Gotowa bibliografia na temat „Aster de microtubules”

The mitotic spindle positions the cytokinesis furrow. The cytokinesis furrow then forms and ingresses at the site of the mitotic spindle, between the spindle poles. Two populations of spindle microtubules are implicated in cytokinesis furrow positioning: radial microtubule arrays called asters and bundled non-kinetochore microtubules called the spindle midzone. Here I will discuss our recent results that provided examples of how aster-positioned and midzone-positioned cytokinesis can be mechanically and genetically separated. These experiments illustrate how asters and midzone contribute to cytokinesis. ASS (asymmetric spindle severing) is a mechanical way to spatially separate the aster and midzone signals. In Caenorhabditis elegans embryos, asters and midzone provide two consecutive signals that position the cytokinesis furrow. The first signal is positioned midway between the microtubule asters; the second signal is positioned over the spindle midzone. Aster and midzone contribution can also be genetically separated. Mutants in spd-1 have no detectable midzone and are defective in midzone-positioned but not aster-positioned cytokinesis. Disruption of the function of LET-99 and the heterotrimeric G-proteins GOA-1/GPA-16 and their regulator GPR-1/2 causes defects in aster-positioned cytokinesis but not in midzone-positioned cytokinesis. In order to understand aster-positioned cytokinesis we have to understand how microtubule asters spatially control the activity of LET-99, GPR-1/2 and GOA-1/GPA-16 and how the activity of these G-protein pathway components control the assembly of a contractile ring.

Coordinated interplay of the microtubule and actin cytoskeletons has long been known to be crucial for many cellular processes including cell migration and cytokinesis. However, interactions between these two systems have been difficult to document by conventional approaches, for a variety of technical reasons. Here the distribution of f-actin and microtubules were analyzed in the absence of fixation using Xenopus egg extracts as an in vitro source of microtubules and f-actin, demembranated Xenopus sperm to nucleate microtubule asters, fluorescent phalloidin as a probe for f-actin, and fluorescent tubulin as a probe for microtubules. F-actin consistently colocalized in a lengthwise manner with microtubules of asters subjected to extensive washing in flow chambers. F-actin-microtubule association was heterogenous within a given aster, such that f-actin is most abundant toward the distal (plus) ends of microtubules, and microtubules heavily labeled with f-actin are found in close proximity to microtubules devoid of f-actin. However, this distribution changed over time, in that 5 minute asters had more f-actin in their interiors than did 15 minute asters. Microtubule association with f-actin was correlated with microtubule bending and kinking, while elimination of f-actin resulted in straighter microtubules, indicating that the in vitro interaction between f-actin and microtubules is functionally significant. F-actin was also found to associate in a lengthwise fashion with microtubules in asters centrifuged through 30% sucrose, and microtubules alone (i.e. microtubules not seeded from demembranated sperm) centrifuged through sucrose, indicating that the association cannot be explained by flow-induced trapping and alignment of f-actin by aster microtubules. Further, cosedimentation analysis revealed that microtubule-f-actin association could be reconstituted from microtubules assembled from purified brain tubulin and f-actin assembled from purified muscle actin in the presence, but not the absence, of Xenopus oocyte microtubule binding proteins. The results provide direct evidence for an association between microtubules and f-actin in vitro, indicate that this interaction is mediated by one or more microtubule binding proteins, and suggest that this interaction may be responsible for the mutual regulation of the microtubule and actomyosin cytoskeletons observed in vivo.

Assembly of a cytokinetic contractile ring is a form of cell polarization in which the equatorial cell cortex becomes differentiated from the polar regions. Microtubules direct cytokinetic polarization via the central spindle and astral microtubules. The mechanism of central spindle–directed furrow formation is reasonably well understood, but the aster-directed pathway is not. In aster-directed furrowing, cytoskeletal factors accumulate to high levels at sites distal to the asters and at reduced levels at cortical sites near the asters. In this paper, we demonstrate that the cytoskeletal organizing protein anillin (ANI-1) promotes the formation of an aster-directed furrow in Caenorhabditis elegans embryos. Microtubule-directed nonmuscle myosin II polarization is aberrant in embryos depleted of ANI-1. In contrast, microtubule-directed polarized ANI-1 localization is largely unaffected by myosin II depletion. Consistent with a role in the induction of cortical asymmetry, ANI-1 also contributes to the polarization of arrested oocytes. Anillin has an evolutionarily conserved capacity to associate with microtubules, possibly providing an inhibitory mechanism to promote polarization of the cell cortex.

"Spiral asters" composed of swirls of subcortical microtubules were recently described in fertilized eggs of the sea urchin Strongylocentrotus purpuratus. In our study, these structures did not occur at culture temperatures below 16 degrees C. When the culture temperature was elevated, however, "spiral asters" routinely appeared during a susceptible period before mitotic prophase when the sperm aster-diaster normally exists. A massive and protracted rotation of the cytoplasm (excluding an immobile cortex and perinuclear region) began within 1 min of exposure to elevated temperature. Fibrils of the "spiral aster" could be seen within this rotating mass even by bright-field microscopy. The identity of microtubules in these structures was confirmed by indirect immunofluorescence microscopy. A mechanistic association between "spiral aster" formation and cytoplasmic rotation was indicated by the simultaneous inhibitory effects of microtubule and dynein poisons. Inhibitors of microfilaments, however, had no effect. We infer that elevated temperature induces unique changes in the microtubules of the pre-prophase sperm aster-diaster, resulting in cytoplasmic rotation and the spiral configuration of microtubules. Comparative cytological evidence supports the idea that "spiral asters" do not normally occur in fertilized sea urchin eggs. Biogeographic evidence for S. purpuratus indicates that fertilization and development naturally occur below 15 degrees C, hence "spiral asters" in eggs of this species should be regarded as abnormalities induced in the laboratory by unnaturally elevated temperatures.

Mechanisms of spindle pole formation rely on minus-end-directed motor proteins. γ-Tubulin is present at the centre of poles, but its function during pole formation is completely unknown. To address the role of γ-tubulin in spindle pole formation, we overexpressed GFP (green fluorescent protein)-fused γ-tubulin (γ-Tu-GFP) in Xenopus oocytes and produced self-assembled mitotic asters in the oocyte extracts. γ-Tu-GFP associated with endogenous α-, β- and γ-tubulin, suggesting that it acts in the same manner as that of endogenous γ-tubulin. During the process of aster formation, γ-Tu-GFP aggregated as dots on microtubules, and then the dots were translocated to the centre of the aster along microtubules in a manner dependent on cytoplasmic dynein activity. Inhibition of the function of γ-tubulin by an anti-γ-tubulin antibody resulted in failure of microtubule organization into asters. This defect was restored by overexpression of γ-Tu-GFP, confirming the necessity of γ-tubulin in microtubule recruitment for aster formation. We also examined the effects of truncated γ-tubulin mutants, which are difficult to solubly express in other systems, on aster formation. The middle part of γ-tubulin caused abnormal organization of microtubules in which minus ends of microtubules were not tethered, but dispersed. An N-terminus-deleted mutant prevented recruitment of microtubules into asters, similar to the effect of the anti-γ-tubulin antibody. The results indicate possible roles of γ-tubulin in spindle pole formation and show that the system developed in the present study could be useful for analysing roles of many proteins that are difficult to solubly express.

Prc1E and Kif4A prune out anti-parallel microtubules in the huge asters that position cleavage furrows in Xenopus eggs. Within asters, this promotes radial order in the face of the randomizing effect of nucleation away from centrosomes. At boundaries between asters, it blocks growth of a microtubule from one aster into its neighbor.

Anti-tubulin antibodies and confocal immunofluorescence microscopy were used to examine the organization and regulation of cytoplasmic and cortical microtubules during the first cell cycle of fertilized Xenopus eggs. Appearance of microtubules in the egg cortex temporally coincided with the outgrowth of the sperm aster. Microtubules of the sperm aster first reached the animal cortex at 0.25, (times normalized to first cleavage), forming a radially organized array of cortical microtubules. A disordered network of microtubules was apparent in the vegetal cortex as early as 0.35. Cortical microtubule networks of both animal and vegetal hemispheres were reorganized at times corresponding to the cortical rotation responsible for specification of the dorsal-ventral (D-V) axis. Optical sections suggest that the cortical microtubules are continuous with the microtubules of the sperm aster in fertilized eggs, or an extensive activation aster in activated eggs. Neither assembly and organization, nor disassembly of the cortical microtubules coincided with MPF activation during mitosis. However, cycloheximide or 6-dimethylaminopurine, which arrest fertilized eggs at interphase, blocked cortical microtubule disassembly. Injection of p13, a protein that specifically inhibits MPF activation, delayed or inhibited cortical microtubule breakdown. In contrast, eggs injected with cyc delta 90, a truncated cyclin that arrest eggs in M-phase, showed normal microtubule disassembly. Finally, injection of partially purified MPF into cycloheximide-arrested eggs induced cortical microtubule breakdown. These results suggest that, despite a lack of temporal coincidence, breakdown of the cortical microtubules is dependent on the activation of MPF.

In many eukaryotic cells going through M-phase, a bipolar spindle is formed by microtubules nucleated from centrosomes. These microtubules, in addition to being "captured" by kinetochores, may be stabilized by chromatin in two different ways: short-range stabilization effects may affect microtubules in close contact with the chromatin, while long-range stabilization effects may "guide" microtubule growth towards the chromatin (e.g., by introducing a diffusive gradient of an enzymatic activity that affects microtubule assembly). Here, we use both meiotic and mitotic extracts from Xenopus laevis eggs to study microtubule aster formation and microtubule dynamics in the presence of chromatin. In "low-speed" meiotic extracts, in the presence of salmon sperm chromatin, we find that short-range stabilization effects lead to a strong anisotropy of the microtubule asters. Analysis of the dynamic parameters of microtubule growth show that this anisotropy arises from a decrease in the catastrophe frequency, an increase in the rescue frequency and a decrease in the growth velocity. In this system we also find evidence for long-range "guidance" effects, which lead to a weak anisotropy of the asters. Statistically relevant results on these long-range effects are obtained in "high-speed" mitotic extracts in the presence of artificially constructed chromatin stripes. We find that aster anisotropy is biased in the direction of the chromatin and that the catastrophe frequency is reduced in its vicinity. In this system we also find a surprising dependence of the catastrophe and the rescue frequencies on the length of microtubules nucleated from centrosomes: the catastrophe frequency increase and the rescue frequency decreases with microtubule length.

Tipulid spermatocytes form normally functioning bipolar spindles after one of the centrosomes is experimentally dislocated from the nucleus in late diakinesis (Dietz, R., 1959, Z. Naturforsch., 14b:749-752; Dietz, R., 1963, Zool. Anz. Suppl., 23:131-138; Dietz, R., 1966, Heredity, 19:161-166). The possibility that dissociated pericentriolar material (PCM) is nevertheless responsible for the formation of the spindle in these cells cannot be ruled out based on live observation. In studying serial sections of complete cells and of lysed cells, it was found that centrosome-free spindle poles in the crane fly show neither pericentriolar-like material nor aster microtubules, whereas the displaced centrosomes appear complete, i.e., consist of a centriole pair, aster microtubules, and PCM. Exposure to a lysis buffer containing tubulin resulted in an increase of centrosomal asters due to aster microtubule polymerization. Aster-free spindle poles did not show any reaction, also indicating the absence of PCM at these poles. The results favor the hypothesis of chromosome-induced spindle pole formation at the onset of prometaphase and the dispensability of PCM in Pales.

Taxol, a microtubule stabilizing drug, induces the formation of numerous microtubule asters in the cytoplasm of mitotic cells (De Brabander, M., G. Geuens, R. Nuydens, R. Willebrords, J. DeMey. 1981. Proc. Natl. Acad. Sci. USA. 78:5608-5612). The center of these asters share with spindle poles some characteristics such as the presence of centrosomal material and calmodulin. We have recently reproduced the assembly of taxol asters in a cell-free system (Buendia, B., C. Antony, F. Verde, M. Bornens, and E. Karsenti. 1990. J. Cell Sci. 97:259-271) using extracts of Xenopus eggs. In this paper, we show that taxol aster assembly requires phosphorylation, and that they do not grow from preformed centers, but rather by a reorganization of microtubules first crosslinked into bundles. This process seems to involve sliding of microtubules along each other and we show that cytoplasmic dynein is required for taxol aster assembly. This result provides a possible functional basis to the recent findings, that dynein is present in the spindle and enriched near spindle poles (Pfarr, C. M., M. Cove, P. M. Grissom, T. S. Hays, M. E. Porter, and J. R. McIntosh. 1990. Nature (Lond.). 345:263-265; Steuer, E. R., L. Wordeman, T. A. Schroer, and M. P. Sheetz. 1990. Nature (Lond.). 345:266-268).

Au cours de la division cellulaire, la mise en place d’un fuseau bipolaire correct est nécessaire au maintien de la stabilité génomique, en permettant une ségrégation égale des chromosomes dans les deux cellules filles. Les microtubules, éléments constitués d’un assemblage de tubuline sous une forme cylindrique et creuse, sont les constituants de base de ce fuseau mitotique. Lors de l’assemblage du fuseau, les centrosomes se séparent, et des changements surviennent au niveau de la dynamique des microtubules. Cependant, sans les propriétés fonctionnelles d’autres protéines venant réguler cette dynamique, les microtubules ne seraient pas capables d’accomplir seuls les tâches qui leurs sont attribuées. Les protéines responsables de cette régulation sont les Protéines associées aux Microtubules ou MAPs. Des variations de la stabilité et de la dynamique des microtubules sont susceptibles d’atteindre la formation et l’organisation du fuseau mitotique ainsi que l’attachement des chromosomes, et peuvent provoquer des problèmes d’ instabilité génétique et la cancérogenèse. Nous avons identifié au laboratoire une nouvelle protéine humaine associée aux microtubules que nous avons appelé ASAP pour ASter-Associated Protein. La protéine ASAP est associée au fuseau mitotique. Sa surexpression entraîne des mitoses aberrantes et abortives (fuseaux multipolaires et cellules multinucléées avec une amplification des centrosomes, fuseaux monopolaires avec non séparation/migration des centrosomes). Ces phénotypes sont fréquemment observés dans des cancers. Sa déplétion provoque des fuseaux anormaux, retarde la mitose en entraînant des défauts de congression et de ségrégation des chromosomes et aboutit à des cytokinèses abortives ou à la mort cellulaire. ASAP est donc une nouvelle MAP jouant un rôle crucial au cours du cycle cellulaire. Une étude plus ciblée sur son implication dans la régulation de la dynamique et de l’organisation des microtubules a ensuite été entreprise afin de mieux comprendre l’impact d’ASAP au niveau du réseau de microtubules. Nous avons ainsi pu mettre en évidence une implication étendue de la protéine, tant au niveau des différentes phases dynamiques de la polymérisation, qu’au niveau de l’assemblage et de l’organisation des microtubules. Nous avons également clairement mis en évidence la présence d’ASAP aux centrosomes, avec la possibilité pour cette protéine de participer à la nucléation des microtubules, un rôle qu’il reste encore cependant à démontrer in vivo.

La division cellulaire asymétrique est un processus essentiel du développement. Ce processus ainsi que sa régulation ont fait l’objet de nombreuses études chez l’embryon de Caenorhabditis elegans. La division asymétrique de l'embryon unicellulaire est un processus conservé à travers les espèces de nématodes, cependant les caractéristiques cellulaires menant à la division sont étonnamment variables. Au cours de mon doctorat, j'ai voulu étudier ces différences en utilisant deux embryons non-C. elegans : Diploscapter pachys et Pristionchus pacificus. D. pachys est le parent parthénogénétique le plus proche de C. elegans. La polarité étant induite par le sperme chez C. elegans, on ne peut expliquer ce qui brise la symétrie chez D. pachys. Mes résultats montrent que le noyau occupe le plus souvent l’hémisphère de D. pachys qui deviendra le pole postérieur. Dans les embryons où il est astreint à un pôle par centrifugation, le noyau fini par revenir à son pôle préférentiel. Même si l’embryon est polarisé, l’agitation corticale et le cytosquelette d’actine semblent identiques aux deux pôles. D’autre part, la position du fuseau méiotique est corrélée avec la future cellule postérieure. Dans certains ovocytes, on observe des structures de microtubules émanant du fuseau méiotique combiné à un faible enrichissement en actine au future pôle postérieur. Finalement, mon principal projet de thèse montre que la polarité de D. pachys est atteinte durant la méiose, au cours de laquelle le fuseau méiotique pourrait jouer un rôle par un mécanisme présent mais inhibé chez C. elegans. Chez P. pacificus, la transgénèse biolistique a été récemment utilisée avec succès. Toutefois, par manque d’un marqueur de sélection fiable, il était illusoire de poursuivre cette approche. En conclusion, les résultats de ma thèse contribuent à une meilleure compréhension de l’embryogénèse hors C. elegans. Ils soulignent l’importance de ces espèces dans l’optique d’études comparatives
Asymmetric cell division is an essential process of development. The process and its regulation have been studied extensively in the Caenorhabditis elegans embryo. Asymmetric division of the single-cell embryo is a conserved process in nematode species, however, the cellular features leading up to division are surprisingly variable. During my PhD, I aimed to study these differences by using two non-C. elegans embryos: Diploscapter pachys and Pristionchus pacificus. D. pachys is the closest parthenogenetic relative to C. elegans. Since the polarity cue in C. elegans is brought by the sperm, how polarity is triggered in D. pachys remains unknown. My results show that the nucleus inhabits principally the hemisphere of the D. pachys embryo that will become the posterior pole. Moreover, in embryos where the nucleus is forced to one pole by centrifugation, it returns to its preferred pole. Although the embryo is polarized, cortical ruffling and actin cytoskeleton at both poles appear identical. Interestingly, the location of the meiotic spindle also correlates with the future posterior cell. In some oocytes, a slight actin enrichment along with unusual microtubule structures emanating from the meiotic spindle are observed at the future posterior pole. Overall, my main PhD project shows that polarity of the D. pachys embryo is attained during meiosis wherein the meiotic spindle could potentially be playing a role by a mechanism that may be present but suppressed in C. elegans. For P. pacificus, biolistic transgenesis has been shown recently successful. However, due to a lack of a stringent selection marker, the continuation of this project was unfeasible during my PhD. Altogether, the results of my PhD add to the understanding of non-C. elegans early embryogenesis and emphasizes on the importance of using these species for comparative studies

Thesis advisor: David R. Burgess
Centration of the nucleus after fertilization is an essential step for setting-up cell division and proper embryonic development in many proliferating cells such as the sea urchin. The sperm aster must capture the female pronucleus for fusion as well as the nucleus becoming positioned at the center of the cell. Microtubules (MTs) are known to play a role in this centration but the exact mechanism remains unknown. This begins to investigate current models of nuclear centration and the role of various interactions. Three phases of migration were observed as the male aster migrated with support in independent movements of the male and female pronuclei. Dimpling affects present that altered the morphology of the cell were observed when engagement occurred between the male and female pronuclei. It was discovered that this dimpling effect was a result of an interaction between MTs and the cortex, as confirmed by visualization of sheared cells in which only the cortex remained. Stemming from previous and current research in the lab, the role of post-translational modifications (PMTs) in nuclear centration was investigated for the different forces exerted due to various factors. Tyrosinated and detyrosinated populations were observed with and without the presence of parthenolide (PTL), an agent that inhibits detyrosination. PTL was observed to not only prevent the proper migration, but also that it expanded tyrosination of tubulin – which would further disrupt the force vectors created through the PMTs promotion of dyneins and kinesins. The results have lead to a new hypothesis to be furthered in order to gain an in-depth understanding in the mechanism(s) for pronuclear migration
Thesis (BS) — Boston College, 2018
Submitted to: Boston College. College of Arts and Sciences
Discipline: Departmental Honors
Discipline: Biology

Thesis advisor: David R. Burgess
Microtubule (MT) asters are radial arrays of MTs nucleated from a microtubule organizingcenter (MTOC) such as the centrosome. Within many cell types, which display highly diverse size and shape, MT asters orchestrate spatial positioning of organelles to ensure proper cellular function throughout the cell cycle and development. Therefore, asters have adopted a wide variety of sizes and morphologies, which are directly affects how they migrate and position within the cell. In large cells, for example during embryonic development, asters growth to sizes on the scales of hundreds of microns to millimeters. Due to this relatively enormous size scale, it is widely accepted that MT asters migrate primarily through pulling mechanisms driven by dynein located in the cytoplasm and/or the cell cortex. Moreover, prior to this dissertation, significant contributions from pushing forces as a result of aster growth and expansion against the cell cortex have not been detected in large cells. Here we have reinvestigated sperm aster growth, morphology, and positioning of MT asters using the large interphase sperm aster of the sea urchin zygote, which is historically a powerful system due to long range migration of the sperm aster to the geometric cell center following fertilization. First, through live-cell quantification of sperm aster growth and geometry, chemical manipulation of aster geometry, inhibition of dynein, and targeted chemical ablation, we show that the sperm aster migrates to the zygote center predominantly through a pushing-based mechanism that appears to largely independent of proposed pulling models. Second, we investigate the fundamental principles for how sperm aster size is determined during growth and centration. By physically manipulating egg size, we obtain samples of eggs displaying a wide range of diameters, all of which are at identical developmental stages. Using live-cell and fluorescence microscopy, we find strong preliminary evidence that aster diameter and migration rates show a direct, linear scaling to cell diameter. Finally, we hypothesize that a collective growth model for aster growth, or centrosome independent MT nucleation, may explain how the sperm aster of large sea urchin zygotes overcomes the proposed physical limitations of a pushing mechanism during large aster positioning. By applying two methods of super resolution microscopy, we find support for this collective growth model in the form of MT branching. Together, we present a model in which growth of astral MTs, potentially through a collective growth model, pushes the sperm aster to the zygote center
Thesis (PhD) — Boston College, 2020
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

The large cells in early vertebrate embryos are organized by radial arrays of microtubules called asters. Asters grow, interact, and move to precisely position the cleavage planes of for each cell division. Cell-spanning dimensions are presumably required for interphase asters to explore the size and shape of the large cytoplasm. It has been unclear whether asters grow to fill the enormous egg according to the standard model of aster growth proposed in smaller somatic cells, or whether special mechanisms are required. In this dissertation, I combine biochemical reconstitution and biophysical modeling to propose a new model of aster growth that involves autocatalytic microtubule nucleation. By imaging asters in a cell-free system derived from frog eggs, I measure the number and positions of microtubules over time and find that most microtubules were nucleated away from the centrosome. I also find the interphase egg cytoplasm supports spontaneous nucleation after a time lag. Given these observations, I construct a biophysical model that describes aster growth from the interplay of microtubule polymerization dynamics and autocatalytic nucleation. This leads to the concept of a critical nucleation rate, which defines the quantitative conditions that predicts either (i) a growing aster characterized by a linear increase radius without dilution of microtubule density at the periphery, or (ii) a steady-state aster with small, constant radius. By combining theory and experiments, I propose a scenario where unbounded aster growth consists of individual microtubules that are themselves bounded in length. This offers a mechanistic explanation to how cells might differentially regulate aster size during the cell cycle. In summary, aster growth is a collective phenomenon of microtubules providing us with insight to how cells self-organize.
Systems Biology