scholarly journals Separate roles of IQGAP Rng2p in forming and constricting the Schizosaccharomyces pombe cytokinetic contractile ring

2013 ◽  
Vol 24 (12) ◽  
pp. 1904-1917 ◽  
Author(s):  
Irene R. Tebbs ◽  
Thomas D. Pollard
Keyword(s):  
Ring Formation ◽  
Actin Binding ◽  
Deletion Mutants ◽  
Alternative Mechanism ◽  
Adapter Proteins ◽  
Contractile Ring ◽  
Systematic Analysis ◽  
Septum Formation ◽  
Normal Ring ◽  
Ring Constriction

Eukaryotic cells require IQGAP family multidomain adapter proteins for cytokinesis, but many questions remain about how IQGAPs contribute to the process. Here we show that fission yeast IQGAP Rng2p is required for both the normal process of contractile ring formation from precursor nodes and an alternative mechanism by which rings form from strands of actin filaments. Our work adds to previous studies suggesting a role for Rng2p in node and ring formation. We demonstrate that Rng2p is also required for normal ring constriction and septum formation. Systematic analysis of domain-deletion mutants established how the four domains of Rng2p contribute to cytokinesis. Contrary to a previous report, the actin-binding calponin homology domain of Rng2p is not required for viability, ring formation, or ring constriction. The IQ motifs are not required for ring formation but are important for ring constriction and septum formation. The GTPase-activating protein (GAP)–related domain is required for node-based ring formation. The Rng2p C-terminal domain is the only domain essential for viability. Our studies identified several distinct functions of Rng2 at multiple stages of cytokinesis.

scholarly journals The F-BAR Cdc15 promotes contractile ring formation through the direct recruitment of the formin Cdc12

2015 ◽  
Vol 208 (4) ◽  
pp. 391-399 ◽  
Author(s):  
Alaina H. Willet ◽  
Nathan A. McDonald ◽  
K. Adam Bohnert ◽  
Michelle A. Baird ◽  
John R. Allen ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Binding Proteins ◽  
Molecular Mechanisms ◽  
Ring Formation ◽  
Scaffolding Protein ◽  
Actin Binding ◽  
Contractile Ring ◽  
Bar Domain ◽  
Medial Cortex ◽  
N Terminus

In Schizosaccharomyces pombe, cytokinesis requires the assembly and constriction of an actomyosin-based contractile ring (CR). Nucleation of F-actin for the CR requires a single formin, Cdc12, that localizes to the cell middle at mitotic onset. Although genetic requirements for formin Cdc12 recruitment have been determined, the molecular mechanisms dictating its targeting to the medial cortex during cytokinesis are unknown. In this paper, we define a short motif within the N terminus of Cdc12 that binds directly to the F-BAR domain of the scaffolding protein Cdc15. Mutations preventing the Cdc12–Cdc15 interaction resulted in reduced Cdc12, F-actin, and actin-binding proteins at the CR, which in turn led to a delay in CR formation and sensitivity to other perturbations of CR assembly. We conclude that Cdc15 contributes to CR formation and cytokinesis via formin Cdc12 recruitment, defining a novel cytokinetic function for an F-BAR domain.

scholarly journals Myosin-independent cytokinesis inGiardiautilizes flagella to coordinate force generation and direct membrane trafficking

2017 ◽  
Vol 114 (29) ◽  
pp. E5854-E5863 ◽  
Author(s):  
William R. Hardin ◽  
Renyu Li ◽  
Jason Xu ◽  
Andrew M. Shelton ◽  
Germain C. M. Alas ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Myosin Ii ◽  
Live Cell ◽  
Actin Binding ◽  
Alternative Mechanism ◽  
Membrane Tension ◽  
Contractile Ring

Devoid of all known canonical actin-binding proteins, the prevalent parasiteGiardia lambliauses an alternative mechanism for cytokinesis. Unique aspects of this mechanism can potentially be leveraged for therapeutic development. Here, live-cell imaging methods were developed forGiardiato establish division kinetics and the core division machinery. Surprisingly,Giardiacytokinesis occurred with a median time that is ∼60 times faster than mammalian cells. In contrast to cells that use a contractile ring, actin was not concentrated in the furrow and was not directly required for furrow progression. Live-cell imaging and morpholino depletion of axonemal Paralyzed Flagella 16 indicated that flagella-based forces initiated daughter cell separation and provided a source for membrane tension. Inhibition of membrane partitioning blocked furrow progression, indicating a requirement for membrane trafficking to support furrow advancement. Rab11 was found to load onto the intracytoplasmic axonemes late in mitosis and to accumulate near the ends of nascent axonemes. These developing axonemes were positioned to coordinate trafficking into the furrow and mark the center of the cell in lieu of a midbody/phragmoplast. We show that flagella motility, Rab11, and actin coordination are necessary for proper abscission. Organisms representing three of the five eukaryotic supergroups lack myosin II of the actomyosin contractile ring. These results support an emerging view that flagella play a central role in cell division among protists that lack myosin II and additionally implicate the broad use of membrane tension as a mechanism to drive abscission.

scholarly journals The ARP2/3 complex prevents excessive formin activity during cytokinesis

2019 ◽  
Vol 30 (1) ◽  
pp. 96-107 ◽  
Author(s):  
Fung-Yi Chan ◽  
Ana M. Silva ◽  
Joana Saramago ◽  
Joana Pereira-Sousa ◽  
Hailey E. Brighton ◽  
...  
Keyword(s):  
Cell Division ◽  
Caenorhabditis Elegans ◽  
Actin Filament ◽  
Ring Formation ◽  
Contractile Ring ◽  
Present Evidence ◽  
Daughter Cells ◽  
Ring Constriction ◽  
Filament Network ◽  
Kinetics Of

Cytokinesis completes cell division by constriction of an actomyosin contractile ring that separates the two daughter cells. Here we use the early Caenorhabditis elegans embryo to explore how the actin filament network in the ring and the surrounding cortex is regulated by the single cytokinesis formin CYK-1 and the ARP2/3 complex, which nucleate nonbranched and branched filaments, respectively. We show that CYK-1 and the ARP2/3 complex are the predominant F-actin nucleators responsible for generating distinct cortical F-actin architectures and that depletion of either nucleator affects the kinetics of cytokinesis. CYK-1 is critical for normal F-actin levels in the contractile ring, and acute inhibition of CYK-1 after furrow ingression slows ring constriction rate, suggesting that CYK-1 activity is required throughout ring constriction. Surprisingly, although the ARP2/3 complex does not localize in the contractile ring, depletion of the ARP2 subunit or treatment with ARP2/3 complex inhibitor delays contractile ring formation and constriction. We present evidence that the delays are due to an excess in formin-nucleated cortical F-actin, suggesting that the ARP2/3 complex negatively regulates CYK-1 activity. We conclude that the kinetics of cytokinesis are modulated by interplay between the two major actin filament nucleators.

scholarly journals Actin filament severing by cofilin is more important for assembly than constriction of the cytokinetic contractile ring

2011 ◽  
Vol 195 (3) ◽  
pp. 485-498 ◽  
Author(s):  
Qian Chen ◽  
Thomas D. Pollard
Keyword(s):  
Fission Yeast ◽  
Computer Simulations ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Binding ◽  
Wild Type ◽  
Contractile Ring ◽  
Ring Constriction ◽  
Release Model ◽  
Mutant Cells

We created two new mutants of fission yeast cofilin to investigate why cytokinesis in many organisms depends on this small actin-binding protein. These mutant cofilins bound actin monomers normally, but bound and severed ADP-actin filaments much slower than wild-type cofilin. Cells depending on mutant cofilins condensed nodes, precursors of the contractile ring, into clumps rather than rings. Starting from clumped nodes, mutant cells slowly assembled rings from diverse intermediate structures including spiral strands containing actin filaments and other contractile ring proteins. This process in mutant cells depended on α-actinin. These slowly assembled contractile rings constricted at a normal rate but with more variability, indicating ring constriction is not very sensitive to defects in severing by cofilin. Computer simulations of the search-capture-pull and release model of contractile ring formation predicted that nodes clump when the release step is slow, so cofilin severing of actin filament connections between nodes likely contributes to the release step.

scholarly journals CYK-4 regulates Rac, but not Rho, during cytokinesis

2017 ◽  
Vol 28 (9) ◽  
pp. 1258-1270 ◽  
Author(s):  
Yelena Zhuravlev ◽  
Sophia M. Hirsch ◽  
Shawn N. Jordan ◽  
Julien Dumont ◽  
Mimi Shirasu-Hiza ◽  
...  
Keyword(s):  
Alternative Model ◽  
Single Cell Analysis ◽  
Myosin Ii ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Filamentous Actin ◽  
Contractile Ring ◽  
Division Plane ◽  
Ring Constriction

Cytokinesis is driven by constriction of an actomyosin contractile ring that is controlled by Rho-family small GTPases. Rho, activated by the guanine-nucleotide exchange factor ECT-2, is upstream of both myosin-II activation and diaphanous formin-mediated filamentous actin (f-actin) assembly, which drive ring constriction. The role for Rac and its regulators is more controversial, but, based on the finding that Rac inactivation can rescue cytokinesis failure when the GTPase-activating protein (GAP) CYK-4 is disrupted, Rac activity was proposed to be inhibitory to contractile ring constriction and thus specifically inactivated by CYK-4 at the division plane. An alternative model proposes that Rac inactivation generally rescues cytokinesis failure by reducing cortical tension, thus making it easier for the cell to divide when ring constriction is compromised. In this alternative model, CYK-4 was instead proposed to activate Rho by binding ECT-2. Using a combination of time-lapse in vivo single-cell analysis and Caenorhabditis elegans genetics, our evidence does not support this alternative model. First, we found that Rac disruption does not generally rescue cytokinesis failure: inhibition of Rac specifically rescues cytokinesis failure due to disruption of CYK-4 or ECT-2 but does not rescue cytokinesis failure due to disruption of two other contractile ring components, the Rho effectors diaphanous formin and myosin-II. Second, if CYK-4 regulates cytokinesis through Rho rather than Rac, then CYK-4 inhibition should decrease levels of downstream targets of Rho. Inconsistent with this, we found no change in the levels of f-actin or myosin-II at the division plane when CYK-4 GAP activity was reduced, suggesting that CYK-4 is not upstream of ECT-2/Rho activation. Instead, we found that the rescue of cytokinesis in CYK-4 mutants by Rac inactivation was Cdc42 dependent. Together our data suggest that CYK-4 GAP activity opposes Rac (and perhaps Cdc42) during cytokinesis.

scholarly journals Synthetic lethality screen identifies a novel yeast myosin I gene (MYO5): myosin I proteins are required for polarization of the actin cytoskeleton.

1996 ◽  
Vol 133 (6) ◽  
pp. 1277-1291 ◽  
Author(s):  
H V Goodson ◽  
B L Anderson ◽  
H M Warrick ◽  
L A Pon ◽  
J A Spudich
Keyword(s):  
Actin Cytoskeleton ◽  
Synthetic Lethality ◽  
Critical Role ◽  
Neck Region ◽  
Actin Binding ◽  
Cell Physiology ◽  
Deletion Mutants ◽  
Tail Domain ◽  
Amino Terminal ◽  
Myosin I

The organization of the actin cytoskeleton plays a critical role in cell physiology in motile and nonmotile organisms. Nonetheless, the function of the actin based motor molecules, members of the myosin superfamily, is not well understood. Deletion of MYO3, a yeast gene encoding a "classic" myosin I, has no detectable phenotype. We used a synthetic lethality screen to uncover genes whose functions might overlap with those of MYO3 and identified a second yeast myosin 1 gene, MYO5. MYO5 shows 86 and 62% identity to MYO3 across the motor and non-motor regions. Both genes contain an amino terminal motor domain, a neck region containing two IQ motifs, and a tail domain consisting of a positively charged region, a proline-rich region containing sequences implicated in ATP-insensitive actin binding, and an SH3 domain. Although myo5 deletion mutants have no detectable phenotype, yeast strains deleted for both MYO3 and MYO5 have severe defects in growth and actin cytoskeletal organization. Double deletion mutants also display phenotypes associated with actin disorganization including accumulation of intracellular membranes and vesicles, cell rounding, random bud site selection, sensitivity to high osmotic strength, and low pH as well as defects in chitin and cell wall deposition, invertase secretion, and fluid phase endocytosis. Indirect immunofluorescence studies using epitope-tagged Myo5p indicate that Myo5p is localized at actin patches. These results indicate that MYO3 and MYO5 encode classical myosin I proteins with overlapping functions and suggest a role for Myo3p and Myo5p in organization of the actin cytoskeleton of Saccharomyces cerevisiae.

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

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.

scholarly journals Rho-dependent control of the Citron kinase, Sticky, drives midbody ring maturation

2019 ◽  
Vol 30 (17) ◽  
pp. 2185-2204 ◽  
Author(s):  
Nour El-amine ◽  
Sabrya C. Carim ◽  
Denise Wernike ◽  
Gilles R. X. Hickson
Keyword(s):  
Function Analysis ◽  
Mechanisms Of Action ◽  
Ring Formation ◽  
Ring Closure ◽  
S2 Cells ◽  
Contractile Ring ◽  
Subsequent Formation ◽  
Control Assembly ◽  
Citron Kinase ◽  
Guide Ring

Rho-dependent proteins control assembly of the cytokinetic contractile ring, yet it remains unclear how those proteins guide ring closure and how they promote subsequent formation of a stable midbody ring. Citron kinase is one important component required for midbody ring formation but its mechanisms of action and relationship with Rho are controversial. Here, we conduct a structure–function analysis of the Drosophila Citron kinase, Sticky, in Schneider’s S2 cells. We define two separable and redundant RhoGEF/Pebble-dependent inputs into Sticky recruitment to the nascent midbody ring and show that each input is subsequently required for retention at, and for the integrity of, the mature midbody ring. The first input is via an actomyosin-independent interaction between Sticky and Anillin, a key scaffold also required for midbody ring formation. The second input requires the Rho-binding domain of Sticky, whose boundaries we have defined. Collectively, these results show how midbody ring biogenesis depends on the coordinated actions of Sticky, Anillin, and Rho.

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