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Immobile myosin-II plays a scaffolding role during cytokinesis in budding yeast.

ABSTRACT: Core components of cytokinesis are conserved from yeast to human, but how these components are assembled into a robust machine that drives cytokinesis remains poorly understood. In this paper, we show by fluorescence recovery after photobleaching analysis that Myo1, the sole myosin-II in budding yeast, was mobile at the division site before anaphase and became immobilized shortly before cytokinesis. This immobility was independent of actin filaments or the motor domain of Myo1 but required a small region in the Myo1 tail that is thought to be involved in higher-order assembly. As expected, proteins involved in actin ring assembly (tropomyosin and formin) and membrane trafficking (myosin-V and exocyst) were dynamic during cytokinesis. Strikingly, proteins involved in septum formation (the chitin synthase Chs2) and/or its coordination with the actomyosin ring (essential light chain, IQGAP, F-BAR, etc.) displayed Myo1-dependent immobility during cytokinesis, suggesting that Myo1 plays a scaffolding role in the assembly of a cytokinesis machine.

SUBMITTER: Wloka C

PROVIDER: S-EPMC3563683 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Immobile myosin-II plays a scaffolding role during cytokinesis in budding yeast.

Wloka Carsten C Vallen Elizabeth A EA Thé Lydia L Fang Xiaodong X Oh Younghoon Y Bi Erfei E

The Journal of cell biology 20130128 3

Core components of cytokinesis are conserved from yeast to human, but how these components are assembled into a robust machine that drives cytokinesis remains poorly understood. In this paper, we show by fluorescence recovery after photobleaching analysis that Myo1, the sole myosin-II in budding yeast, was mobile at the division site before anaphase and became immobilized shortly before cytokinesis. This immobility was independent of actin filaments or the motor domain of Myo1 but required a sma ...[more]

PMID: 23358243

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Project description:BackgroundThe mitotic exit network (MEN) is required for events at the end of mitosis such as degradation of mitotic cyclins and cytokinesis. Bub2 and its binding partner Bfa1 act as a GTPase activating protein (GAP) to negatively regulate the MEN GTPase Tem1. The Bub2/Bfa1 checkpoint pathway is required to delay the cell cycle in response to mispositioned spindles. In addition to its role in mitotic exit, Tem1 is required for actomyosin ring contraction.ResultsTo test the hypothesis that the Bub2 pathway prevents premature actin ring assembly, we compared the timing of actin ring formation in wild type, bub2Delta, mad2Delta, and bub2Deltamad2Delta cells both with and without microtubules. There was no difference in the timing of actin ring formation between wild type and mutant cells in a synchronized cell cycle. In the presence of nocodazole, both bub2Delta and mad2Delta cells formed rings after a delay of the same duration. Double mutant bub2Deltamad2Delta and bfa1Deltamad2Delta cells formed rings at the same time with and without nocodazole. To determine if Bub2 has an effect on actomyosin ring contraction through its regulation of Tem1, we used live cell imaging of Myo1-GFP in a bub2Delta strain. We found a significant decrease in the total time of contraction and an increase in rate of contraction compared to wild type cells. We also examined myosin contraction using Myo1-GFP in cells overexpressing an epitope tagged Bub2. Surprisingly, overexpression of Bub2 also led to a significant increase in the rate of contraction, as well as morphological defects. The chained cell phenotype caused by Bub2 overexpression could be rescued by co-overexpression of Tem1, and was not rescued by deletion of BFA1.ConclusionOur data indicate that the Bub2 checkpoint pathway does not have a specific role in delaying actin ring formation. The observed increase in the rate of myosin contraction in the bub2Delta strain provides evidence that the MEN regulates actomyosin ring contraction. Our data suggest that the overexpression of the Bub2 fusion protein acts as a dominant negative, leading to septation defects by a mechanism that is Tem1-dependent.

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Immobile myosin-II plays a scaffolding role during cytokinesis in budding yeast.

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Immobile myosin-II plays a scaffolding role during cytokinesis in budding yeast.

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