The reorganization of microtubules and microfilaments in differentiating keratinocytes

Using immunofluorescence techniques, we have examined the microtubules and microfilaments in colonies of terminally differentiating human keratinocytes in tissue culture. The undifferentiated keratinocytes contained numerous microtubules, which radiated from a centrosomal organization center (MTOC). Differentiating keratinocytes, which leave the basal layer and begin to synthesize involucrin, displayed an altered cytoskeleton. Thick mats and coils of microtubules formed throughout the cytoplasm of the differentiated squames, and microfilaments were no longer visible after staining with phalloidin. Instead, only scattered stipples of phalloidin-stained material were observed. The results suggest that the terminal differentiation of epidermal cells involves a reorganization not only of the keratin filaments but of the entire cytoskeleton.     

The distribution and requirements of microtubules and microfilaments in bovine oocytes during in vitro maturation

Microtubules and microfilaments are major cytoskeletal components and important modulators for chromosomal movement and cellular division in mammalian oocytes. In this study we observed microtubule and microfilament organisation in bovine oocytes by laser scanning confocal microscopy, and determined requirements of their assembly during in vitro maturation. After germinal vesicle breakdown, small microtubular asters were observed near the condensed chromatin. The asters appeared to elongate and encompass condensed chromatin particles. At the metaphase stage, microtubules were observed in the second meiotic spindle at the metaphase stage. The meiotic spindle was a symmetrical, barrel-shaped structure containing anastral broad poles, located peripherally and radially oriented. Treatment with nocodazole did not inhibit germinal vesicle breakdown. However, progression to metaphase failed to occur in oocytes treated with nocodazole. In contrast, microfilaments were observed as a relatively thick uniform area around the cell cortex and overlying chromatin following germinal vesicle breakdown. Treatment with cytochalasin B inhibited microfilament polymerisation but did not prevent either germinal vesicle breakdown or metaphase formation. However, movement of chromatin to the proper position was inhibited in oocytes treated with cytochalasin B. These results suggest that both microtubules and microfilaments are closely associated with reconstruction and proper positioning of chromatin during meiotic maturation in bovine oocytes.     

Dynamic reorganization of microtubules and microfilaments in flax cells during the resistance response to flax rust infection

The cytoskeleton in plant cells is a dynamic structure that can rapidly respond to extracellular stimuli. Alteration of the organization of microtubules and actin microfilaments was examined in mesophyll cells of flax, Linum usitatissimum L., during attempted infection by the flax rust fungus, Melampsora lini (Ehrenb.) Lev. Flax leaves that had been inoculated with either a compatible (yielding a susceptible reaction) or an incompatible (yielding a resistant reaction) strain of M. lini were embedded in butyl-methylmethacrylate resin; sections of this material were immunofluorescently labelled with anti-tubulin or anti-actin and examined using confocal laser scanning microscopy. In uninfected leaves, microtubules in the mesophyll cells formed a transverse array in the cell cortex. Microfilaments radiated through the cytoplasm from the nucleus. In an incompatible interaction, microtubules and microfilaments were extensively reorganized in mesophyll cells that were in contact with fungal infection hyphae or haustorial mother cells before penetration of the cell by the infection peg. After the initiation of haustorium development, microtubules disappeared from the infected cells, and growth of the haustoria ceased. In an incompatible interaction, hypersensitive cell death occurred in more than 70% of infected cells but occurred in less than 20% of cells in compatible interactions. After the infected cell had undergone hypersensitive cell death, the cytoskeleton in neighbouring cells became focused on the walls shared with the necrotic cell. In compatible interactions, reorganization of the cytoskeleton was either not observed at all or was observed much less frequently up to 48 h after inoculation.Abbreviations FITC fluorescein isothiocyanate - WGA wheatgerm agglutinin We thank Dr. G.J. Lawrence for providing valuable discussions and materials.     

The role of microtubules and microfilaments in the micronucleus ofParamecium bursaria during mitosis

Summary A unique spindle apparatus develops during mitosis in the micronucleus ofParamecium bursaria. During interphase the micronucleus contains short microtubule profiles and clumps of condensed chromatin. Throughout mitosis the nuclear envelope remains intact. During prophase, cup-shaped structures termed microlamellae develop in close association with regions of condensed chromatin. Each micromella consists of an outer sublamella, an inner sublamellae, and ring-shaped structures termed microsepta that join the two sublamellae. Microtubules elongate parallel to the division axis. During metaphase, the microlamellae appear to act as kinetochorelike structures that aid in the alignment of the chromosomes. The microlamellae appear conical and join to a meshwork of microfilaments at their apices. Further toward the polar regions the microfilaments join with microtubules that converge and terminate near the nuclear envelope. During metaphase-anaphase and anaphase the chromosomes are apparently moved by the microfilaments pulling on the kinetochorelike microlamellae. Also during metaphase-anaphase, extranuclear microtubules join the nuclear envelope of the micronucleus to microtubule elements of the cell cortex. By anaphasetelophase, microlamellae and the microfilament meshwork degenerate and microtubules represent the only spindle elements. The evidence of this report supports the hypothesis that microfilaments can participate with microtubules in the movement of chromosomes.This report is part of a Ph.D. Thesis presented by the senior author at Fordham University.     

The role of centrosomes and astral microtubules during asymmetric division of Drosophila neuroblasts

Drosophila neuroblasts are stem cells that divide asymmetrically to produce another large neuroblast and a smaller ganglion mother cell (GMC). During neuroblast division, several cell fate determinants, such as Miranda, Prospero and Numb, are preferentially segregated into the GMC, ensuring its correct developmental fate. The accurate segregation of these determinants relies on proper orientation of the mitotic spindle within the dividing neuroblast, and on the correct positioning of the cleavage plane. In this study we have analyzed the role of centrosomes and astral microtubules in neuroblast spindle orientation and cytokinesis. We examined neuroblast division in asterless (asl) mutants, which, although devoid of functional centrosomes and astral microtubules, form well-focused anastral spindles that undergo anaphase and telophase. We show that asl neuroblasts assemble a normal cytokinetic ring around the central spindle midzone and undergo unequal cytokinesis. Thus, astral microtubules are not required for either signaling or positioning cytokinesis in Drosophila neuroblasts. Our results indicate that the cleavage plane is dictated by the positioning of the central spindle midzone within the cell, and suggest a model on how the central spindle attains an asymmetric position during neuroblast mitosis. We have also analyzed the localization of Miranda during mitotic division of asl neuroblasts. This protein accumulates in morphologically regular cortical crescents but these crescents are mislocalized with respect to the spindle orientation. This suggests that astral microtubules mediate proper spindle rotation during neuroblast division.     

Study of a lysis medium stabilizing microfilaments and microtubules in vitro and in vivo

Determination of experimental conditions which allow the evaluation of the variations in the ratio of non polymerized and polymerized forms of actin and tubulin during the reorganization of the cytoskeletal cell system is of most valuable importance. In order to prepare cell homogenates which would reflect the in vivo situation, we tested in vitro a lysis medium which stabilized both microfilaments and microtubules, which were determined by DNase inhibition assays and colchicine binding assays respectively. This lysis medium containing 10 mM potassium phosphate, 1mM magnesium chloride, 5 mM EGTA, 1 M hexylene glycol, 1% Triton X-100, pH 6.4, used at 4 degrees C a) diffused rapidly into the cells; b) did not denature actin and tubulin; c) did not displace the equilibrium between non polymerized and polymerized forms of actin and tubulin, allowing biochemical assays on cell homogenates; d) blocked the evolution of the cytoskeletal system and permitted structural studies; e) and allowed the decoration of microfilaments by heavy meromyosin.     

Dynamic reorganization of microfilaments and microtubules is necessary for the expression of non-host resistance in barley coleoptile cells

To show the involvement of microfilaments and microtubules in non-host resistance of barley, partially dissected coleoptiles which had been inoculated with a non-pathogen, Erysiphe pisi, were treated with several actin and tubulin inhibitors. If the coleoptiles were not treated with any of the inhibitors, the non-pathogen always failed to penetrate the coleoptile cells. However, when coleoptiles were treated with actin or tubulin polymerization or depolymerization inhibitors, the non-pathogen was able to penetrate successfully and to form haustoria in coleoptile cells of a non-host plant, barley. Actin polymerization inhibitors, cytochalasins, were more effective in causing an increase in penetration efficiency of E. pisi than tubulin inhibitors. The effects of cytochalasins depended on the kind of cytochalasin; the strength of the actin depolymerizing activity correlated significantly with the efficiency of increasing the penetration of the non-pathogen. When both actin and tubulin inhibitors were added simultaneously, the polarization of defense-related responses, such as massive cytoplasmic aggregation, deposition of papillae and accumulation of autofluorescent compounds, at fungal penetration sites was suppressed. Actin inhibitors did not affect arrangement and stability of microtubules and vice versa, and a double treatment of coleoptile cells with both microfilament and microtubule inhibitors showed an additive effect in increasing the penetration efficiency of E. pisi. Furthermore, cytochalasin A treatment allowed other non-pathogens, Colletotrichum lagenarium and Alternaria alternata, to penetrate successfully into the non-host barley cells. These results strongly suggest that microfilaments and microtubules might play important roles in the expression of non-host resistance of barley.     

Cortical microtubules rearrange during differentiation of vascular cambial derivatives, microfilaments do not

The plant cytoskeleton has been implicated in a variety of morphogenetic events in higher plants. Most of this work, however, has concentrated on epidermal cells or primary tissues. We have investigated the cortical microtubular (CMT) and microfilament (MF) components of the cytoskeleton in a secondary tissue  –  active vascular cambium of Aesculus hippocastanum L. (horse-chestnut)  –  and followed the changes in these components during the early stages of differentiation of fusiform cambial derivatives to axial elements of the secondary vascular system. A correlative approach was used employing indirect immunofluorescence microscopy of α-tubulin on 6 μm sections, and transmission electron microscopy of 60 nm sections. The study has demonstrated a rearrangement of the CMT cytoskeleton, from random to helical, as fusiform vascular cambial cells begin to differentiate as secondary phloem vascular tissue. A similar CMT rearrangement is seen as fusiform cambial cells begin to differentiate as secondary xylem fibres. This rearrangement is interpreted as evidence of determination of cambial derivatives towards vascular development. Axially-oriented MF bundles are present in fusiform cambial cells and their axial orientation is retained in the vascular derivatives at early stages of their development even though the CMTs have become rearranged. Received: 5 August 1996 /  Accepted: 23 September 1996     

Roles of microfilaments and microtubules in paxillin dynamics

We investigated the roles of microfilaments and microtubules in the localization and tyrosine phosphorylation of paxillin, a focal adhesion-associated signaling molecule, in bovine aortic endothelial cells (BAECs). Paxillin tyrosine phosphorylation is inhibited by cytochalasin D (CD), but slightly increased by colchicine and paclitaxol (taxol). CD also caused an overall disassembly of paxillin-containing focal adhesions (paxillin-FAs) and translocation of paxillin to the cytoplasm and perinuclear region with a diffuse distribution. Meanwhile, colchicine and taxol caused a disassembly of paxillin-FAs from cell periphery and lamellipodia, and their assembly in cell center. These results indicate that actin filaments are important in paxillin assembly in the FAs of the whole ECs and that microtubules are critical in paxillin assembly in cell periphery and lamellipodia; thus the microfilaments and microtubules play differential roles in the dynamics of paxillin assembly/disassembly. Our findings also suggest that tyrosine phosphorylation is an important element in paxillin dynamics at FAs.     

Polarity of microtubules nucleated by centrosomes and chromosomes of Chinese hamster ovary cells in vitro

The structural and growth polarities of centrosomal and chromosomal microtubules were studied by analyzing the kinetics of growth of these microtubules and those initiated by flagellar seeds. By comparing rates of elongation of centrosomal and flagellar-seeded microtubules, we determined whether the centrosomal microtubules were free to grow at their plus ends only, minus ends ony, or at both ends. Our results show that centrosomal microtubules elongate at a rate corresponding to the addition of subunits at the plus end only. The depolymerization rate was also equivalent to that for the plus end only. Chromosomal microtubule elongation was similar to the centrosome-initiated growth. Since the data do not support the hypothesis that both ends of these spindle microtubules are able to interact with monomer in solution, then growth must occur only distal or only proximal to the organizing centers, implying tha the opposite ends in unavailable for exchange of subunits. Experiments with flagellar-seeded microtubules serving as internal controls indicated that the inactivity of the minus end could not be accounted for by a diffusible inhibitor, suggesting a structural explanation. Since there is no apparent way in which the distal ends may be capped, whereas the proximal ends are embedded in the pericentriolar cloud, we conclude that centrosomal microtubules are oriented with their plus ends distal to the site of nucleation. A similar analysis for chromosomal microtubules suggests that they too must be oriented with their plus ends distal to the site of initiation.     

The distribution of centrosomes in migrating endothelial cells during wound healing in situ.

We have examined the distribution of centrioles in rabbit thoracic aortic endothelial cells induced to migrate by wounding the endothelium in situ. Following denudation of the endothelium from a segment of the aorta with a balloon catheter, a wound edge was created from which endothelial cells began to migrate onto the denuded surface. In this in situ model of cell migration, the position of centrioles was determined in cells along the wound edge by immunofluorescence and antibodies which specifically label these cell organelles, and then they were classified in relation to the nucleus and the direction of cell migration as being oriented toward the wound, in the center, or away from wound. At time 0, as in normal unwounded adult rabbit aorta, no preferential orientation of centrioles was evident. Within 12 h after wounding, the centrioles in about 53% of endothelial cells near the wound edge were oriented toward the wound, while in less than 20% of the cells they were oriented away from wound. At 24 h, in cells up to 800 microns from the wound edge, centrioles in only about 10% of the endothelial cells were oriented away from wound, while in about 52% of cells they were found in the center and in 38% of the cells they remained oriented toward the wound. At 48 h, up to 2000 microns from the wound edge, the majority of endothelial cells had their centrioles in the center, possibly as a result of an increase in mitotic index as cells replicate to reestablish an intact endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vitrification of immature and in vitro matured pig oocytes: study of distribution of chromosomes, microtubules, and actin microfilaments

Studies were conducted to compare viability of immature and mature porcine oocytes vitrified in ethylene glycol (EG) using open-pulled straws (OPS). Oocytes that had been allowed to mature for 12 h (germinal vesicle group; GV) and 40 h (metaphase II group; MII) were divided into three treatments: (1) control; (2) treated with cytochalasin B and exposed to EG; and (3) treated with cytochalasin B and vitrified by stepwise exposure to EG in OPS. After warming, a sample of oocytes was fixed and evaluated by specific fluorescent probes before visualization using confocal microscopy. The remaining oocytes were fertilized and cleavage rate was recorded. Exposure of GV oocytes to EG or vitrification had a dramatic effect on spindle and chromosome configurations and no cleavage was obtained after in vitro fertilization. When MII oocytes were exposed to EG or were vitrified, 18 and 11% of oocytes, respectively, maintained the spindle structure and either EG exposure or vitrification resulted in substantial disruption in microfilament organization. The cleavage rates of mature oocytes after being exposed to EG or after vitrification were similar (14 and 13%, respectively) but were significantly less than that of control oocytes (69%). These results indicate that porcine oocytes at different meiotic stages respond differently to cryopreservation and MII porcine oocytes had better resistance to cryopreservation than GV stage oocytes.     

Cortical microtubules and cortical microfilaments in the green alga,Micrasterias pinnatifida

Summary Cortical microtubules and cortical microfilaments were visualized in cells ofMicrasterias pinnatifida treated by freeze-substitution, and the pattern of their distribution was reconstructed from serial sections. Most cortical microtubules accompanied the long microfilaments that ran parallel to the microtubules. Cortical microfilaments not accompanied by the microtubules were also found. They were short and slightly curved. Both types of cortical microfilament were not grouped into bundles, and were 6–7 nm in diameter, a value that corresponds to the diameter of filaments of F-actin.     

Intermediate filaments,microtubules and microfilaments in epidermis of sea urchin tube foot

Summary Tube foot epidermal cells of the sea urchin Strongylocentrotus purpuratus were examined by transmission electron microscopy and fluorescence microscopy to identify the chemical nature of prominent bundles of cytoplasmic filaments. Cross sections revealed filaments of roughly 7–8 nm in diameter closely packed into dense bundles. These bundles, in turn, were each surrounded by a loose sheath of microtubules. The filament size and negative reaction with the fluorescent F-actin binding drug NBD-phallacidin indicated that they were not actin. Indirect immunofluorescence microscopy of whole tissues and frozen sections revealed a strong reaction of the filaments with a monoclonal antibody prepared against porcine stomach desmin. In SDS-polyacrylamide gels of whole tube foot protein, a band of apparent molecular weight around 50 000 daltons reacted with the anti-desmin monoclonal antibody. The combined data provide evidence that the epidermal filament bundles are related to vertebrate intermediate filaments, but further biochemical studies will be necessary to assign them to a particular class of filament proteins.     

Actomyosin transports microtubules and microtubules control actomyosin recruitment during Xenopus oocyte wound healing

BACKGROUND: Interactions between microtubules and actin filaments (F-actin) are critical for cellular motility processes ranging from directed cell locomotion to cytokinesis. However, the cellular bases of these interactions remain poorly understood. We have analyzed the role of microtubules in generation of a contractile array comprised of F-actin and myosin-2 that forms around wounds made in Xenopus oocytes. RESULTS: After wounding, microtubules are transported to the wound edge in association with F-actin that is itself recruited to wound borders via actomyosin-powered cortical flow. This transport generates sufficient force to buckle and break microtubules at the wound edge. Transport is complemented by local microtubule assembly around wound borders. The region of microtubule breakage and assembly coincides with a zone of actin assembly, and perturbation of the microtubule cytoskeleton disrupts this zone as well as local recruitment of the Arp2/3 complex and myosin-2. CONCLUSIONS: The results reveal transport of microtubules in association with F-actin that is pulled to wound borders via actomyosin-based contraction. Microtubules, in turn, focus zones of actin assembly and myosin-2 recruitment at the wound border. Thus, wounding triggers the formation of a spatially coordinated feedback loop in which transport and assembly of microtubules maintains actin and myosin-2 in close proximity to the closing contractile array. These results are surprisingly reminiscent of recent findings in locomoting cells, suggesting that similar feedback interactions may be generally employed in a variety of fundamental cell motility processes.     

Making microtubules and mitotic spindles in cells without functional centrosomes

Centrosomes are considered to be the major sites of microtubule nucleation in mitotic cells (reviewed in ), yet mitotic spindles can still form after laser ablation or disruption of centrosome function . Although kinetochores have been shown to nucleate microtubules, mechanisms for acentrosomal spindle formation remain unclear. Here, we performed live-cell microscopy of GFP-tubulin to examine spindle formation in Drosophila S2 cells after RNAi depletion of either gamma-tubulin, a microtubule nucleating protein, or centrosomin, a protein that recruits gamma-tubulin to the centrosome. In these RNAi-treated cells, we show that poorly focused bipolar spindles form through the self-organization of microtubules nucleated from chromosomes (a process involving gamma-tubulin), as well as from other potential sites, and through the incorporation of microtubules from the preceding interphase network. By tracking EB1-GFP (a microtubule-plus-end binding protein) in acentrosomal spindles, we also demonstrate that the spindle itself represents a source of new microtubule formation, as suggested by observations of numerous microtubule plus ends growing from acentrosomal poles toward the metaphase plate. We propose that the bipolar spindle propagates its own architecture by stimulating microtubule growth, thereby augmenting the well-described microtubule nucleation pathways that take place at centrosomes and chromosomes.     

Microtubule flux in mitosis is independent of chromosomes, centrosomes, and antiparallel microtubules.

We investigated the mechanism of poleward microtubule flux in the mitotic spindle by generating spindle subassemblies in Xenopus egg extracts in vitro and assaying their ability to flux by photoactivation of fluorescence and low-light multichannel fluorescence video-microscopy. We find that monopolar intermediates of in vitro spindle assembly (half-spindles) exhibit normal poleward flux, as do astral microtubule arrays induced by the addition of dimethyl sulfoxide to egg extracts in the absence of both chromosomes and conventional centrosomes. Immunodepletion of the kinesin-related microtubule motor protein Eg5, a candidate flux motor, suggests that Eg5 is not required for flux. These results suggest that poleward flux is a basic element of microtubule behavior exhibited by even simple self-organized microtubule arrays and presumably underlies the most elementary levels of spindle morphogenesis.