Microtubule dynamics and chromosome motion visualized in living anaphase cells

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.     

Microtubular cytoskeletons of the trophic cells of five eumycetozoans

Summary The trophic cells of the protostelids,Ceratiomyxella tahitiensis, Cavostelium apophysatum, Planoprotostelium aurantium, andClastostelium recurvatum, and the reduced myxomycete,Echinostelium bisporum, were examined with indirect immunofluorescence to determine the overall structure of the microtubular cytoskeletons of each type of cell. All five species have a distinct flagellar apparatus in the amoeboflagellate state, but they vary with respect to the presence of body microtubules, those microtubules which do not focus on the flagellar apparatus. The obligate amoebae ofC. tahitiensis, C. apophysatum andC. recurvatum all have extensive microtubular cytoskeletons, but each is unique to its respective species. The obligate amoeba ofC. tahitiensis has scattered microtubules which often describe curved paths. The microtubules never appear to focus on MTOCs. The microtubular cytoskeleton ofC. apophysatum consists of relatively straight, rigid appearing microtubules. Small subpopulations of these microtubules radiate from MTOCs near the nucleus. The obligate amoeba ofC. recurvatum has a cytoskeleton consisting of numerous microtubules which radiate from a perinuclear MTOC and fill the body of the cell. These results correlate well with earlier ultrastructural observations which suggest that the amoeboflagellate state is homologous in these mycetozoans and that the obligate amoebae, when present, are unique to the various lineages in which they appear.     

Cytoskeletal Architecture of Dermal Chromatophores of the Freshwater Teleost Oryzias latipes

Cytoskeletal construction of dermal chromatophores of Orgzias latipes was studied by immunofluorescence microscopy. A microtubule system was most prominent in melanophores where a large number of microtubules emanated from the center of the cell. Xanthophores had an arrangement basically similar to that of melanophores, though the radial pattern became more irregular in the peripheral region where intersecting wavy microtubules were quite frequent. Oval-shaped leucophores exhibited the least-developed microtubule system, where the limited number of microtubules formed a loose basket-like architecture. Intermediate filaments were ubiquitously present in all types of chromatophores and were found to be vimentin-immunoreactive. Examination of doubly-labeled cells indicated that vimentin filaments had similar distribution patterns with microtubules. Orderly arranged bundles of actin filaments were found only in xanthophores, while in melanophores and xanthophores, actin expression was diffuse without displaying a conspicuous filamentous organization. Colchicine treatment induced depolymerization of microtubules and retraction of dendrites in varying degrees in cells in culture and in situ. Melanophores in culture are very sensitive to the treatment while xanthophores appeared to be more resistant in respect to the maintenance of cell morphology.     

Organization of tubulin in normal and transformed rat kidney cells

We have carried out a quantitative biochemical and ultrastructural study of tubulin and microtubules in a normal rat kidney (NRK) cell line and its viral transformant (442) in culture. Under equivalent culture conditions, both cell lines contain the same amount of tubulin according to a colchicine-binding assay. The normal and transformed cells differ significantly, however, with respect to the state of organization of their tubulin. Counts of microtubules in sectioned cells indicate that NRK cells have almost twice as many microtubules per unit area of cytoplasm as the 442 cells. Centrifugation studies, on the other hand, show that 442 cells have almost twice as much pelletable tubulin as the NRK cells. We propose, therefore, that the transformed cells contain a large amount of tubulin which is in some alternative aggregate form that is not morphologically detectable as microtubles in the cytoplasm     

Evidence for rapid structural and functional changes of the melanophore microtubule-organizing center upon pigment movements

Melanophores of the angelfish, pterophyllum scalare, have previously been shown to display approximately 2,400 microtubules in cells wih pigment dispersed; these microtubules radiate from a presumptive organizing center, the central apparatus (CA), and their number is reduced to approximately 1,000 in the state with aggregated pigment (M. Schliwa and U. Euteneuer, 1978, J. Supramol. Struct. 8:177-190). In an attempt to elucidate the factors controlling this rapid reorganization of the microtubule apparatus, structure and function of the CA have been investigated under different physiological conditions. As a function of the state of pigment distribution, melanophores differ markedly with respect to CA organization. A complex of dense amorphous aggregates and associated fuzzy material, several micrometers in diameter, surrounds the centrioles in cells with pigment dispersed, and numerous microtubules emanate from this complex in a radial fashion. In the aggregated state, on the other hand, few microtubules are observed in the pericentiolar region, and the amount of fibrous material is greatly reduced. These changes in CA morphology as a function of the state of pigment distribution are associated with a marked difference in its capacity to initiatiate the assembly of microtubules from exogenous pure porcine brain tubulin in lysed cell preparations. After complete removal of preexisting microtubules, cells lysed in the dispersed state into a solution of 1-2 mg/ml pure tubulin have numerous microtubules associated with the CA in radial fashion, while cells lysed in the aggregated state nucleate the assembly of only a few microtubules. We conclude that it is the activity of the CA that basically regulates the expression of microtubules. This regulation is achieved through a variation in the capacity to initiate microtubule assembly. Increase or decrease in the amount of dense material, as readily observed in the cell system studied here, seems to be a morphologic expression of such a physiologic function.     

Appearances of microtubules after various fixative procedures, and comparison with the appearances of tobacco mosaic virus.

Microtubules in crane-fly spermatids appeared altered when the glutaraldehyde-fixed cells were not postfixed with osmium tetroxide. The cytoplasmic microtubules were altered more than the doublet microtubules. Addition of osmium tetroxide after dehydration did not produce appearances identical with those of microtubules postfixed directly after glutaraldehyde, and thus at least some alterations occurred during dehydration, possibly due to extraction of microtubule-associated lipid. The omission of osmium tetroxide postfixation did not cause drastic alterations in the appearances of either tobacco mosaic virus (TMV), or polymerized tobacco mosaic virus protein (without RNA), suggesting that microtubule stability is different from TMV stability (with respect to the embedment procedure). The electron-dense stain associated with embedded-sectioned TMV is predominantly outside the TMV protein, as demonstrated by the known distribution of TMV protein compared with the dimensions of sectioned TMV and negatively stained TMV. The same might hold true for microtubules, as evidenced by the dimensions of negatively stained, isolated brain microtubules compared with those of embedded and sectioned brain microtubules.     

Microtubule Formation in Regenerating Terminal Buds of the Silurid Fish, Corydoras aeneus

Regenerating terminal buds of Corydoras aeneus were observed by electron microscopy to determine how terminal buds developed with respect to microtubule formation. After surgical removal of the fish barbel, it and the terminal bud began to regenerate 1.5 weeks later at 25°C. The regenerating terminal buds were ovoid in shape and contained three types of cells. The first type of cell had extended cellular processes which contained numerous microtubules and tubules. A bundle of three or four microtubules ran parallel to the long axis of the cellular process. Receptor villi protruded from the cell two weeks later, suggesting that it is a receptor cell. The second cell type, which appeared 1.5 weeks after barbel removal, had numerous microtubules oriented along the long axis of the cellular process; and numerous dense granules appeared two weeks later, suggesting that it is a supporting cell. The third type of cell observed was a basal cell without cellular processes. These results suggest that microtubule formation plays an important role in the elongation of regenerating terminal buds.     

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.     

Identification with cellular microtubules of one of the co-assemlbing microtubule-associated proteins.

In this paper we describe a procedure for detecting proteins associated with cytoplasmic microtubules in vivo. Detergent-extracted cytoskeletons of NIL8 hamster cells are prepared under conditions which preserve the microtubules. The cytoskeletons are then extracted in the presence of calcium, which depolymerizes the microtubules and quantitatively extracted cytoskeletons are prepared from cells that have been incubated with colchicine. The cytoskeletons from these cells contain no microtubules or tubulin. Electrophoretic analysis of the calcium extracts of the colchicine-treated and untreated cells reveals several radioactively labeled polypeptides. There is, however, no apparent quantitative or qualitative difference between the two extracts other than the tubulin polypeptides. Each of the extracts is mixed with an excess of unlabeled calf brain microtubule protein and carried through cycles of temperature-dependent microtubule assembly. Distinct species from each extract co-assemble at a constant ratio, but only one polypeptide is uniquely derived from cells containing intact microtubules. The molecular weight of this polypeptide is similar to that proposed for the tau species detected in brain microtubule preparations.     

The mitotic spindle and actin tails

To segregate their chromosomes, eukaryotic cells rely on a dynamic structure made of microtubules: the mitotic spindle. This structure can form in cells lacking centrosomes, because their chromosomes also nucleate microtubules. This second assembly pathway is observed even in some cells that naturally have centrosomes, for example when the centrosomes are ablated by laser surgery. Recent results have started to address the complementary question of whether centrosome-nucleated microtubules alone could sustain the formation of a functional mitotic spindle. We wonder in this respect whether lower eukaryotes such as yeasts are different from higher eukaryotes such as vertebrates.     

The dynamics of reconstitution of microtubules around the cell center after cooling

In interphase PE cells, after cooling (2 h at 0 degree C), some microtubules are retained in the cytoplasm. After the transfer of the cells to a thermostat (37 degrees C), the reconstitution of the microtubule network begins near the cell center. At this time in most of the cells around the center one can see the electron-dense foci of convergence of microtubules which then disappear. The number of microtubules diverging radially from the mother centriole reaches a maximum after 15 to 16 min, that of microtubules growing from the daughter centriole 12 min after the cells are placed at 37 degrees C. 45 min after the heating started the number of radially diverging microtubules somewhat exceeds the control level. These data show that microtubules are associated with the centers only during their growth. The mature microtubule is separated from the center and may be replaced by a new one. Thus, most, of not all, microtubules originate from the cell center, but at any moment only some of the microtubules are associated with it.     

ULTRASTRUCTURAL ANALYSIS OF MITOTIC SPINDLE ELONGATION IN MAMMALIAN CELLS IN VITRO : Direct Microtubule Counts

The mitotic spindle of many mammalian cells undergoes an abrupt elongation at anaphase. In both cultured rat kangaroo (strain PtK₁) and Chinese hamster (strain Don-C) fibroblasts, the distance from pole to pole at metaphase doubles during anaphase and telophase. In order to determine the organization and distribution of spindle microtubules during the elongation process, cells were fixed and flat embedded in Epon 812. Selected cells were photographed with the phase-contrast microscope and then serially sectioned perpendicular to the major spindle axis. Microtubule profiles were counted in selected sections, and the number was plotted with respect to position along the spindle axis. Interpretation of the distribution profiles indicated that not all interpolar microtubules extended from pole to pole. It is estimated that 55–70% of the interpolar microtubules are overlapped at the cell equator while 30–45% extend across the equator into both half spindles. This arrangement appeared to persist from early anaphase (before elongation) until telophase after the elongation process. Although sliding or shearing of microtubules may occur in the spindle, such appears not to be the mechanism by which the spindle elongates in anaphase. Instead, our data support the hypothesis that spindle elongation occurs by growth of prepositioned microtubules which "push" the poles apart.     

Early events in rice somatic embryogenesis: Changes in cortical microtubule distribution and orientation

Summary The changes in the microtubular pattern during the first few days of somatic embryo induction were studied using the rice scutellum epithelium system. A clear difference in the distribution and orientation of the microtubules could be found as early as 12 h after culturing. In the control treatment, the microtubules tended to congregate near the terminal part of the cells and had an oblique to transverse orientation with respect to the long axis of the cells. In the hormone-treated explants, the microtubules were more evenly distributed throughout the cells as compared to the control treatment. The microtubules initially had an oblique orientation similar to the control. However, by 16 h, the majority of microtubules were aligned parallel to the long axis of the cells. The scutellum epithelial cells in both the control- and hormone-treated explants divided periclinally at about 48 h. In the control treatment, the scutellum epithelial cells gradually became vacuolated over time, while the hormone-treated explants remained densely cytoplasmic. The fluorescence staining intensity gradually declined in the control, whereas a distinct staining pattern of microtubules remained in the hormone-treated explants.     

The distribution of tau and HMW microtubule-associated proteins in different cell types

To investigate the distribution of the tau and HMW microtubule-associated proteins (MAPS) and their relationship to microtubules in vivo, we have examined a wide variety of avian and mammalian cell types by immunofluorescence with antisera to these two proteins. Anti-HMW serum stains cytoplasmic microtubules in all mammalian cell types so far examined. However, anti-tau serum did not stain cytoplasmic microtubules in rat glial cells or in pig kidney cells. In mammalian neurons, fibroblasts and neuroblastoma cells, the staining of microtubules with both sera was similar. Anti-HMW serum did not stain primary cilia or cilia on isolated tracheal epithelial cells, whereas anti-tau serum did stain these ciliary microtubules. We believe these results indicate that some types of microtubules may be associated with only the tau or the HMW protein, whereas others may be associated with both tau and HMW protein. With respect to avian cells, anti-HMW serum did not stain microtubules in any of the three cell types examined, whereas the anti-tau serum stained them in two cell types. Furthermore, double diffusion tests indicated that anti-pig tau serum will precipitate both pig brain tau and tau protein isolated from chick brain, whereas anti-HMW serum will precipitate only pig brain and not chick brain HMW protein. We believe tau protein is antigenically similar in both avian and mammalian cells, whereas the HMW protein from these two sources is antigenically distinct.     

Effect of colchicine on rat mast cells

In the mast cell, a well-developed array of microtubules is centered around the centrioles. Complete loss of microtubules is observed when mast cells are treated with 10(-5) M colchicine for 4 h at 37 degrees C. The loss of ultrastructurally evident microtubules is associated with a marked change in the shape of mast cells from spheroids to highly irregular, frequently elongated forms with eccentric nuclei. In colchicine-treated cells the association of nucleus, Golgi apparatus, and centrioles is also lost. Mast cells exposed to 10(-5) M colchicine for 4 h at 37 degrees C retain 80% of their capacity to release histamine when stimulated by polymyxin B. Exocytosis is evident in stimulated cells pretreated with colchicine and lacking identifiable microtubules. When the conditions of exposure of mast cells to colchicine are varied with respect to the concentration of colchicine, the length of exposure, and the temperature of exposure, dissociation between deformation of cell shape and inhibition of histamine secretion is observed. These observations indicate that microtubules are not essential for mast cell histamine release and bring into question the assumption that the inhibitory effect of colchicine on mast cell secretion depends on interference with microtubule integrity.     

The oral apparatus of Tetrahymena pyriformis, strain WH-6. IV. Observations on the organization of microtubules and filaments in the isolated oral apparatus and the differential effect of potassium chloride on the stability of oral apparatus microtubules.

This report is an ultrastructural analysis of the organization of the isolated oral apparatus of Tetrahymena pyriformis, strain WH-6, syngen 1. Attention has been focused on the organization of microtubules and filaments in oral apparatus membranelles. Oral apparatus membranellar basal bodies were characterized with respect to structural differentiations at the distal and proximal ends. The distal region of membranellar basal bodies contains the basal plate, accessory microtubules and filaments. The proximal end contains a dense material from which emanate accessory microtubules and filaments. There are at least two possibly three different arrangements of accessory structures at the proximal end of membranellar basal bodies. All membranellar basal bodies appear to have a dense material at the proximal end from which filaments emanate. Some of these basal bodies have accessory microtubules and filaments emanating from this dense material. A possible third arrangement is represented by basal bodies which have lateral projections, from the proximal end, of accessory microtubules and filaments which constitute cross or peripheral connectives. There are at least three examples of direct associations between oral apparatus microtubules and filaments: (1) filaments which form links between basal body triplet microtubules, (2) filaments which link the material of the basal plate to internal basal body microtubules, (3) filaments which link together microtubule bundles from membranellar connectives. KCl extraction of the isolated oral apparatus resulted in the selective solubilization of oral apparatus basal bodies, remnants of ciliary axonemes and fused basal plates. Based on their response to KCl extraction two distinct sets of morphologically similar micro tubules can be identified: (a) microtubules which constitute the internal structure of basal bodies and ciliary axonemes, (b) microtubules which constitute the fiber connectives between basal bodies.     

Formation and maintenance of viroplasmic centers in Tipula iridescent virus-infected mosquito cells with deranged cytoskeletons

We have examined the role of cytoskeletal elements with respect to the formation and maintenance of viroplasmic centers (VCs) in Tipula iridescent virus (TIV)-infected mosquito Aedes albopictus (C6/36) cells. Filamentous systems consisting of microtubules and microfilaments were detected by immunofluorescence microscopy. Inoculation of cells with TIV resulted in an alteration of microtubule and microfilament organization whether or not VCs developed. The formation of short arrays of microtubules induced by taxol or the depolymerization of microtubules by colchicine, as observed by immunofluorescence microscopy, had no apparent effect upon the development of VCs as detected by Hoechst staining and electron microscopy. The dissolution of the actin-containing filamentous system by cytochalasin B also had no effect upon development. We conclude from these results that microtubules and microfilaments are not involved in the formation or maintenance of VCs in TIV-infected A. albopictus (C6/36) cells.     

Mechanics of microtubule bundles in pillar cells from the inner ear.

The mechanical properties of cross-linked microtubule bundles were measured from outer pillar cells isolated from the mammalian inner ear. Measurements were made using a three-point bending test and were incorporated into a mathematical model designed to distinguish between the stiffness contributions from microtubules and their cross-linking proteins. Outer pillar cells were composed of 1000-3000 parallel bundled microtubules in a square array that was interdigitated and cross-linked with actin filaments. The average midpoint bending stiffness of intact cells was 7 x 10(-4) N/m. After removal of both the actin filaments and cross-links with detergent in the presence of DNase I, the square array was disrupted and the stiffness decreased by a factor of 4, to 1.7 x 10(-4) N/m. The bending modulus for individual microtubules was calculated to be 7 x 10(-23) Nm2, and the Young's modulus for these 15 protofilament microtubules was 2 x 10(9) Pa. The shear modulus between microtubules in intact cells was calculated to be 10(3) Pa. It was concluded that cross-linking proteins provided shear resistance between microtubules, which resulted in a fourfold increase in stiffness. The model can be used to estimate the mechanical properties of cross-linked microtubule bundles in cells from which direct measurements are not available.     

The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.     

Loss of Spatial Control of the Mitotic Spindle Apparatus in a Chlamydomonas Reinhardtii Mutant Strain Lacking Basal Bodies

The bld2-1 mutation in the green alga Chlamydomonas reinhardtii is the only known mutation that results in the loss of centrioles/basal bodies and the loss of coordination between spindle position and cleavage furrow position during cell division. Based on several different assays, bld2-1 cells lack basal bodies in >99% of cells. The stereotypical cytoskeletal morphology and precise positioning of the cleavage furrow observed in wild-type cells is disrupted in bld2-1 cells. The positions of the mitotic spindle and of the cleavage furrow are not correlated with respect to each other or with a specific cellular landmark during cell division in bld2-1 cells. Actin has a variable distribution during mitosis in bld2-1 cells, but this aberrant distribution is not correlated with the spindle positioning defect. In both wild-type and bld2-1 cells, the position of the cleavage furrow is coincident with a specialized set of microtubules found in green algae known as the rootlet microtubules. We propose that the rootlet microtubules perform the functions of astral microtubules and that functional centrioles are necessary for the organization of the cytoskeletal superstructure critical for correct spindle and cleavage furrow placement in Chlamydomonas.