtubule stability is too simplistic to apply to human cells.     

Biotin-tubulin incorporates into kinetochore fiber microtubules during early but not late anaphase

The dynamic behavior of kinetochore fiber microtubules has been examined in PtK1 cells during anaphase of mitosis. Cells in anaphase were injected with biotin-tubulin and, at various intervals after injection, fixed for light or electron microscopic immunolocalization of biotin-tubulin-containing microtubules. When cells in early to mid anaphase were injected with biotin-tubulin and fixed 1-2 min later, fluorescence was observed throughout the spindle, including the region of the kinetochore fibers. Electron microscopy of early to mid anaphase cells, after processing with immunogold methods, revealed both labeled and unlabeled microtubules in the kinetochore fibers; some labeled microtubules contacted the kinetochores. When late anaphase cells were injected with biotin-tubulin, and fixed a few minutes later, little fluorescence was observed in the kinetochore fibers. Electron microscopy confirmed that kinetochore fibers in late anaphase cells were refractory to tubulin incorporation. The results of these experiments demonstrate that the kinetochore fiber incorporates new microtubules during early anaphase but that this incorporation ceases in mid to late anaphase. Thus, microtubule turnover within the kinetochore fiber does not abruptly cease at the onset of anaphase and anaphase kinetochore fiber microtubules are more dynamic than previously suspected.     

Spindle microtubule dynamics: modulation by metabolic inhibitors

Recent experiments have shown that spindle microtubules are exceedingly dynamic. Measurements of fluorescence recovery after photobleaching (FRAP), in cells previously microinjected with fluorescent tubulin, provide quantitative information concerning the rate of turnover, or exchange, of tubulin subunits with the population of microtubules in living cells at steady state. In an effort to elucidate the pathways and factors that regulate tubulin exchange with microtubules in living cells, we have investigated the energy requirements for tubulin turnover as measured by FRAP. Spindle morphology was not detectably altered in cells incubated with 5 mM sodium azide and 1 mM 2-deoxyglucose (Az/DOG) for 5 minutes, as assayed by polarized light microscopy and antitubulin immunofluorescence. In FRAP experiments on these ATP-depleted cells, the average rate of recovery and the average percent of bleached fluorescence recovered were reduced to 37% and 30% of controls, respectively. When the inhibitors were removed, cells continued through mitosis, and rapid FRAP was restored. In the presence of azide and glucose, the rate of recovery and percent of fluorescence recovered were only slightly reduced, demonstrating that energy production via glycolysis can support microtubule turnover. Longer incubations with Az/DOG altered the microtubule organization in mitotic cells: astral microtubules lengthened and spindle fibers shortened. Furthermore, both astral and spindle microtubules became resistant to nocodazole-induced disassembly under these conditions. Together these observations indicate that microtubule dynamics require ATP and suggest a relationship between microtubule organization and turnover.     

The total length of spindle microtubules depends on the number of chromosomes present

We extracted chromosomes by micromanipulation from Melanoplus differentialis spermatocytes, producing metaphase spindles with only one or a few chromosomes instead of the usual complement of 23. Cells with various numbers of chromosomes were prepared for electron microscopy, and spindle microtubule length was measured. A constant increment of microtubule length was lost upon the removal of each chromosome; we estimate that only approximately 40% of the original length would remain in the total absence of chromosomes. Unexpectedly, kinetochore microtubules were not the only ones affected when chromosomes were removed: nonkinetochore microtubules accounted for a substantial fraction of the total length lost. No compensatory increase in microtubule length outside the spindle was found. Studies by others show that the kinetochore microtubules of extracted chromosomes are left behind in the cell and dissassemble. The resulting increase in subunit concentration would be expected from in vitro studies to drive microtubule assembly until the original total microtubule length was restored, but that did not happen in these living cells. We conclude that the assembly of a certain, large fraction of microtubule subunits into stable microtubules is dependent on the presence of chromosomes. Possible explanations include (a) limits on microtubule length that prevent any net assembly of the subunits released after chromosomes are removed or (b) a promotion of microtubule assembly by chromosomes, which therefore is reduced in their absence. Chromosome-dependent regulation of microtubule length may account for some features of normal mitosis.     

Chromosome fiber dynamics and congression oscillations in metaphase PtK2 cells at 23 degrees C

A bioriented chromosome is tethered to opposite spindle poles during congression by bundles of kinetochore microtubules (kMts). At room temperature, kinetochore fibers are a dominant component of mitotic spindles of PtK2 cells. PtK2 cells at room temperature were injected with purified tubulin covalently bound to DTAF and congression movements of individual chromosomes were recorded in time lapse. Congression movements of bioriented chromosomes between the poles occur over distances of 4.5 microns or greater. DTAF-tubulin injection had no effect on either the velocity or extent of these movements. Other cells were lysed, fixed, and the location of DTAF-tubulin incorporation was detected from digitally processed images of indirect immunofluorescence of an antibody to DTAF. Microtubules were labeled with an anti-beta tubulin antibody. At 2-5 minutes after injection, concentrated DTAF-tubulin staining was seen in the kinetochore fibers proximal to the kinetochores; a low concentration of DTAF-tubulin staining occurred at various sites through the remaining length of the fibers toward the pole. Kinetochore fibers in the same cell displayed different lengths (0.2 to 4 microns) of concentrated DTAF-tubulin incorporation proximal to the kinetochore, as did sister kinetochore fibers. Ten minutes after injection, the lengths of DTAF-containing chromosomal fibers were greater than expected if incorporation resulted solely from the lengthening of kinetochore microtubules due to congression movements of the chromosomes. Besides incorporation as a result of chromosome movement, two other mechanisms might explain the length of the DTAF-containing segments: 1) a poleward flux of tubulin subunits (Mitchison, 1989) or 2) capture of DTAF-containing nonkinetochore microtubules.     

Polarity of kinetochore microtubules in Chinese hamster ovary cells after recovery from a colcemid block

The polarity of kinetochore microtubules was determined in a system for which kinetochore-initiated microtubule assembly has been demonstrated. Chinese hamster ovary cells were treated with 0.3 micrograms/ml colcemid for 8 h and then released from the block. Prior to recovery, microtubules were completely absent from the cells. The recovery was monitored using light and electron microscopy to establish that the cells progress through anaphase and that the kinetochore fibers are fully functional. Since early stages of recovery are characterized by short microtubule segments that terminate in the kinetochore fibrous corona rather than on the outer disk, microtubule polarity was determined at later stages of recovery when longer kinetochore bundles had formed, allowing us to establish unambiguously the spatial relationship between microtubules, kinetochores, and chromosomes. The cells were lysed in a detergent mixture containing bovine brain tubulin under conditions that allowed the formation of polarity-revealing hooks. 20 kinetochore bundles were assayed for microtubule polarity in either thick or thin serial sections. We found that 95% of the decorated kinetochore microtubules had the same polarity and that, according to the hook curvature, the plus ends of the microtubules were at the kinetochores. Hence, the polarity of kinetochore microtubules in Chinese hamster ovary cells recovering from a colcemid block is the same as in normal untreated cells. This result suggests that microtubule polarity is likely to be important for spindle function since kinetochore microtubules show the same polarity, regardless of the pattern of spindle formation.     

hNuf2 inhibition blocks stable kinetochore-microtubule attachment and induces mitotic cell death in HeLa cells

Identification of proteins that couple kinetochores to spindle microtubules is critical for understanding how accurate chromosome segregation is achieved in mitosis. Here we show that the protein hNuf2 specifically functions at kinetochores for stable microtubule attachment in HeLa cells. When hNuf2 is depleted by RNA interference, spindle formation occurs normally as cells enter mitosis, but kinetochores fail to form their attachments to spindle microtubules and cells block in prometaphase with an active spindle checkpoint. Kinetochores depleted of hNuf2 retain the microtubule motors CENP-E and cytoplasmic dynein, proteins previously implicated in recruiting kinetochore microtubules. Kinetochores also retain detectable levels of the spindle checkpoint proteins Mad2 and BubR1, as expected for activation of the spindle checkpoint by unattached kinetochores. In addition, the cell cycle block produced by hNuf2 depletion induces mitotic cells to undergo cell death. These data highlight a specific role for hNuf2 in kinetochore-microtubule attachment and suggest that hNuf2 is part of a molecular linker between the kinetochore attachment site and tubulin subunits within the lattice of attached plus ends.     

Spindle microtubules and their mechanical associations after micromanipulation in anaphase

Micromanipulation of living grasshopper spermatocytes in anaphase has been combined with electron microscopy to reveal otherwise obscure features of spindle organization. A chromosome is pushed laterally outside the spindle and stretched, and the cell is fixed with a novel, agar-treated glutaraldehyde solution. Two- and three-dimensional reconstructions from serial sections of seven cells show that kinetochore microtubules of the manipulated chromosome are shifted outside the confusing thicket of spindle microtubules and mechanical associations among microtubules are revealed by bent or shifted microtubules. These are the chief results: (a) The disposition of microtubules invariably is consistent with a skeletal role for spindle microtubules. (b) The kinetochore microtubule bundle is composed of short and long microtubules, with weak but recognizable mechanical associations among them. Some kinetochore microtubules are more tightly linked to one other microtubule within the bundle. (c) Microtubules of the kinetochore microtubule bundle are firmly connected to other spindle microtubules only near the pole, although some nonkinetochore microtubules of uncertain significance enter the bundle nearer to the kinetochore. (d) The kinetochore microtubules of adjacent chromosomes are mechanically linked, which provides an explanation for interdependent chromosome movement in "hinge anaphases." In the region of the spindle open to analysis after chromosome micromanipulation, microtubules may be linked mechanically by embedment in a gel, rather than by dynein or other specific, cross-bridging molecules.     

Kinetochore microtubule dynamics and attachment stability are regulated by Hec1

Mitotic cells face the challenging tasks of linking kinetochores to growing and shortening microtubules and actively regulating these dynamic attachments to produce accurate chromosome segregation. We report here that Ndc80/Hec1 functions in regulating kinetochore microtubule plus-end dynamics and attachment stability. Microinjection of an antibody to the N terminus of Hec1 suppresses both microtubule detachment and microtubule plus-end polymerization and depolymerization at kinetochores of PtK1 cells. Centromeres become hyperstretched, kinetochore fibers shorten from spindle poles, kinetochore microtubule attachment errors increase, and chromosomes severely mis-segregate. The N terminus of Hec1 is phosphorylated by Aurora B kinase in vitro, and cells expressing N-terminal nonphosphorylatable mutants of Hec1 exhibit an increase in merotelic attachments, hyperstretching of centromeres, and errors in chromosome segregation. These findings reveal a key role for the Hec1 N terminus in controlling dynamic behavior of kinetochore microtubules.     

Dynamics of a fluorescent calmodulin analog in the mammalian mitotic spindle at metaphase

We have compared the exchange kinetics of fluorescein-labeled calmodulin and tubulin in the spindles of living mitotic cells at metaphase. Cultured mammalian cells in early stages of mitosis were microinjected with labeled calmodulin or tubulin and returned to an incubator to allow equilibration of the fluorescent protein with the endogenous protein pools. Calmodulin becomes concentrated in the mitotic spindle, and treatments with inhibitors of tubulin assembly show that this concentration is dependent on the presence of microtubules. The steady-state exchange rates of both tubulin and calmodulin were measured by an analysis of fluorescence redistribution after photobleaching (FRAP), using cells pre-equilibrated to either 26 +/- 2 degrees C or 36 +/- 2 degrees C. A pulse of laser light focused to a 5-microns diameter column was used to destroy the fluorescence at one pole of a metaphase mitotic spindle. Ratios of fluorescence intensity from the two half-spindles and from the two polar regions were calculated for each image in a post-bleach time series to determine the rates and extents of FRAP. For tubulin, we confirm earlier observations concerning the temperature dependence of the extent of FRAP, but our data do not show a significant temperature dependence for the rate of FRAP. We hypothesize that the reduced extent of tubulin FRAP at the lower temperatures is a result of microtubules that are stable to depolymerization at 26 degrees C and are thus less likely to exchange subunits. Calmodulin's FRAP, however, does not exhibit any of the temperature dependence observed with fluorescent tubulin. At 26 +/- 2 degrees C calmodulin exchanges rapidly with the relatively stable population of microtubules, suggesting that calmodulin is bound, either directly or indirectly, to microtubule walls.     

Immunofluorescence analysis of sucrose-induced changes in spindle morphology

Metaphase and anaphase PtK1 cells show spindle elongation without concomitant chromosome motion when treated with culture medium containing 0.5 M sucrose. Electron microscopy has shown sucrose-induced changes in microtubule (MT) organization, changes in trilaminar kinetochore structure, and specific kinetochore-MT associations which may account for these results. In this paper we employ double-label immunofluorescence techniques using antibodies against tubulin and the kinetochore to analyze changes in spindle microtubule and kinetochore distribution produced by sucrose treatment. Cells treated from prometaphase through anaphase with 0.5 M sucrose from 10 min to 2 h showed spindle elongation and a distinct rearrangement of spindle microtubules into bundles, with a pronounced increase in length of interpolar microtubule bundles. In sucrose-treated mitotic cells kinetochores remained as antigenically distinct structures, similar to those found in untreated interphase cells. Kinetochore determinants remained positioned within a diffuse chromatin mass, but the orientation of sister kinetochores to opposite spindle poles was lost. Instead, kinetochore pairs were found in lateral association with microtubule bundles, with several pairs of determinants associated with a single bundle in many instances. Cells released from 0.5 M sucrose treatment showed a return of the spindle to a pretreatment arrangement for both the microtubules and kinetochore determinants.     

The interplay of the N- and C-terminal domains of MCAK control microtubule depolymerization activity and spindle assembly

Spindle assembly and accurate chromosome segregation require the proper regulation of microtubule dynamics. MCAK, a Kinesin-13, catalytically depolymerizes microtubules, regulates physiological microtubule dynamics, and is the major catastrophe factor in egg extracts. Purified GFP-tagged MCAK domain mutants were assayed to address how the different MCAK domains contribute to in vitro microtubule depolymerization activity and physiological spindle assembly activity in egg extracts. Our biochemical results demonstrate that both the neck and the C-terminal domain are necessary for robust in vitro microtubule depolymerization activity. In particular, the neck is essential for microtubule end binding, and the C-terminal domain is essential for tight microtubule binding in the presence of excess tubulin heterodimer. Our physiological results illustrate that the N-terminal domain is essential for regulating microtubule dynamics, stimulating spindle bipolarity, and kinetochore targeting; whereas the C-terminal domain is necessary for robust microtubule depolymerization activity, limiting spindle bipolarity, and enhancing kinetochore targeting. Unexpectedly, robust MCAK microtubule (MT) depolymerization activity is not needed for sperm-induced spindle assembly. However, high activity is necessary for proper physiological MT dynamics as assayed by Ran-induced aster assembly. We propose that MCAK activity is spatially controlled by an interplay between the N- and C-terminal domains during spindle assembly.