Cortical recruitment of nonmuscle myosin II in early syncytial Drosophila embryos: its role in nuclear axial expansion and its regulation by Cdc2 activity

The nuclei of early syncytial Drosophila embryos migrate dramatically toward the poles. The cellular mechanisms driving this process, called axial expansion, are unclear, but myosin II activity is required. By following regulatory myosin light chain (RLC)–green fluorescent protein dynamics in living embryos, we observed cycles of myosin recruitment to the cortex synchronized with mitotic cycles. Cortical myosin is first seen in a patch at the anterocentral part of the embryo at cycle 4. With each succeeding cycle, the patch expands poleward, dispersing at the beginning of each mitosis and reassembling at the end of telophase. Each cycle of actin and myosin recruitment is accompanied by a cortical contraction. The cortical myosin cycle does not require microtubules but correlates inversely with Cdc2/cyclinB (mitosis-promoting factor) activity. A mutant RLC lacking inhibitory phosphorylation sites was fully functional with no effect on the cortical myosin cycle, indicating that Cdc2 must be modulating myosin activity by some other mechanism. An inhibitor of Rho kinase blocks the cortical myosin recruitment cycles and provokes a concomitant failure of axial expansion. These studies suggest a model in which cycles of myosin-mediated contraction and relaxation, tightly linked to Cdc2 and Rho kinase activity, are directly responsible for the axial expansion of the syncytial nuclei

Recruitment of Mad2 to the Kinetochore Requires the Rod/Zw10 Complex

SummaryCompromising the activity of the spindle checkpoint permits mitotic exit in the presence of unattached kinetochores and, consequently, greatly increases the rate of aneuploidy in the daughter cells [1–3]. The metazoan checkpoint mechanism is more complex than in yeast in that it requires additional proteins and activities besides the classical Mads and Bubs. Among these are Rod, Zw10, and Zwilch, components of a 700 Kdal complex (Rod/Zw10) [4–6] that is required for recruitment of dynein/dynactin to kinetochores [7, 8] but whose role in the checkpoint is poorly understood. The dynamics of Rod and Mad2, examined in different organisms, show intriguing similarities as well as apparent differences [7, 9] . Here we simultaneously follow GFP-Mad2 and RFP-Rod and find they are in fact closely associated throughout early mitosis. They accumulate simultaneously on kinetochores and are shed together along microtubule fibers after attachment. Their behavior and position within attached kinetochores is distinct from that of BubR1; Mad2 and Rod colocalize to the outermost kinetochore region (the corona), whereas BubR1 is slightly more interior. Moreover, Mad2, but not BubR1, Bub1, Bub3, or Mps1, requires Rod/Zw10 for its accumulation on unattached kinetochores. Rod/Zw10 thus contributes to checkpoint activation by promoting Mad2 recruitment and to checkpoint inactivation by recruiting dynein/dynactin that subsequently removes Mad2 from attached kinetochores

A molecular analysis of three unstable alleles in drosophila : (transposable elements, mutable alleles, white locus)

We have determined the structure of several unstable mutant alleles of the white locus in Drosophila melanogaster. The white ivory (w[superscript i]) allele is a moderately unstable allele, which gave rise to the highly unstable white-crimson (w[superscript C]) allele. We have determined that the w[superscript i] mutation is due to the duplication of 2.9 kilobases (kb) of DNA within the white locus, and that reversion of w[superscript i] to wild type usually occurs by simple loss of one copy of the duplication. We have also analyzed two highly unstable alleles of the white locus, we and white dominant zeste-like (w[superscript DZL]) and have shown that both are insertion mutations. The w[superscript C] mutation results from the insertion of 10 kb of DNA into the w[superscript i] duplication, and the w[superscript DZL] mutation results from the insertion of 13 kb of DNA at or near the right end of the white locus. The w[superscript C] and w[superscript DZL] insertions are structurally related, but not identical, and are related to a previously characterized family of transposable elements, the fold back(FB) elements. The we insertion consists of a single FB elementwith a low eopy number sequence between the moderately repetitive terminal inverted repeats. The wDZL insertion contains two FB elements which flank a single copy sequence in the middle of the insertion. Reversion of w[superscript C] to w[superscript i] is mediated by an apparently precise excision event, while reversion of w[superscript DZL] to wild type occurs by an imprecise excision of the insertion. We suggest that structural differences in the two insertions may account for these different modes of reversion.Mary Collins, Robert Levis, Roger Karess, and Gerald M. Rubin, Department of Embryology Carnegie Institute of Washington, 115 West University Parkway, Baltimore, Maryland

Cloning of rough deal

Mutations in the rough deal (rod) gene of Drosophila greatly increase the missegregation of sister chromatids during mitosis, suggesting a role for this gene product in spindle or kinetochore function. The activity provided by rod also appears to be necessary for the recruitment of two known kinetochore components, Zw10 and cytoplasmic dynein. In this paper we describe the cloning of rough deal and an initial cytological characterization of its product. The Rod protein shares no identifiable structural motif with other known proteins, although apparent homologs exist in the genomes of nematode and man. By immunocytochemistry we show that Rod displays a dynamic intracellular staining pattern, localizing first to kinetochores in prometaphase, but moving to kinetochore microtubules at metaphase. Early in anaphase the protein is once again restricted to the kinetochores, where it persists until the end of telophase. This behavior is in all respects similar to that described for Zw10, and suggests that the proteins function together

Rôle de la myosine II non musculaire dans la cytocinèse et l'embryogénèse précoce chez la drosophile

Lors des premiers cycles de division de l'embryon de drosophile, les noyaux, formant un syncytium, migrent vers les pôles par un mécanisme encore mal défini. En suivant la dynamique de la protéine chimère: chaîne légère de la myosine-GFP (RLC-GFP) dans les embryons vivants, nous avons observé des cycles de recrutement cortical de la myosine en coordination avec les cycles mitotiques. La myosine corticale apparaît au cycle 5 sous forme d'une tâche au tiers antérieur de l'embryon. À chaque nouveau cycle, cette tâche s'étale vers les pôles en se dispersant à chaque début de mitose et en réapparaissant en fin de télophase. Chaque recrutement de la myosine s'accompagne d'une contraction du cortex. Le recrutement cortical de la myosine ne dépend ni des microtubules ni de l'actine, mais dépend de l'activité du MPF. Une RLC mutée sur ses sites de phosphorylation inhibiteurs reste fonctionnelle et n'a aucun effet sur les cycles corticaux de la myosine. Le MPF module donc l'activité de la myosine par un autre mécanisme que via la phosphorylation de ces sites. Nous proposons un modèle de l'expansion axiale des noyaux où les cycles de contraction du cortex provoqués par la myosine et régulés par l'activité du MPF, sont responsables du mouvement des noyaux syncitiaux vers les pôles de l'embryon. L'embryogenèse précoce est également caractérisée par deux évènements d'invagination de membrane présentant des similitudes avec la cytocinèse: la formation du pseudo sillon de clivage et la cellularisation. Grâce à la RLC-GFP, nous avons suivi la dynamique de la formation des sillons et le comportement de la myosine, dans les conditions normales et après injection de drogues. Ces observations nous ont conduit à développer un nouveau modèle de la cellularisation qui divise cet évènement en deux processus distincts: l'invagination du sillon perpendiculaire à la surface de l'embryon dépendante des microtubules, et la fermeture basale des cellules qui requiert l'actine et la myosine.In early syncytial Drosophilae embryos, the nuclei undergo a dramatic migration towards the poles. The cellular mechanisms driving this process are unclear. By following regulatory myosin light chain-GFP (RLC-GFP) dynamics in living embryos, we observed cycles of myosin recruitment to the cortex, synchronized with mitotic cycles. Cortical myosin is first seen in a patch at the antero-central part of the embryo at cycle 5. With each succeeding cycle the patch expands poleward, dispersing at the beginning of each mitosis, and reassembling at the end of telophase. Each cycle of myosin recruitment is accompanied by a cortical contraction. The cortical myosin cycle does not depend on F-actin, nor microtubules, but depends on MPF activity. A mutant RLC lacking inhibitory phosphorylation sites was fully functional, with no effect on the cortical myosin cycle. MPF therefore must be modulating myosin activity through a mechanism other than phosphorylation at these sites. These studies suggest a model in which cycles of myosin-mediated contraction and relaxation, tightly linked to MPF activity, are directly responsible for the axial expansion of the syncytial nuclei. The syncytial embryo is also characterizes by two kinds of membrane invagination events believed to be mechanistically related to conventional cytokinesis: metaphase furrow formation and cellularization. We have examined the role of myosin in these cytokinesis-related processes. With the myosine marker RLC-GFP, we refine the dynamics of furrow invagination, in both normal and experimentally perturbed embryos, and we test the influence of actin and tubulin on myosin behavior. We find that the actual forces creating these membrane invaginations are independent of myosin activity. Finally we propose a model for cellularisation, dividing it into two distinct processes, furrow ingression, which requires microtubules; and an independently regulated basal closure of the columnar cells, requiring myosin and actin.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

CARACTERISATION DU ROLE DE LA PROTEINE ROUGH DEAL AU COURS DE LA SEGREGATION DES CHROMOSOMES CHEZ LA DROSOPHILE

LE CYCLE CELLULAIRE EST UNE SUITE D'EVENEMENTS ORDONNES ET ETROITEMENT CONTROLES. DES MECANISMES DE SURVEILLANCE ASSURENT QUE CHAQUE ETAPE DU CYCLE EST ACCOMPLIE CORRECTEMENT ET PERMETTENT LE PASSAGE A L'ETAPE SUIVANTE. NOUS NOUS INTERESSONS AUX MECANISMES QUI CONTROLENT QUE LA SEGREGATION DES CHROMOSOMES SE FAIT CORRECTEMENT EN MITOSE ET EN MEIOSE. DANS CE BUT, NOUS ETUDIONS DES MUTATIONS DE LA MOUCHE DU VINAIGRE, DROSOPHILA MELANOGASTER, QUI PERTURBENT LA SEGREGATION DES CHROMOSOMES. NOUS NOUS SOMMES PARTICULIEREMENT INTERESSES A L'ETUDE DE LA MUTATION ROUGHDEAL. DANS LES MOUCHES MUTANTES POUR CE GENE, DES DEFAUTS DE SEGREGATION DES CHROMOSOMES A L'ANAPHASE ENTRAINENT L'ACCUMULATION DE CELLULES ANEUPLOIDES. EN OUTRE, LES CERVEAUX MUTANTS TRAITES A LA COLCHICINE, UNE DROGUE DE DEPOLYMERISATION DES MICROTUBULES QUI ARRETENT LES CELLULES EN PROMETAPHASE, PRESENTENT UNE SEPARATION PRECOCE DES CHROMATIDES SURS. AU COURS DE MA THESE, J'AI CONTRIBUE AU CLONAGE DU GENE ROUGH DEAL (ROD) ET INITIE L'ETUDE MOLECULAIRE DE LA PROTEINE ROD. ROD PRESENTE UNE DISTRIBUTION DYNAMIQUE AU COURS DES DIVISIONS MITOTIQUES ET MEIOTIQUES, S'ASSOCIANT SUCCESSIVEMENT AVEC LES KINETOCHORES EN PROMETAPHASE, LES FIBRES KINETOCHORIENNES EN METAPHASE ET A NOUVEAU LES KINETOCHORES EN ANAPHASE. LE COMPORTEMENT DE LA PROTEINE ROD HUMAINE OBSERVE PAR IMMUNOCYTOCHIMIE EST SIMILAIRE A CELUI DE LA PROTEINE DE DROSOPHILE. L'INACTIVATION DE L'HOMOLOGUE ROD DU NEMATODE PROVOQUE DES DEFAUTS DE SEGREGATION DES CHROMOSOMES COMPARABLES A CEUX OBSERVES CHEZ LE MUTANT DE DROSOPHILE. LA PROTEINE ROD EXISTE DANS UN LARGE COMPLEXE PROTEIQUE CONTENANT ZW10, UNE AUTRE PROTEINE IMPLIQUEE DANS LA SEGREGATION DES CHROMOSOMES. NOUS PENSONS QUE CE COMPLEXE JOUE UN ROLE DANS LE CONTROLE DE LA TRANSITION METAPHASE ANAPHASE EN BLOQUANT SPECIFIQUEMENT L'ENTREE EN ANAPHASE TANT QUE TOUS LES CHROMOSOMES NE SONT PAS CORRECTEMENT ATTACHES AUX DEUX POLES DU FUSEAU.PARIS-BIUSJ-Thèses (751052125) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF