Microtubule-kinetosome relationships in the motile cells of the Blastocladiales

Summary Motile cells of four members of the fungal order Blastocladiales have been examined with respect to distribution of intracellular microtubules. In all cases, the cellular microtubules originate from a sleeve of electron opaque material which surrounds the proximal third of the kinetosome. The microtubules run forward in the zoospores, ensheathing the nucleus and the nuclear cap before terminating at the anterior ends of the cells. Each cell contains 27 microtubules which are arranged in 9 groups of 3 tubules each. The significance of these observations with respect to centers of organization for microtubules is discussed.This work was supported by a grant (GB-4529) from the National Science Foundation.     

Signals from the spindle midzone are required for the stimulation of cytokinesis in cultured epithelial cells.

The interaction between the mitotic spindle and the cellular cortex is thought to play a critical role in stimulating cell cleavage. However, little is understood about the nature of such interactions, particularly in tissue culture cells. We have investigated the role of the spindle midzone in signaling cytokinesis by creating a barrier in cultured epithelial cells with a blunted needle, to block signals that may emanate from this region. When the barrier was created during metaphase or early anaphase, cleavage took place only on the sides of the cortex facing the mitotic spindle. Microtubules on the cleaving side showed organization typical of that in normal dividing cells. On the noncleaving side, most microtubules passed from one side of the equator into the other without any apparent organization, and actin filaments failed to organize in the equatorial region. When the barrier was created after the first minute of anaphase, cells showed successful cytokinesis, with normal organization of microtubules and actin filaments on both sides of the barrier. Our study suggests that transient signals from the midzone of early anaphase spindles are required for equatorial contraction in cultured cells and that such signaling may involve the organization of microtubules near the equator.     

Microtubule reorganization,cell wall synthesis and establishment of the axis of elongation in regenerating protoplasts of the algaMougeotia

Summary Microtubule reorganization and cell wall deposition have been monitored during the first 30 hours of regeneration of protoplasts of the filamentous green algaMougeotia, using immunofluorescence microscopy to detect microtubules, and the cell-wall stain Tinopal LPW to detect the orientation of cell wall microfibrils. In the cylindrical cells of the alga, cortical microtubules lie in an ordered array, transverse to the long axis of the cells. In newly formed protoplasts, cortical microtubules exhibit some localized order, but within 1 hour microtubules become disordered. However, within 3 to 4 hours, microtubules are reorganized into a highly ordered, symmetrical array centered on two cortical foci. Cell wall synthesis is first detected during early microtubule reorganization. Oriented cell wall microfibrils, co-aligned with the microtubule array, appear subsequent to microtubule reorganization but before cell elongation begins. Most cells elongate in the period between 20 to 30 hours. Elongation is preceded by the aggregation of microtubules into a band intersecting both foci, and transverse to the incipient axis of elongation. The foci subsequently disappear, the microtubule band widens, and microfibrils are deposited in a band which is co-aligned with the band of microtubules. It is proposed that this band of microfibrils restricts lateral expansion of the cells and promotes elongation. Throughout the entire regeneration process inMougeotia, changes in microtubule organization precede and are paralleled by changes in cell wall organization. Protoplast regeneration inMougeotia is therefore a highly ordered process in which the orientation of the rapidly reorganized array of cortical microtubules establishes the future axis of elongation.     

Organization of neuronal microtubules in the nematode Caenorhabditis elegans

We have studied the organization of microtubules in neurons of the nematode Caenorhabditis elegans. Six neurons, which we call the microtubule cells, contain bundles of darkly staining microtubules which can be followed easily in serial-section electron micrographs. Reconstruction of individual microtubules in these cells indicate that most, if not all, microtubules are short compared with the length of the cell process. Average microtubule length varies characteristically with cell type. The arrangement of microtubules gives an overall polarity to each bundle: the distal ends of the microtubles are on the outside of the bundle, whereas the proximal ends are preferentially inside. The distal and proximal ends each have a characteristic appearance indicating that these microtubules may have a polarity of their own. Short microtubules in processes of other neurons in C. elegans have also been observed.     

Microtubules of guard cells are light sensitive

Guard cells of stomata are characterized by ordered bundles of microtubules radiating from the ventral side toward the dorsal side of the cylindrical cell. It was suggested that microtubules play a role in directing the radial arrangement of the cellulose micro-fibrils of guard cells. However, the role of microtubules in daily cycles of opening and closing of stomata is not clear. The organization of microtubules in guard cells of Commelina communis leaves was studied by analysis of three-dimensional immunofluorescent images. It was found that while guard cell microtubules in the epidermis of leaves incubated in the light were organized in parallel, straight and dense bundles, in the dark they were less straight and oriented randomly near the stomatal pore. The effect of blue and red light on the organization of guard cell microtubules resembled the effects of white light and dark respectively. When stomata were induced to open in the dark with fusicoccin, microtubules remained in the dark configuration. Furthermore, when incubated in the light, guard cell microtubules were more resistant to oryzalin. Similarly, microtubules of Arabidopsis guard cells, expressing green fluorescent protein-tubulin alpha 6, were disorganized in the dark, but were organized in parallel arrays in the presence of white light. The dynamics of microtubule rearrangement upon transfer of intact leaves from dark to light was followed in single stomata, showing that an arrangement of microtubules typical for light conditions was obtained after 1 h in the light. Our data suggest that microtubule organization in guard cells is responsive to light signals.     

Microtuble organization in Xenopus eggs during the first cleavage and its role in cytokinesis

It has been suggested that the organization of microtubules during mitosis plays an important role in cytokinesis in animal cells. We studied the organization of microtubules during the first cleavage and its role in cytokinesis of Xenopus eggs. First, we examined the immunofluorescent localization of microtubules in Xenopus eggs at various stages during the first cleavage. The astral microtubules that extend from each of the two centrosomes towards the division plane meet and connect with each other at the division plane as cytokinesis proceeds. The microtubular connection thus advances from the animal pole to the vegetal pole, and its leading edge is located approximately beneath the leading edge of the cleavage furrow. Furthermore, an experiment using nocodazole suggests that microtubules have an essential role in advancement of the cleavage furrow, but neither in contraction nor maintenance of the already formed contractile ring which underlies the cleavage furrow membrane. These results suggest that the astral microtubules play an important role in controlling the formation of the contractile ring in Xenopus eggs.     

NuMA is required for the organization of microtubules into aster-like mitotic arrays

NuMA (Nuclear protein that associates with the Mitotic Apparatus) is a 235-kD intranuclear protein that accumulates at the pericentrosomal region of the mitotic spindle in vertebrate cells. To determine if NuMA plays an active role in organizing the microtubules at the polar region of the mitotic spindle, we have developed a cell free system for the assembly of mitotic asters derived from synchronized cultured cells. Mitotic asters assembled in this extract are composed of microtubules arranged in a radial array that contain NuMA concentrated at the central core. The organization of microtubules into asters in this cell free system is dependent on NuMA because immunodepletion of NuMA from the extract results in randomly dispersed microtubules instead of organized mitotic asters, and addition of the purified recombinant NuMA protein to the NuMA-depleted extract fully reconstitutes the organization of the microtubules into mitotic asters. Furthermore, we show that NuMA is phosphorylated upon mitotic aster assembly and that NuMA is only required in the late stages of aster assembly in this cell free system consistent with the temporal accumulation of NuMA at the polar ends of the mitotic spindle in vivo. These results, in combination with the phenotype observed in vivo after the prevention of NuMA from targeting onto the mitotic spindle by antibody microinjection, suggest that NuMA plays a functional role in the organization of the microtubules of the mitotic spindle.     

Electron-microscopic and immunohistochemical study of the reorganization of the microtubule system in granular cells of frog urinary bladder after water transport induction]

Structural changes in organization of the microtubule system in granular cells of frog urinary bladder after water transport induction by vasopressin were studied by methods of electron microscopy and immunocytochemistry. It is shown that in steady-state conditions microtubules form a wide network equally distributed in the whole cytoplasm of granular cells. After vasopressin action, the amount of microtubules increases in the apical region of the cytoplasm. A predominant orientation of microtubules, perpendicular to the apical membrane direction, appears. A structural association of microtubules with specific granules and large vacuoles was observed. A supposition is advanced about association of the described microtubule system reorganization with the activation of vectorial intracellular transport occurring after transepithelial water transport induction.     

Injury toNitella internodal cells alters microtubule organization but microtubules are not involved in the wound response

Summary The arrangement and relative stability of cortical microtubules during and after wound induction in internodal cells ofNitella flexilis andNitella pseudoflabellata were examined by immunofluorescence and by microinjection of fluorescently tagged tubulin. The formation of cellulosic wall appositions (wound walls), induced by treatment with 5×10^–2MCaCl₂, was identicalin young, growing cells and older non-growing internodes, suggesting that the initial microtubule pattern, which differs in growing and non-growing cells, does not influence wound wall formation. Depolymerization of microtubules with oryzalin did not alter wound wall morphology and microtubules were not detected during wound wall formation. After cessation of wound wall growth, microtubules were once again found in the wound site but these were always randomly oriented, even in young cells where the surrounding microtubules were organized into transverse arrays. Microtubules were similarly randomized in chloroplast-free windows induced by laser irradiation. Analysis of microtubule organization in living cells revealed that the microtubules in wound sites are less stable than the microtubules of adjacent transversely oriented arrays. The results indicate that although wounding can alter the relative stability and spatial organization of cortical microtubules, microtubules are neither involved in vesicle transport nor the construction of cellulosic wound walls.Abbreviations AFW artificial fresh water - BSA bovine serum albumin - DMSO dimethyl sulfoxide - FITC fluorescein isothiocyanate - PBS phosphate-buffered saline     

Tubulin-specific antibody and the expression of microtubules in 3T3 cells after attachment to a substratum : Further evidence for the polar growth of cytoplasmic microtubules in vivo

Mouse 3T3 cells were allowed to attach to and spread on glass. The expression of cytoplasmic microtubules during the respreading process was monitored by immunofluorescence microscopy using monospecific antibody against tubulin. During radial attachment of the cells a ring of flattened cytoplasm is seen around the nucleus. Cytoplasmic microtubules then enter this spreading ring from the perinuclear region and elongate toward the plasma membrane. At later times microtubules appear perpendicular to the plasma membrane and seem to be in intimate contact with it giving the impression that they “stretch” the cytoplasm. When the cells assume their typical fibroblastic shape numerous microtubules are seen. They traverse the cytoplasm. Some come close to the plasma membrane and some bend to conform to the shape of the cell. Changes in microtubular organization correlate well with changes in cell shape. These results together with our previous observations on the assembly of cytoplasmic microtubules upon recovery from colcemid treatment suggest that microtubules may grow as polar structures from a microtubular organizing center towards the plasma membrane. The hypothesis that cytoplasmic microtubules might confer polarity on the cell is discussed.     

Microtubule organization and the effects of GFP-tubulin expression in dictyostelium discoideum

We have labeled microtubules in living Dictyostelium amoebae by incorporation of a GFP-alpha-tubulin fusion protein. The GFP-alpha-tubulin incorporates into microtubules and, as reported by others [Neujahr et al., 1998], the labeled microtubules are highly motile. Electron microscopy (EM) analysis of the distribution and organization of microtubules in the amoebae shows that some cytoplasmic microtubules form close associations. These associations could allow motor proteins attached to one microtubule to walk along an adjacent microtubule and thus generate some of the observed motility. Protein blot analysis indicates that the GFP-alpha-tubulin incorporates into microtubules at a lower efficiency than does the endogenous alpha-tubulin. EM and immunofluorescence (IF) analyses suggest that the GFP-alpha-tubulin interferes with microtubule nucleation. We have also observed an increased sensitivity of the GFP-alpha-tubulin expressing cells to blue light, as compared to wild-type cells. These results suggest that although GFP-alpha-tubulin can be used as a marker for microtubules in living cells, the use of this marker is not recommended for certain types of studies such as assembly dynamics.     

Identification of multiple microtubule initiating sites in mouse neuroblastoma cells.

Mouse neuroblastoma N-18 cells can be induced by serum deprivation to sprout multiple neurite-like processes which contain many microtubules. Mitotic drugs such as colcemid and colchicine depolymerize these microtubules and the cells lose their processes. Reappearance of microtubules after removal of the drugs was followed by immunofluorescence microscopy using tubulin specific antibodies. At early recovery times multiple star-like structures which contained tubulin were detected in the perinuclear are and in the cytoplasm of individual cells. The mean number seen per cell as approximately 5. Their formation preceeded the organization of the complex microtubular networks typical of N-18 cells. The probable action of these structures as microtubular organization centers (MTOCs) is discussed. Multiple structures were detected during recovery from the influence of mitotic drugs both in previously induced and non-induced N-18 cells, suggesting that N-18 cells harbour the potential of formation of multiple organization centers even without previous induction. We discuss the possibility that differentiation of neuroblastoma N-18 cells may require microtubular organization centers.     

Role of the centrosome in organizing the interphase microtubule array: properties of cytoplasts containing or lacking centrosomes

To study the role of the centrosome in microtubule organization in interphase cells, we developed a method for obtaining cytoplasts (cells lacking a nucleus) that did or did not contain centrosomes. After drug- induced microtubule depolymerization, cytoplasts with centrosomes made from sparsely plated cells reconstituted a microtubule array typical of normal cells. Under these conditions cytoplasts without centrosomes formed only a few scattered microtubules. This difference in degree of polymerization suggests that centrosomes affect not only the distribution but the amount of microtubules in cells. To our surprise, the extent of microtubules assembled increased with the cell density of the original culture. At confluent density, cytoplasts without centrosomes had many microtubules, equivalent to cytoplasts with centrosomes. The additional microtubules were arranged peripherally and differed from the centrosomal microtubules in their sensitivity to nocodazole. These and other results suggest that the centrosome stabilizes microtubules in the cell, perhaps by capping one end. Microtubules with greater sensitivity to nocodazole arise by virtue of change in the growth state of the cell and may represent free or uncapped polymers. These experiments suggest that the spatial arrangement of microtubules may change by shifting the total tubulin concentration or the critical concentration for assembly.     

朱顶红精细胞超微结构观察

精细胞是双受精作用的直接参与者,是生殖生物学中的重点研究对象之一。以往的研究表明,应用连续超薄切片和计算机辅助三维重组技术,结合免疫荧光定位,发现两个精细胞在体积和细胞器含量上存在着差异,即精子的二型性,而且与营养细胞核三者构成紧密功能单位即雄性生殖单位（ＭＧＵ）,微管对精细胞的性状的确定、运动和维持ＭＧＵ的动态结构稳定具有重要的作用。本文应用透射电镜,详细观察了朱顶红花粉管中精细胞的超微结构,并着重微管结构及其分布的观察。朱顶红成熟花粉为两细胞型。成熟花粉于２６℃、黑暗条件下,在液体培养基（含１０％蔗糖和１００ｐｐｍ硼酸）中培养１３－１８小时,然后收集花粉管,固定,供电镜观察并照相。朱顶红成熟花粉培养１３小时后,生殖细胞在花粉管中完成核分裂和胞质分裂等两个过程。形成两个精细胞。初形成的两个精细胞前后排列,营养核前导并靠近花粉管顶端。领头的精细胞的细胞质以很大的表面与营养核相互贴合（图版Ⅰ－１,２）,有时营养核与两个精细胞彼此穿插、缠绕（图版Ⅰ－３）。两精细胞之间的共同壁上具有很多胞质通道和含均质电子密度中等的基质（图版Ⅱ－４）。精细胞质在核与共同壁之间的区域染色较深,经高倍放大,观察到此处含丰富的微管,基     

Microtubule organization in germinated pollen of the conifer Picea abies (Norway spruce,Pinaceae)

The organization of microtubules in germinated pollen of the conifer Picea abies (Norway spruce, Pinaceae) was examined using primarily confocal microscopy. Pollination in conifers differs from angiosperms in the number of mitotic divisions between the microspore and the sperm and in the growth rate of the pollen tube. These differences may be orchestrated by the cytoskeleton, and this study finds that there are important functional differences in microtubule organization within conifer pollen compared to the angiosperm model systems. Pollen from P. abies contains two degenerated prothallial cells, a body cell, a stalk cell, and a vegetative cell. The body cell produces the sperm. In the vegetative cell, microtubules form a continuous network from within the pollen grain, out through the aperture, and down the length of the tube to the elongating tip. Within the grain, this network extends from the pollen grain wall to the body and stalk cell complex. Microtubules within the body and stalk cells form a densely packed array that enmeshes amyloplasts and the nucleus. Microtubule bundles can be traced between the body and stalk cells from the cytoplasm of the body cell to the adjoining cell wall and into the cytoplasm of the stalk cell. Body and stalk cells are connected by plasmodesmata. The organization of microtubules and the presence of plasmodesmata suggest that microtubules form a path for intercellular communication by projecting from the cytoplasm to interconnecting plasmodesmata. Microtubules in the elongating tube form a net axial array that ensheathes the vegetative nucleus. Microtubules are enriched at the elongating tip, where they form an array beneath the plasma membrane that is perpendicular to the direction of tube growth. This enriched region extends back 20 μm from the tip. There is an abrupt transition from a net perpendicular to a net axial organization at the edge of the enriched region. In medial sections, microtubules are present in the core of the elongating tip. The organization of microtubules in the tip differs from that seen in angiosperm pollen tubes.     

Microtubule organization in three-dimensional confined geometries: evaluating the role of elasticity through a combined in vitro and modeling approach

Microtubules or microtubule bundles in cells often grow longer than the size of the cell, which causes their shape and organization to adapt to constraints imposed by the cell geometry. We test the reciprocal role of elasticity and confinement in the organization of growing microtubules in a confining box-like geometry, in the absence of other (active) microtubule organizing processes. This is inspired, for example, by the cortical microtubule array of elongating plant cells, where microtubules are typically organized in an aligned array transverse to the cell elongation axis. The method we adopt is a combination of analytical calculations, in which the polymers are modeled as inextensible filaments with bending elasticity confined to a two-dimensional surface that defines the limits of a three-dimensional space, and in vitro experiments, in which microtubules are polymerized from nucleation seeds in microfabricated chambers. We show that these features are sufficient to organize the polymers in aligned, coiling configurations as for example observed in plant cells. Though elasticity can account for the regularity of these arrays, it cannot account for a transverse orientation of microtubules to the cell's long axis. We therefore conclude that an additional active, force-generating process is necessary to create a coiling configuration perpendicular to the long axis of the cell.     

Microtubule organization in lymphoid malignancies.

The abnormal organization of actin-containing microfilaments and vimentin-containing intermediate filaments in neoplastic lymphocytes of T and B cell origin has been described. We investigated microtubules of pathologic cells from 34 lymphoid malignancies, by immunofluorescence microscopy, using monoclonal tubulin antibody. In most cases, apart from two cases of lymphoma, one T cell lymphoma and one B cell lymphoma, interphase leukemia cells, lymphoma cells, and myeloma cells were shown to contain well-organized microtubules which were associated with a microtubule organization center at one end. In the cells of a patient with T cell lymphoma, although microtubules were not visible in the lymphoma cells from lymph nodes, they became visible after 72 hours in culture with concanavalin A (Con A) and interferon alpha. Cap formation was observed with antitubulin monoclonal antibody in the peripheral blood lymphocytes from a chronic lymphocytic leukemia patient, but well-developed microtubules were observed on other occasions in the same patient. There were no obvious structural differences between microtubules in T and B cell lymphoid malignancies, but leukemia cells and lymphoma cells with irregularly shaped nuclei, such as adult T cell leukemia cells and B cell lymphoma cells with cleaved nuclei, had complicated microtubules surrounding their irregular nuclei. In general, after blastogenic stimuli with phytohemagglutinin-P (PHA-P), Con A, and pokeweed mitogen (PWM), the development of the microtubules was proportional to the incorporation of 3H thymidine (3H-TDR). In most cases, after incubation with granulocyte colony-stimulating factor (G-CSF) and interferon alpha, the number of intact cells decreased and the number of degenerated cells increased, but the intact cells had intact microtubules.     

Opposing motor activities are required for the organization of the mammalian mitotic spindle pole

We use both in vitro and in vivo approaches to examine the roles of Eg5 (kinesin-related protein), cytoplasmic dynein, and dynactin in the organization of the microtubules and the localization of NuMA (Nu-clear protein that associates with the Mitotic Apparatus) at the polar ends of the mammalian mitotic spindle. Perturbation of the function of Eg5 through either immunodepletion from a cell free system for assembly of mitotic asters or antibody microinjection into cultured cells leads to organized astral microtubule arrays with expanded polar regions in which the minus ends of the microtubules emanate from a ring-like structure that contains NuMA. Conversely, perturbation of the function of cytoplasmic dynein or dynactin through either specific immunodepletition from the cell free system or expression of a dominant negative subunit of dynactin in cultured cells results in the complete lack of organization of microtubules and the failure to efficiently concentrate the NuMA protein despite its association with the microtubules. Simultaneous immunodepletion of these proteins from the cell free system for mitotic aster assembly indicates that the plus end- directed activity of Eg5 antagonizes the minus end-directed activity of cytoplasmic dynein and a minus end-directed activity associated with NuMA during the organization of the microtubules into a morphologic pole. Taken together, these results demonstrate that the unique organization of the minus ends of microtubules and the localization of NuMA at the polar ends of the mammalian mitotic spindle can be accomplished in a centrosome-independent manner by the opposing activities of plus end- and minus end-directed motors.     

Heat stress affects the organization of microtubules and cell division in Nicotiana tabacum cells

To investigate the effects of heat stress on the plant cytoskeleton, the structure of microtubule arrays in N. tabacum suspension cells incubated at 38 or 42°C was analysed. Whilst incubation at 42 °C resulted in the disruption of the majority of cellular microtubules after 30 min, in cells exposed to 38 °C all the microtubule arrays were preserved even after 12 h of incubation, although their organization was altered. The most susceptible were the microtubules of the mitotic spindle and the phragmoplast. Several abnormalities were observed: (i) splitting of the spindle into several parts; (ii) elongation of the spindles; (iii) formation of microtubule asters in mitotic cells, and (iv) elongation of phragmoplast microtubules. Exposure of cells to 38 °C caused a decrease in the mitotic index but an accumulation of telophase cells. The recovery of normal microtubule organization occurred after 12 h. Treatment of the cells subjected to heat stress conditions with an inhibitor of protein synthesis, cycloheximide, did not prevent either the alterations of microtubule organization or accumulation of cells containing phragmoplasts. Therefore, heat shock proteins do not seem to be directly responsible for the microtubule disorganization induced by heat stress.     

Higher plant cortical microtubule array analyzed in vitro in the presence of the cell wall

Plant morphogenesis depends on an array of microtubules in the cell cortex, the cortical array. Although the cortical array is known to be essential for morphogenesis, it is not known how the array becomes organized or how it functions mechanistically. Here, we report the development of an in vitro model that provides good access to the cortical array while preserving the array's organization and, importantly, its association with the cell wall. Primary roots of maize (Zea mays) are sectioned, without fixation, in a drop of buffer and then incubated as desired before eventual fixation. Sectioning removes cytoplasm except for a residuum comprising cortical microtubules, vesicles, and fragments of plasma membrane underlying the microtubules. The majority of the cortical microtubules remain in the cut-open cells for more than 1 h, fully accessible to the incubation solution. The growth zone or more mature tissue can be sectioned, providing access to cortical arrays that are oriented either transversely or obliquely to the long axis of the root. Using this assay, we report, first, that cortical microtubule stability is regulated by protein phosphorylation; second, that cortical microtubule stability is a function of orientation, with divergent microtubules within the array depolymerizing within minutes of sectioning; and third, that the polarity of microtubules in the cortical array is not uniform. These results suggest that the organization of the cortical array involves random nucleation followed by selective stabilization of microtubules formed at the appropriate orientation, and that the signal specifying alignment must treat orientations of +/- 180 degrees as equivalent.