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Many cell types contain a subset of long-lived, ‘stable’ microtubules that differ from dynamic microtubules in that they are enriched in post-translationally detyrosinated tubulin (Glu-tubulin). Elevated Glu tubulin does not stabilize the microtubules and the mechanism for the stability of Glu microtubules is not known. We used detergent-extracted cell models to investigate the nature of Glu microtubule stability. In these cell models, Glu microtubules did not incorporate exogenously added tubulin subunits on their distal ends, while >70% of the bulk microtubules did. Ca2+-generated fragments of Glu microtubules incorporated tubulin, showing that Glu microtubule ends are capped. Consistent with this, Glu microtubules in cell models were resistant to dilution-induced breakdown. Known microtubule end-associated proteins (EB1, APC, p150Glued and vinculin focal adhesions) were not localized on Glu microtubule ends. ATP, but not nonhydrolyzable analogues, induced depolymerization of Glu microtubules in cell models. Timelapse and photobleaching studies showed that ATP triggered subunit loss from the plus end. ATP breakdown of Glu microtubules was inhibited by AMP-PNP and vanadate, but not by kinase or other inhibitors. Additional experiments showed that conventional kinesin or kif3 were not involved in Glu microtubule capping. We conclude that Glu microtubules are stabilized by a plus-end cap that includes an ATPase with properties similar to kinesins.

Turnover is important for the maintenance and remodeling of the cytoskeleton during the processes of cell morphogenesis, mitosis and motility. Microtubule (MT) turnover is thought to occur by dynamic instability, growth and shortening at distal (plus) ends. Recent observation of MT release from the centrosome and depolymerization from proximal (minus) ends indicates the existence of a minus end pathway. To evaluate the relative contributions of plus and minus end pathways to turnover, we analyzed MT dynamics in a model system, the fish melanophore, a large non-motile cell with a regular radial array of long MTs. MT ends were tracked in digital fluorescence time-lapse sequences and life histories of individual MTs were analyzed using random walk theory generalized to the case of diffusion with drift. Analysis of plus end dynamics gave an apparent diffusion coefficient of D=7.5 microm2/minute. The random walk model predicts that the half-time for turnover driven solely by plus end dynamics will depend strongly on position in the cell. Based on the experimentally determined value of D, turnover of MTs near the center of a typical melanophore of radius 70 microm was calculated to require over 5 hours, a paradoxically long time. To examine MT behavior deep in the cytoplasm, we developed a novel, sequential subtraction mode of image analysis. This analysis revealed a subpopulation of MTs which shortened from their minus ends, presumably after constitutive release from the centrosome. Given the relative slowness of plus end dynamics to turn over the root of a long MT, the turnover of MTs near the cell center is determined primarily by the minus-end pathway. MTs released from the centrosome become replaced by newly nucleated ones. The relative contributions of plus and minus end pathways was estimated from the diffusion coefficient, D, for the plus end, the length distribution of MTs, t he frequency of free minus ends, and the rate of minus-end shortening. We conclude that, in large animal cells with a centrosomally focussed array of MTs, turnover occurs by a combination of plus and minus end pathways, the plus end dominating at the cell periphery and the minus end dominating near the cell center.

During the movement of chromosomes in anaphase, microtubules that extend between the kinetochores and the poles shorten. We sought to determine where subunits are lost from these microtubules during their shortening. Prophase or prometaphase cells on coverslips were injected with fluoresceinated tubulin and allowed to progress through mitosis. Immediately after the onset of anaphase, a bar-shaped beam of laser light was used to mark a domain on the kinetochore fibers by photobleaching a band, approximately 1.0 micron wide, across the spindle. In different cells, spindles were photobleached at varying distances from the chromosomes. Cells were allowed to continue in anaphase until the chromosomes had further separated. They were then lysed, fixed, and prepared for double-label immunofluorescence with an antibody to fluorescein that does not bind appreciably to bleached fluorescein, and with an antibody to tubulin. Photobleached domains of microtubules appeared as bands of reduced fluorescence in the anti-fluorescein image. However, the anti-tubulin labeling revealed that microtubules were present and continuous through the photobleached domains. In all cases, the chromosomes approached and invaded the bleached domain while the bleached domain itself remained stationary with respect to the near pole. These results demonstrate that the chromosomes move along stationary kinetochore microtubules and that depolymerization of these microtubules during anaphase takes place at the kinetochore. In contrast to the generally accepted older view that chromosomes are passive objects pulled by "traction fibers," we suggest that the kinetochore is an active participant in generating the motive force that propels the chromosome to the pole.

Clarification of the mechanism of microtubule dynamics requires an analysis of the microtubule pattern at two time points in the same cell with single fiber resolution. Single microtubule resolution was obtained by microinjection of haptenized tubulin (fluorescein-tubulin) and subsequent indirect immunofluorescence with an antifluorescein antibody. The two time points in a single cell were, first, the time of photobleaching fluorescein-tubulin, and second, the time of fixation. The pattern of fluorescence replacement in the bleached zone during this time interval revealed the relevant mechanisms. In fibroblasts, microtubule domains in the bleached zone are replaced microtubule by microtubule and not by mechanisms that affect all microtubules simultaneously. Of the models we consider, treadmilling and subunit exchange along the length do not account for this observation, but dynamic instability can since it suggests that growing and shrinking microtubules coexist. In addition, we show that the half-time for microtubule replacement is shortest at the leading edge. Dynamic instability accounts for this observation if in general microtubules do not catastrophically disassemble from the plus end, but instead have a significant probability of undergoing a transition to the growing phase before they depolymerize completely. This type of instability we call tempered rather than catastrophic because, through limited disassembly followed by regrowth, it will preferentially replace polymer domains at the ends of microtubules, thus accounting for the observation that the half-time of microtubule domain replacement is shorter with proximity to the leading edge.

Protein phosphorylation has been suggested as an important control mechanism for the events leading toward the initiation and completion of mitosis. Using a monoclonal antibody recognizing a class of phosphoproteins abundant in mitotic cells, we demonstrated the localization of a subset of these phosphoproteins to several discrete mitotic structures. Patchy immunofluorescence was present in the interphase nuclei, but a significant increase in nuclear immunofluorescence was apparent at prophase. Subsequent mitotic stages demonstrated that immunoreactive material was particularly apparent at microtubule organizing centers, namely, centrosomes, kinetochores, and midbodies. Intense centrosomal localization occurred at the prophase-prometaphase transition and persisted until the reformation of the nuclear membrane in early G1. The cytoplasm of mitotic cells also contained immunoreactive material in sharp contrast to interphase cells that exhibited no cytoplasmic fluorescent staining. Much of the diffuse immunofluorescent cytoplasmic material was removed by a brief lysis of the cells with 0.15% Triton X-100 prior to fixation. The localization of the remaining immunoreactive material after detergent lysis to mitotic microtubule organizing centers suggests that they contain phosphoprotein structural components important, perhaps, in the mitotic phase-interphase transition.

Swiss 3T3 fibroblasts and LLC-PK epithelial cells in prometaphase or metaphase were either injected with fura-2 or loaded with the acetoxymethyl ester derivative of fura-2 (fura-2 AM) and monitored by microspectrofluorimetry. With both methods of loading, we observed two aspects of intracellular free calcium (Cai) metabolism. (a) Most fibroblasts and epithelial cells exhibited a gradual rise from 75 nM in metaphase to 185 nM during cleavage, returning to baseline by early G1. (b) Mitotic Swiss 3T3 cells exhibited rapid transient Cai changes, similar to those previously reported [Poenie, M., J. Alderton, R. Y. Tsien, R. A. Steinhardt. 1985. Nature (Lond.). 315:147-149; Poenie, M., J. Alderton, R. Steinhardt, and R. Tsien. 1986. Science (Wash. DC). 233:886-889; Ratan, R., and M. L. Shelanski. 1988. J. Cell Biol. 107:993]. These Cai transients occurred repetitively, often beginning in metaphase and continuing long after daughter cell formation. Eliminating serum or calcium from the medium abolished the transients, but delayed neither the gradual Cai elevation nor anaphase onset. Co-injection of EGTA or 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) with fura-2 in calcium-free medium, but not in calcium containing medium, blocked both anaphase and the sustained Cai elevation in almost all cases. Blocked cells were rescued by returning calcium to the medium, whereupon Cai slowly but steadily rose as the cell entered anaphase. Spindle microtubules persisted through the EGTA block. Depolymerization of spindle microtubules by nocodazole also reversibly blocked anaphase onset and the sustained Cai elevation, but did not block transients. This study has revealed the following: (a) anaphase in mammalian fibroblasts and epithelial cells is not triggered by brief calcium transients; (b) anaphase is a calcium-modulated event, usually accompanied by a sustained elevation of Cai above 50 nM; (c) the elevation of Cai is dependent upon an intact spindle; and (d) fibroblasts progress through mitosis by drawing upon either intracellular or extracellular sources of calcium.

Spindle pole bodies (SPBs) were isolated from the yeast Saccharomyces cerevisiae by an adaptation of the Kleinschmidt monolayer technique. Spheroplasts prepared from the cells were lysed on an air-water interface. Spread preparations were picked up on grids, transferred to experimental test solutions, and prepared for whole-mount electron microscopy. Using purified exogenous tubulin from porcine brain tissue, the isolated SPBs were shown to nucleate the assembly of microtubules in vitro. Microtubule growth was directional and primarily onto the intranuclear face of the SPB. Neither the morphology nor the microtubule-initiating capacity of the SPB was affected by treatment with the enzymes DNase, RNase, or phospholipase although both properties were sensitive to trypsin. Analysis of SPBs at various stages of the cell cycle showed that newly replicated SPBs had the capacity to nucleate microtubules. SPBs isolated from exponentially growing cells initiated a subset of the yeast spindle microtubules equivalent to the number of pole-to-pole microtubules seen in vivo. However, SPBs isolated from cells in stationary phase and therefore arrested in G1 nucleated a number of microtubules equal to the total chromosomal and pole-to-pole tubules in the yeast spindle. This may mean that in G1-arrested cells, the SPB is associated with microtubule attachment sites of the yeast chromatin.

The origin of non-centrosomal microtubules was investigated in a variety of animal cells in culture by means of time-lapse digital fluorescence microscopy. A previous study (Keating et al. (1997) Proc. Nat. Acad. Sci. USA 94, 5078-5083) demonstrated a pathway for formation of non-centrosomal microtubules by release from the centrosome. Here we show a parallel pathway not dependent upon the centrosome. Correlative immunostaining with anti-tubulin antibodies and electron microscopy established that apparent free microtubules observed in vivo were not growing ends of long stable microtubules. Free microtubules appeared spontaneously in the cytoplasm and occasionally by breakage of long microtubules. Estimates of the frequencies of free microtubule formation suggest that it can be a relatively common rather than exceptional event in PtK1 cells and may represent a significant source of non-centrosomal microtubules. The observation of free microtubules permitted analysis of the microtubule minus end. Unlike the plus end which showed dynamic instability, the minus end was stable or depolymerized. Breakage of long microtubules generated nascent plus and minus ends; the nascent minus end was generally stable while the plus end was always dynamic. The stability of microtubule minus ends in vivo apparently provides the necessary condition for free microtubule formation in the cytoplasm. Parameters of the dynamic instability of plus ends of free microtubules were similar to those for the distal ends of long microtubules, indicating that the free microtubules were not exceptional in their dynamic behavior. Random walk analysis of microtubule end dynamics gave apparent diffusion coefficients for free and long microtubules which permitted an estimate of turnover half-times. The results support the concept that, in PtK1 cells, a pathway other than plus end dynamics is needed to account for the rapidity of microtubule turnover.

The polarity of actin filaments is fundamental for the subcellular mechanics of actin-myosin interaction; however, little is known about how actin filaments are oriented with respect to myosin in non-muscle cells and how actin polarity organization is established and maintained. Here we approach these questions by investigating changes in the organization and polarity of actin relative to myosin II during actin filament translocation. Actin and myosin II reorganization was followed both kinetically, using microinjected fluorescent analogs of actin and myosin, and ultrastructurally, using myosin S1 decoration and immunogold labelling, in cultured fibroblasts that were induced to contract by treatment with cytochalasin D. We observed rapid (within 15 minutes) formation of ordered actin filament arrays: short tapered bundles and aster-like assemblies, in which filaments had uniform polarity with their barbed ends oriented toward the aggregate of myosin II at the base of a bundle or in the center of an aster. The resulting asters further interacted with each other and aggregated into bigger asters. The arrangement of actin in asters was in sharp contrast to the mixed polarity of actin filaments relative to myosin in non-treated cells. At the edge of the cell, actin filaments became oriented with their barbed ends toward the cell center; that is, the orientation was opposite to what was observed at the edge of nontreated cells. This rearrangement is indicative of relative translocation of actin and myosin II and of the ability of myosin II to sort actin filaments with respect to their polarity during translocation. The results suggest that the myosin II-actin system of non-muscle cells is organized as a dynamic network where actin filament arrangement is defined in the course of its interaction with myosin II.

Implicit to all models for mitotic spindle assembly is the view that centrosomes are essentially permanent structures. Yet, immunofluorescence revealed that spindles in larval brains of urchin mutants in Drosophila were frequently monastral but bipolar; the astral pole contained a centrosome while the opposing anastral pole showed neither gamma tubulin nor a radial array of astral microtubules. Thus, mutations in the urchin gene seem to uncouple centrosome organization and spindle bipolarity in mitotic cells. Hypomorphic mutants showed a high frequency of monastral bipolar spindles but low frequencies of polyploidy, suggesting that monastral bipolar spindles might be functional. To test this hypothesis, we performed pedigree analysis of centrosome distribution and spindle structure in the four mitotic divisions of gonial cells. Prophase gonial cells showed two centrosomes, suggesting cells entered mitosis with the normal number of centrosomes and that centrosomes separated during prophase. Despite a high frequency of monastral bipolar spindles, the end products of the four mitotic divisions were equivalent in size and chromatin content. These results indicate that monastral bipolar spindles are functional and that the daughter cell derived from the anastral pole can assemble a functional bipolar spindle in the subsequent cell cycle. Cell proliferation despite high frequencies of monastral bipolar spindles can be explained if centrosome structure in mitotic cells is dynamic, allowing transient and benign disorganization of pericentriolar components. Since urchin proved to be allelic to KLP61F which encodes a kinesin related motor protein (Heck et al. (1993) J. Cell Biol. 123, 665-671), our results suggest that motors influence the dynamic organization of centrosomes.

We have developed a new method for determining levels of tubulin polymer, based on quantitative fluorescence detection of x-rhodamine tubulin microinjected into living cells and we have applied this method to analysis of the mitosis-interphase transition. LLC-PK cells in interphase and mitosis were microinjected, then cooled and rewarmed to drive tubulin incorporation. Total tubulin fluorescence in individual, living cells was quantified using a cooled, scientific grade CCD image sensor. Cells were then washed and lysed into a microtubule-stabilizing buffer to extract the soluble pool. Total tubulin polymer fluorescence was determined for the extracted cells in the same way as for living cells. Fluorescence images were corrected by flat-fielding and background subtraction. The ratio of extracted cell fluorescence/living cell fluorescence for individual cells, was taken as the proportion of tubulin as polymer. Cells in M-phase, G1 and random interphase were analyzed. G1 cells had almost the same proportion as random interphase cells. Mitotic cells gave a value of 90 +/- 5% of G1 cells at 37 degrees C. Within M-phase, levels of tubulin as polymer in metaphase and early anaphase were not significantly different. In contrast to the general expectation of microtubule depolymerization at anaphase onset, these results indicate that as cells exit mitosis, the overall proportion of tubulin as polymer does not change dramatically even though the mitotic spindle disassembles. We conclude that the mitosis-interphase transition is accompanied by a redistribution of tubulin at an essentially constant polymer level. Therefore, a global shift to depolymerization conditions is not the driving force for anaphase chromosome movement.

To study the possible involvement of kinesin-like molecules in mitosis a polyclonal antibody against the head domain of Drosophila kinesin heavy chain (HD antibody) was microinjected into PtK1 cells at the prophase-prometaphase transition. Progress of the cell through mitosis was recorded for subsequent detailed analysis. Cells injected with pre-immune IgG progressed through mitosis at rates similar to those for noninjected cells. After HD antibody injections, chromosomes failed to congress to an equatorial plane and cells failed to form a bipolar spindle. Rather, the spindle poles came together, resulting in a monopolar-like configuration with chromosomes arranged about the poles in a rosette. Sometimes the monopolar array moved to the margin of the cell in a way similar to anaphase B movement in normal cells. Antibody-injected cells progressed into the next cell cycle as evidenced by chromosome decondensation and nuclear envelope reformation. Anti-tubulin immunofluorescence confirmed the presence of a radial monopolar array of microtubules in injected cells. HD antibody stained in a punctate pattern in interphase and the spindle region in mitotic PtK1 cells. The antibody also reacted with spindle fibers of isolated mitotic CHO spindles and with kinetochores of isolated CHO chromosomes. Immunoblotting indicated that the major component recognized by the antibody is the 120 kDa kinesin heavy chain. At higher protein loads the antibody recognized also a 34 kDa polypeptide in PtK1 cell extracts, a 135 kDa polypeptide in a preparation of CHO spindles and a 300 kDa polypeptide in a preparation of CHO mitotic chromosomes. We conclude that a kinesin-like molecule is important for the formation and/or maintenance of the structure of mitotic spindle.

Monoclonal antibodies were prepared to identify molecular components specific to the mitotic apparatus of sea urchin eggs. The mitotic apparatus or asters induced within unfertilized eggs by taxol treatment were isolated from Strongylocentrotus purpuratus and used for immunization of mice. After fusion with spleen cells, the supernatant of hybridomas were screened in two stages by indirect immunofluorescence staining, first on isolated sea urchin mitotic spindles in 96-well microtiter plates to identify rapidly potential positive hybridomas, and second, on whole mitotic eggs on coverslips to distinguish between spindle-specific staining and adventitious contamination. Two hybridomas, SU4 and SU5, secreted antibodies reactive to microtubule-containing structures in eggs during the course of development. They preferentially stained the centrosphere both in isolated mitotic apparatus and in whole metaphase eggs, which was further confirmed by staining the isolated centrospheres with these antibodies. SU4 recognized a major 190-kD polypeptide on immunoblots as well as a species at 180 and 20 kD, whereas hybridoma SU5 stained a species at 50 kD. Thus, these polypeptides may be components of the centrosphere.

Chromosome segregation in most animal cells is brought about through two events: the movement of the chromosomes to the poles (anaphase A) and the movement of the poles away from each other (anaphase B). Essential to an understanding of the mechanism of mitosis is information on the relative movements of components of the spindle and identification of sites of subunit loss from shortening microtubules. Through use of tubulin derivatized with X-rhodamine, photobleaching, and digital imaging microscopy of living cells, we directly determined the relative movements of poles, chromosomes, and a marked domain on kinetochore fibers during anaphase. During chromosome movement and pole-pole separation, the marked domain did not move significantly with respect to the near pole. Therefore, the kinetochore microtubules were shortened by the loss of subunits at the kinetochore, although a small amount of subunit loss elsewhere was not excluded. In anaphase A, chromosomes moved on kinetochore microtubules that remained stationary with respect to the near pole. In anaphase B, the kinetochore fiber microtubules accompanied the near pole in its movement away from the opposite pole. These results eliminate models of anaphase in which microtubules are thought to be traction elements that are drawn to and depolymerized at the pole. Our results are compatible with models of anaphase in which the kinetochore fiber microtubules remain anchored at the pole and in which microtubule dynamics are centered at the kinetochore.

A procedure was developed for the conjugation of vimentin with biotin. Biotinylated vimentin was then microinjected into BHK-21 cells and the fate of the labeled protein was determined at various times postinjection by indirect immunofluorescence. Microinjected vimentin could be traced through a specific sequence of morphological changes ultimately resulting in the formation of a filamentous network. The injected protein was first detected in spots dispersed throughout the cytoplasm. Subsequently, these spots appeared to cluster near the nucleus where they merged into a diffuse "cap." This cap coincided with a concentration of endogenous intermediate filaments and eventually gave rise to a filamentous network that was coincident with the endogenous intermediate filament network as determined by double-label immunofluorescence. The results indicate that the incorporation of exogenous vimentin into a filamentous network is initiated in a perinuclear region and progresses in a polarized fashion toward the cell surface.

Microtubule-associated proteins (MAPs) from calf brain were fluorescently labeled with 6-iodoacetamido fluorescein (I-AF). The modified MAPs (especially enriched for MAP2) were fully active in promoting tubulin polymerization in vitro and readily associated with cytoplasmic filaments when microinjected into living cultured cells. Double-labeling experiments indicated that the microinjected AF-MAPs were incorporated predominantly, if not exclusively, into cytoplasmic microtubules in untreated cells or paracrystals induced within vinblastine-treated cells. Similar results were obtained with different cell types (neuronal, epithelial, and fibroblastic) of diverse origin (man, mouse, chicken, and rat kangaroo). Mobility measurements of the microinjected AF-MAPs using the method of fluorescence-photobleaching recovery (FPR) revealed two populations of AF-MAPs with distinct dynamic properties: One fraction represents the soluble pool of MAPs and is mobile with a diffusion coefficient of D = 3 X 10(-9) cm2/s. The other fraction of MAPs is associated with the microtubules and is essentially immobile on the time scale of FPR experiments. However, it showed slow fluorescence recovery with an apparent half time of approximately 5 min. The slow recovery of fluorescence on defined photobleached microtubules occurred most probably by the incorporation of AF-MAPs from the soluble cytoplasmic pool into the bleached area. The bleached spot on defined microtubules remained essentially immobile during the slow recovery phase. These results suggest that MAPs can associate in vivo with microtubules of diverse cell types and that treadmilling of MAP2-containing microtubules in vivo, if it exists, is slower than 4 micron/h.

To investigate the detailed distributions of microtubules in cultured cells and intact tissues we developed a reversible embedment method for antibody labeling of sectioned material. Fixed tissues were infiltrated with fully polymerized polymethylmethacrylate dissolved in an organic solvent. Evaporation of the solvent left the tissue embedded in hard plastic. After sectioning by conventional methods, the plastic was extracted and sections were processed for indirect immunofluorescence to label microtubules. Clear images of microtubules were observed in sections of cultured epithelial cells, intact chick intestinal epithelium, and dividing sea urchin eggs. Microtubules in the differentiated epithelium of the chick intestine generally paralleled the long axis of the cells and did not focus on a microtubule-organizing center. Mitotic cells of the intestinal epithelium appeared similar to the mitotic cells of epithelial lines in culture. In sections of dividing sea urchin eggs detailed images of spindle and astral fibers were revealed. Immunoelectron microscopic labeling for tubulin was performed on sections of Pt K1 cells using secondary antibodies adsorbed to 20-nm gold particles. Semi-thick sections viewed by high-voltage electron microscopy showed both the overall distribution of microtubules and their detailed interactions with other cellular organelles. Mitochondria were often aligned along labeled microtubules. Reversible embedment cytochemistry should provide a general method for high resolution labeling of cells and tissues with affinity probes.