Mutations in sticky lead to defective organization of the contractile ring during cytokinesis and are enhanced by Rho and suppressed by Rac

The contractile ring is a highly dynamic structure, but how this dynamism is accomplished remains unclear. Here, we report the identification and analysis of a novel Drosophila gene, sticky (sti), essential for cytokinesis in all fly proliferating tissues. sti encodes the Drosophila orthologue of the mammalian Citron kinase. RNA interference–mediated silencing of sti in cultured cells causes them to become multinucleate. Components of the contractile ring and central spindle are recruited normally in such STICKY-depleted cells that nevertheless display asymmetric furrowing and aberrant blebbing. Together with an unusual distribution of F-actin and Anillin, these phenotypes are consistent with defective organization of the contractile ring. sti shows opposite genetic interactions with Rho and Rac genes suggesting that these GTPases antagonistically regulate STICKY functions. Similar genetic evidence indicates that RacGAP50C inhibits Rac during cytokinesis. We discuss that antagonism between Rho and Rac pathways may control contractile ring dynamics during cytokinesis.

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Drosophila Citron Kinase Is Required for the Final Steps of Cytokinesis

Molecular Biology of the Cell ◽

10.1091/mbc.e04-06-0536 ◽

2004 ◽

Vol 15 (11) ◽

pp. 5053-5063 ◽

Cited By ~ 50

Author(s):

Valeria Naim ◽

Sara Imarisio ◽

Ferdinando Di Cunto ◽

Maurizio Gatti ◽

Silvia Bonaccorsi

Keyword(s):

Rna Interference ◽

Cleavage Site ◽

Cell Types ◽

Wild Type ◽

Contractile Ring ◽

Type Function ◽

Central Spindle ◽

Intercellular Bridges ◽

Membrane Protrusions ◽

Citron Kinase

The mechanisms underlying completion of cytokinesis are still poorly understood. Here, we show that the Drosophila orthologue of mammalian Citron kinases is essential for the final events of the cytokinetic process. Flies bearing mutations in the Drosophila citron kinase (dck) gene were defective in both neuroblast and spermatocyte cytokinesis. In both cell types, early cytokinetic events such as central spindle assembly and contractile ring formation were completely normal. Moreover, cytokinetic rings constricted normally, leading to complete furrow ingression. However late telophases of both cell types displayed persistent midbodies associated with disorganized F actin and anillin structures. Similar defects were observed in dck RNA interference (RNAi) telophases, which, in addition to abnormal F actin and anillin rings, also displayed aberrant membrane protrusions at the cleavage site. Together, these results indicate that mutations in the dck gene result in morphologically abnormal intercellular bridges and in delayed resolution of these structures, suggesting that the wild-type function of dck is required for abscission at the end of cytokinesis. The phenotype of Dck-depleted cells is different from those observed in most Drosophila cytokinesis mutants but extraordinarily similar to that caused by anillin RNAi, suggesting that Dck and anillin are in the same pathway for completion of cytokinesis.

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Molecular Dissection of Cytokinesis by RNA Interference inDrosophila Cultured Cells

Molecular Biology of the Cell ◽

10.1091/mbc.01-12-0589 ◽

2002 ◽

Vol 13 (7) ◽

pp. 2448-2460 ◽

Cited By ~ 162

Author(s):

Maria Patrizia Somma ◽

Barbara Fasulo ◽

Giovanni Cenci ◽

Enrico Cundari ◽

Maurizio Gatti

Keyword(s):

Cultured Cells ◽

Contractile Ring ◽

Membrane Behavior ◽

Double Stranded Rna ◽

Central Spindle ◽

Culture Cells ◽

Ring Assembly ◽

Tissue Culture Cells ◽

Genome Wide ◽

Spindle Microtubules

We have used double-stranded RNA-mediated interference (RNAi) to study Drosophila cytokinesis. We show that double-stranded RNAs for anillin, acGAP,pavarotti, rho1, pebble,spaghetti squash, syntaxin1A, andtwinstar all disrupt cytokinesis in S2 tissue culture cells, causing gene-specific phenotypes. Our phenotypic analyses identify genes required for different aspects of cytokinesis, such as central spindle formation, actin accumulation at the cell equator, contractile ring assembly or disassembly, and membrane behavior. Moreover, the cytological phenotypes elicited by RNAi reveal simultaneous disruption of multiple aspects of cytokinesis. These phenotypes suggest interactions between central spindle microtubules, the actin-based contractile ring, and the plasma membrane, and lead us to propose that the central spindle and the contractile ring are interdependent structures. Finally, our results indicate that RNAi in S2 cells is a highly efficient method to detect cytokinetic genes, and predict that genome-wide studies using this method will permit identification of the majority of genes involved inDrosophila mitotic cytokinesis.

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RNA Interference-Mediated Inhibition of Wild-Type Torsin A Expression Increases Apoptosis Caused by Oxidative Stress in Cultured Cells

Neurochemical Research ◽

10.1007/s11064-010-0177-4 ◽

2010 ◽

Vol 35 (8) ◽

pp. 1214-1223 ◽

Cited By ~ 5

Author(s):

Xue-Ping Chen ◽

Xiao-Hui Hu ◽

Shu-Hui Wu ◽

Yang-Wei Zhang ◽

Bo Xiao ◽

...

Keyword(s):

Oxidative Stress ◽

Rna Interference ◽

Cultured Cells ◽

Wild Type

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Drosophila F-BAR protein Syndapin contributes to coupling the plasma membrane and contractile ring in cytokinesis

Open Biology ◽

10.1098/rsob.130081 ◽

2013 ◽

Vol 3 (8) ◽

pp. 130081 ◽

Cited By ~ 27

Author(s):

Tetsuya Takeda ◽

Iain M. Robinson ◽

Matthew M. Savoian ◽

John R. Griffiths ◽

Anthony D. Whetton ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Curvature ◽

Cellular Process ◽

Cleavage Furrow ◽

Contractile Ring ◽

Functional Interaction ◽

Cortical Dynamics ◽

Central Spindle ◽

Bar Domain ◽

Spindle Microtubules

Cytokinesis is a highly ordered cellular process driven by interactions between central spindle microtubules and the actomyosin contractile ring linked to the dynamic remodelling of the plasma membrane. The mechanisms responsible for reorganizing the plasma membrane at the cell equator and its coupling to the contractile ring in cytokinesis are poorly understood. We report here that Syndapin, a protein containing an F-BAR domain required for membrane curvature, contributes to the remodelling of the plasma membrane around the contractile ring for cytokinesis. Syndapin colocalizes with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) at the cleavage furrow, where it directly interacts with a contractile ring component, Anillin. Accordingly, Anillin is mislocalized during cytokinesis in Syndapin mutants. Elevated or diminished expression of Syndapin leads to cytokinesis defects with abnormal cortical dynamics. The minimal segment of Syndapin, which is able to localize to the cleavage furrow and induce cytokinesis defects, is the F-BAR domain and its immediate C-terminal sequences. Phosphorylation of this region prevents this functional interaction, resulting in reduced ability of Syndapin to bind to and deform membranes. Thus, the dephosphorylated form of Syndapin mediates both remodelling of the plasma membrane and its proper coupling to the cytokinetic machinery.

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Mitochondrial morphology and activity regulate furrow ingression and contractile ring dynamics in Drosophila cellularization

Molecular Biology of the Cell ◽

10.1091/mbc.e20-03-0177 ◽

2020 ◽

Vol 31 (21) ◽

pp. 2331-2347

Author(s):

Sayali Chowdhary ◽

Somya Madan ◽

Darshika Tomer ◽

Manos Mavrakis ◽

Richa Rikhy

Keyword(s):

Mitochondrial Fission ◽

Cell Formation ◽

Mitochondrial Morphology ◽

Contractile Ring ◽

Drosophila Embryogenesis ◽

Ring Dynamics ◽

Mitochondrial Distribution ◽

Ring Constriction

Drp1-regulated mitochondrial fission is essential for mitochondrial distribution across the cell in cellularization during Drosophila embryogenesis. Loss of mitochondrial fission in Drp1 mutant embryos leads to defects in morphogenetic events of cell formation and contractile ring constriction in cellularization.

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Ultrastructure of blebbing and phagocytosis of blebs by hyperplastic thyroid epithelial cells in vivo.

The Journal of Cell Biology ◽

10.1083/jcb.72.3.584 ◽

1977 ◽

Vol 72 (3) ◽

pp. 584-594 ◽

Cited By ~ 31

Author(s):

J D Zeligs ◽

S H Wollman

Keyword(s):

Thyroid Gland ◽

Epithelial Cells ◽

Cultured Cells ◽

Ultrastructural Organization ◽

Contractile Ring ◽

Phagocytic Process ◽

Different Types ◽

Rat Thyroid

In addition to pseudopods, somewhat pleomorphic blebs were consistently found protruding from the apical surfaces of hyperplastic rat thyroid epithelial cells into the follicular lumens in vivo. Many blebs were knobby, roughly hemispherical protrusions, with a diameter of 2-3 mum. Such blebs had a densely packed microfilamentous core and contained numerous apparent ribosomes. They were morphologically similar to blebs that have been observed in a variety of cultured cells. Other blebs were larger, more elongate, and less knobby, but had a similar ultrastructural organization. Blebs of all sizes appeared to be phagocytosed on some occasions by nearby epithelial cells. The phagocytic process involved partial engulfment of the bleb by a typical epithelial pseudopod, followed by an apparent pinching-off process, presumably resulting in the separation of the bleb from its cells or origin. The pinching-off process was associated with a band of approx. 6-nm diameter microfilaments that developed within the pseudopod cytoplasm surrounding the base of the bleb and is postulated to function as a contractile ring. The finding of blebbing is an intact tissue in vivo indicates that this phenomenon is not restricted to cultured cells, and thus tends to extend the significance of in vitro observations of the process. In relation to their occurrence in the hyperplastic thyroid gland in vivo, possible interconversions are considered between different types of blebs, and between blebs and pseudopods.

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Fascetto interacting protein ensures proper cytokinesis and ploidy

Molecular Biology of the Cell ◽

10.1091/mbc.e18-09-0573 ◽

2019 ◽

Vol 30 (8) ◽

pp. 992-1007 ◽

Cited By ~ 1

Author(s):

Zachary T. Swider ◽

Rachel K. Ng ◽

Ramya Varadarajan ◽

Carey J. Fagerstrom ◽

Nasser M. Rusan

Keyword(s):

Cell Division ◽

Tissue Repair ◽

Complete Separation ◽

Contractile Ring ◽

Interacting Protein ◽

Simultaneous Reduction ◽

Central Spindle ◽

Daughter Cells ◽

Molecular Machinery ◽

The Brain

Cell division is critical for development, organ growth, and tissue repair. The later stages of cell division include the formation of the microtubule (MT)-rich central spindle in anaphase, which is required to properly define the cell equator, guide the assembly of the acto-myosin contractile ring and ultimately ensure complete separation and isolation of the two daughter cells via abscission. Much is known about the molecular machinery that forms the central spindle, including proteins needed to generate the antiparallel overlapping interzonal MTs. One critical protein that has garnered great attention is the protein regulator of cytokinesis 1, or Fascetto (Feo) in Drosophila, which forms a homodimer to cross-link interzonal MTs, ensuring proper central spindle formation and cytokinesis. Here, we report on a new direct protein interactor and regulator of Feo we named Feo interacting protein (FIP). Loss of FIP results in a reduction in Feo localization, rapid disassembly of interzonal MTs, and several defects related to cytokinesis failure, including polyploidization of neural stem cells. Simultaneous reduction in Feo and FIP results in very large, tumorlike DNA-filled masses in the brain that contain hundreds of centrosomes. In aggregate, our data show that FIP acts directly on Feo to ensure fully accurate cell division.

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mDia2 Induces the Actin Scaffold for the Contractile Ring and Stabilizes Its Position during Cytokinesis in NIH 3T3 Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e07-10-1086 ◽

2008 ◽

Vol 19 (5) ◽

pp. 2328-2338 ◽

Cited By ~ 124

Author(s):

Sadanori Watanabe ◽

Yoshikazu Ando ◽

Shingo Yasuda ◽

Hiroshi Hosoya ◽

Naoki Watanabe ◽

...

Keyword(s):

Rna Interference ◽

Mammalian Cell ◽

Equatorial Region ◽

Cleavage Furrow ◽

3T3 Cells ◽

Contractile Ring ◽

Binucleate Cells ◽

Dividing Cells ◽

Nih 3T3 ◽

Nih 3T3 Cells

mDia proteins are mammalian homologues of Drosophila diaphanous and belong to the formin family proteins that catalyze actin nucleation and polymerization. Although formin family proteins of nonmammalian species such as Drosophila diaphanous are essential in cytokinesis, whether and how mDia proteins function in cytokinesis remain unknown. Here we depleted each of the three mDia isoforms in NIH 3T3 cells by RNA interference and examined this issue. Depletion of mDia2 selectively increased the number of binucleate cells, which was corrected by coexpression of RNAi-resistant full-length mDia2. mDia2 accumulates in the cleavage furrow during anaphase to telophase, and concentrates in the midbody at the end of cytokinesis. Depletion of mDia2 induced contraction at aberrant sites of dividing cells, where contractile ring components such as RhoA, myosin, anillin, and phosphorylated ERM accumulated. Treatment with blebbistatin suppressed abnormal contraction, corrected localization of the above components, and revealed that the amount of F-actin at the equatorial region during anaphase/telophase was significantly decreased with mDia2 RNAi. These results demonstrate that mDia2 is essential in mammalian cell cytokinesis and that mDia2-induced F-actin forms a scaffold for the contractile ring and maintains its position in the middle of a dividing cell.

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Sticky/Citron kinase maintains proper RhoA localization at the cleavage site during cytokinesis

The Journal of Cell Biology ◽

10.1083/jcb.201105136 ◽

2011 ◽

Vol 195 (4) ◽

pp. 595-603 ◽

Cited By ~ 40

Author(s):

Zuni I. Bassi ◽

Koen J. Verbrugghe ◽

Luisa Capalbo ◽

Stephen Gregory ◽

Emilie Montembault ◽

...

Keyword(s):

Cleavage Site ◽

Kinase Activity ◽

Rho Kinase ◽

Compact Ring ◽

Contractile Ring ◽

Rhoa Activity ◽

Ring Dynamics ◽

Citron Kinase ◽

Guanosine Triphosphatase

In many organisms, the small guanosine triphosphatase RhoA controls assembly and contraction of the actomyosin ring during cytokinesis by activating different effectors. Although the role of some RhoA effectors like formins and Rho kinase is reasonably understood, the functions of another putative effector, Citron kinase (CIT-K), are still debated. In this paper, we show that, contrary to previous models, the Drosophila melanogaster CIT-K orthologue Sticky (Sti) does not require interaction with RhoA to localize to the cleavage site. Instead, RhoA fails to form a compact ring in late cytokinesis after Sti depletion, and this function requires Sti kinase activity. Moreover, we found that the Sti Citron-Nik1 homology domain interacts with RhoA regardless of its status, indicating that Sti is not a canonical RhoA effector. Finally, Sti depletion caused an increase of phosphorylated myosin regulatory light chain at the cleavage site in late cytokinesis. We propose that Sti/CIT-K maintains correct RhoA localization at the cleavage site, which is necessary for proper RhoA activity and contractile ring dynamics.

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