Project description:To maintain tissue architecture, epithelial cells divide in a planar fashion, perpendicular to their main polarity axis. As the centrosome resumes an apical localization in interphase, planar spindle orientation is reset at each cell cycle. We used three-dimensional live imaging of GFP-labeled centrosomes to investigate the dynamics of spindle orientation in chick neuroepithelial cells. The mitotic spindle displays stereotypic movements during metaphase, with an active phase of planar orientation and a subsequent phase of planar maintenance before anaphase. We describe the localization of the NuMA and LGN proteins in a belt at the lateral cell cortex during spindle orientation. Finally, we show that the complex formed of LGN, NuMA, and of cortically located G?i subunits is necessary for spindle movements and regulates the dynamics of spindle orientation. The restricted localization of LGN and NuMA in the lateral belt is instructive for the planar alignment of the mitotic spindle, and required for its planar maintenance.
Project description:BackgroundIn developing tissues, cell polarity and tissue architecture play essential roles in the regulation of proliferation and differentiation. During cerebral cortical development, adherens junctions link highly polarized radial glial cells in a neurogenic niche that controls their behavior. How adherens junctions regulate radial glial cell polarity and/or differentiation in mammalian cortical development is poorly understood.ResultsConditional deletion of Afadin, a protein required for formation and maintenance of epithelial tissues, leads to abnormalities in radial glial cell polarity and subsequent loss of adherens junctions. We observed increased numbers of obliquely-oriented progenitor cell divisions, increased exit from the ventricular zone neuroepithelium, and increased production of intermediate progenitors.ConclusionsTogether, these findings indicate that Afadin plays an essential role in regulating apical-basal polarity and adherens junction integrity of radial glial cells, and suggest that epithelial architecture plays an important role in radial glial identity by regulating mitotic orientation and preventing premature exit from the neurogenic niche.
Project description:Mitosis is a fundamental process in the development of all organisms. The mitotic spindle guides the cell through mitosis as it mediates the segregation of chromosomes, the orientation of the cleavage furrow, and the progression of cell division. Birth defects and tissue-specific cancers often result from abnormalities in mitotic events. Here, we report a proteomic study of the mitotic spindle from Chinese Hamster Ovary (CHO) cells. Four different isolations of metaphase spindles were subjected to Multi-dimensional Protein Identification Technology (MudPIT) analysis and tandem mass spectrometry. We identified 1155 proteins and used Gene Ontology (GO) analysis to categorize proteins into cellular component groups. We then compared our data to the previously published CHO midbody proteome and identified proteins that are unique to the CHO spindle. Our data represent the first mitotic spindle proteome in CHO cells, which augments the list of mitotic spindle components from mammalian cells.
Project description:The role of centrosomes and centrioles during mitotic spindle assembly in vertebrates remains controversial. In cell-free extracts and experimentally derived acentrosomal cells, randomly oriented microtubules (MTs) self-organize around mitotic chromosomes and assemble anastral spindles. However, vertebrate somatic cells normally assemble a connected pair of polarized, astral MT arrays--termed an amphiaster ("a star on both sides")--that is formed by the splitting and separation of the microtubule-organizing center (MTOC) well before nuclear envelope breakdown (NEB). Whether amphiaster formation requires splitting of duplicated centrosomes is not known. We found that when centrosomes were removed from living vertebrate cells early in their cell cycle, an acentriolar MTOC reassembled, and, prior to NEB, a functional amphiastral spindle formed. Cytoplasmic dynein, dynactin, and pericentrin are all recruited to the interphase aMTOC, and the activity of kinesin-5 is needed for amphiaster formation. Mitosis proceeded on time and these karyoplasts divided in two. However, ~35% of aMTOCs failed to split and separate before NEB, and these entered mitosis with persistent monastral spindles. Chromatin-associated RAN-GTP--the small GTPase Ran in its GTP bound state--could not restore bipolarity to monastral spindles, and these cells exited mitosis as single daughters. Our data reveal the novel finding that MTOC separation and amphiaster formation does not absolutely require the centrosome, but, in its absence, the fidelity of bipolar spindle assembly is highly compromised.
Project description:Proper spindle positioning at anaphase onset is essential for normal tissue organization and function. Here we develop automated spindle-tracking software and apply it to characterize mitotic spindle dynamics in the Xenopus laevis embryonic epithelium. We find that metaphase spindles first undergo a sustained rotation that brings them on-axis with their final orientation. This sustained rotation is followed by a set of striking stereotyped rotational oscillations that bring the spindle into near contact with the cortex and then move it rapidly away from the cortex. These oscillations begin to subside soon before anaphase onset. Metrics extracted from the automatically tracked spindles indicate that final spindle position is determined largely by cell morphology and that spindles consistently center themselves in the XY-plane before anaphase onset. Finally, analysis of the relationship between spindle oscillations and spindle position relative to the cortex reveals an association between cortical contact and anaphase onset. We conclude that metaphase spindles in epithelia engage in a stereotyped "dance," that this dance culminates in proper spindle positioning and orientation, and that completion of the dance is linked to anaphase onset.
Project description:The formation of a metaphase spindle, a bipolar microtubule array with centrally aligned chromosomes, is a prerequisite for the faithful segregation of a cell's genetic material. Using a full-genome RNA interference screen of Drosophila S2 cells, we identified about 200 genes that contribute to spindle assembly, more than half of which were unexpected. The screen, in combination with a variety of secondary assays, led to new insights into how spindle microtubules are generated; how centrosomes are positioned; and how centrioles, centrosomes, and kinetochores are assembled.
Project description:There is an unmet need for new antimitotic drug combinations that target cancer-specific vulnerabilities. Based on our finding of elevated biomolecule oxidation in mitotically arrested cancer cells, we combined Plk1 inhibitors with TH588, an MTH1 inhibitor that prevents detoxification of oxidized nucleotide triphosphates. This combination showed robust synergistic killing of cancer, but not normal, cells that, surprisingly, was MTH1-independent. To dissect the underlying synergistic mechanism, we developed VISAGE, a strategy integrating experimental synergy quantification with computational-pathway-based gene expression analysis. VISAGE predicted, and we experimentally confirmed, that this synergistic combination treatment targeted the mitotic spindle. Specifically, TH588 binding to β-tubulin impaired microtubule assembly, which when combined with Plk1 blockade, synergistically disrupted mitotic chromosome positioning to the spindle midzone. These findings identify a cancer-specific mitotic vulnerability that is targetable using Plk1 inhibitors with microtubule-destabilizing agents and highlight the general utility of the VISAGE approach to elucidate molecular mechanisms of drug synergy.
Project description:We investigated the role of Pav-KLP, a kinesin-6, in the coordination of spindle and cortical dynamics during mitosis in Drosophila embryos. In vitro, Pav-KLP behaves as a dimer. In vivo, it localizes to mitotic spindles and furrows. Inhibition of Pav-KLP causes defects in both spindle dynamics and furrow ingression, as well as causing changes in the distribution of actin and vesicles. Thus, Pav-KLP stabilizes the spindle by crosslinking interpolar microtubule bundles and contributes to actin furrow formation possibly by transporting membrane vesicles, actin and/or actin regulatory molecules along astral microtubules. Modeling suggests that furrow ingression during cellularization depends on: (1) a Pav-KLP-dependent force driving an initial slow stage of ingression; and (2) the subsequent Pav-KLP-driven transport of actin- and membrane-containing vesicles to the furrow during a fast stage of ingression. We hypothesize that Pav-KLP is a multifunctional mitotic motor that contributes both to bundling of interpolar microtubules, thus stabilizing the spindle, and to a biphasic mechanism of furrow ingression by pulling down the furrow and transporting vesicles that deliver new material to the descending furrow.
Project description:The Saccharomyces cerevisiae mitotic spindle in budding yeast is exemplified by its simplicity and elegance. Microtubules are nucleated from a crystalline array of proteins organized in the nuclear envelope, known as the spindle pole body in yeast (analogous to the centrosome in larger eukaryotes). The spindle has two classes of nuclear microtubules: kinetochore microtubules and interpolar microtubules. One kinetochore microtubule attaches to a single centromere on each chromosome, while approximately four interpolar microtubules emanate from each pole and interdigitate with interpolar microtubules from the opposite spindle to provide stability to the bipolar spindle. On the cytoplasmic face, two to three microtubules extend from the spindle pole toward the cell cortex. Processes requiring microtubule function are limited to spindles in mitosis and to spindle orientation and nuclear positioning in the cytoplasm. Microtubule function is regulated in large part via products of the 6 kinesin gene family and the 1 cytoplasmic dynein gene. A single bipolar kinesin (Cin8, class Kin-5), together with a depolymerase (Kip3, class Kin-8) or minus-end-directed kinesin (Kar3, class Kin-14), can support spindle function and cell viability. The remarkable feature of yeast cells is that they can survive with microtubules and genes for just two motor proteins, thus providing an unparalleled system to dissect microtubule and motor function within the spindle machine.
Project description:Taxol functions to suppress the dynamic behavior of individual microtubules, and induces multipolar mitotic spindles. However, little is known about the mechanisms by which taxol disrupts normal bipolar spindle assembly in vivo. Using live imaging of GFP-alpha tubulin expressing cells, we examined spindle assembly after taxol treatment. We find that as taxol-treated cells enter mitosis, there is a dramatic re-distribution of the microtubule network from the centrosomes to the cell cortex. As they align there, the cortical microtubules recruit NuMA to their embedded ends, followed by the kinesin motor HSET. These cortical microtubules then bud off to form cytasters, which fuse into multipolar spindles. Cytoplasmic dynein and dynactin do not re-localize to cortical microtubules, and disruption of dynein/dynactin interactions by over-expression of p50 "dynamitin" does not prevent cytaster formation. Taxol added well before spindle poles begin to form induces multipolarity, but taxol added after nascent spindle poles are visible-but before NEB is complete-results in bipolar spindles. Our results suggest that taxol prevents rapid transport of key components, such as NuMA, to the nascent spindle poles. The net result is loss of mitotic spindle pole cohesion, microtubule re-distribution, and cytaster formation.