Time-varying mobility and turnover of actomyosin ring components during cytokinesis in<i>Schizosaccharomyces pombe</i>

Kamnev, Anton; Palani, Saravanan; Zambon, Paola; Cheffings, T. H.; Burroughs, Nigel John; Balasubramanian, Mohan K.

doi:10.1091/mbc.e20-09-0588

Cited by 7 publications

(4 citation statements)

References 44 publications

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“…However, Mid1 leaves the CR as it constricts ( Wu et al, 2003 ) and CRs can form in the absence of Mid1 ( Chang et al, 1996 ; Sohrmann et al, 1996 ; Huang et al, 2007 ). FRAP analyses indicate that there are both dynamic and immobile populations of Cdc15 in the CR ( Clifford et al, 2008 ; Roberts-Galbraith et al, 2009 ; Roberts-Galbraith and Gould, 2010 ; Laporte et al, 2011 ; McDonald et al, 2015 ; Kamnev et al, 2021 ). It is thus possible that initial Cdc15 condensates develop into more stable structures if assembled with Mid1 or other proteins.…”

Section: Discussionmentioning

confidence: 99%

Multiple polarity kinases inhibit phase separation of F-BAR protein Cdc15 and antagonize cytokinetic ring assembly in fission yeast

Bhattacharjee

Hall

Mangione

et al. 2023

eLife

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The F-BAR protein Cdc15 is essential for cytokinesis in Schizosaccharomyces pombe and plays a key role in attaching the cytokinetic ring (CR) to the plasma membrane (PM). Cdc15’s abilities to bind to the membrane and oligomerize via its F-BAR domain are inhibited by phosphorylation of its intrinsically disordered region (IDR). Multiple cell polarity kinases regulate Cdc15 IDR phosphostate, and of these the DYRK kinase Pom1 phosphorylation sites on Cdc15 have been shown in vivo to prevent CR formation at cell tips. Here, we compared the ability of Pom1 to control Cdc15 phosphostate and cortical localization to that of other Cdc15 kinases: Kin1, Pck1, and Shk1. We identified distinct but overlapping cohorts of Cdc15 phosphorylation sites targeted by each kinase, and the number of sites correlated with each kinases’ abilities to influence Cdc15 PM localization. Coarse-grained simulations predicted that cumulative IDR phosphorylation moves the IDRs of a dimer apart and toward the F-BAR tips. Further, simulations indicated that the overall negative charge of phosphorylation masks positively charged amino acids necessary for F-BAR oligomerization and membrane interaction. Finally, simulations suggested that dephosphorylated Cdc15 undergoes phase separation driven by IDR interactions. Indeed, dephosphorylated but not phosphorylated Cdc15 undergoes liquid–liquid phase separation to form droplets in vitro that recruit Cdc15 binding partners. In cells, Cdc15 phosphomutants also formed PM-bound condensates that recruit other CR components. Together, we propose that a threshold of Cdc15 phosphorylation by assorted kinases prevents Cdc15 condensation on the PM and antagonizes CR assembly.

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Section: Discussionmentioning

confidence: 99%

Multiple polarity kinases inhibit phase separation of F-BAR protein Cdc15 and antagonize cytokinetic ring assembly in fission yeast

Bhattacharjee

Hall

Mangione

et al. 2023

eLife

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“…Numerous FRAP methods have been proposed to evaluate the complex turnover in a normal diffusion-driven case (8–12), anomalous diffusion-driven case (13–16), chemical reaction-driven case (4, 5), and diffusion-reaction case (4, 7, 17). However, because of the current lack of appropriate FRAP methods, the “long-term” turnover driven by stress fiber-associated active dynamics that include both intracellular bulk-like flow and microscopic structural deformation remains highly elusive.…”

Section: Discussionmentioning

confidence: 99%

“…In chemical reaction-driven molecular turnover, for example, the fluorescence intensity is recovered without exhibiting diffusive gradients in space. Reaction rate equation-based models have been used in these cases to determine the kinetics (4, 5), in which the time constant in the single exponential function as a characteristic time gives the dissociation rate (4, 6). In diffusion-driven case where the Brownian motion-based pure-diffusion transports molecules, fluorescence distribution within the bleached region exhibits spatial gradients, meaning that the characteristic time depends on the size of the bleached region (7).…”

Section: Introductionmentioning

confidence: 99%

Analysis of chemical and mechanical behaviors in living cells by continuum mechanics-based FRAP

Saitô

Matsunaga

Deguchi

2022

Preprint

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Fluorescence recovery after photobleaching (FRAP) is a common technique to analyze the turnover of molecules in living cells. Numerous physicochemical models have been developed to quantitatively evaluate the rate of turnover driven by chemical reaction and diffusion that occurs in a few seconds to minutes. On the other hand, they have limitations in interpreting long-term FRAP responses where intracellular active movement inevitably provides target molecular architectures with additional effects other than chemical reaction and diffusion, namely directed transport and structural deformation. To overcome the limitations, we develop a continuum mechanics-based model that allows for decoupling FRAP response into the intrinsic turnover rate and subcellular mechanical characteristics such as displacement vector and strain tensor. Our approach was validated using fluorescently-labeled beta-actin in an actomyosin-mediated contractile apparatus called stress fibers, revealing spatially distinct patterns of the multi-physicochemical events, in which the turnover rate of beta-actin was significantly higher at the center of the cell. We also found that the turnover rate is negatively correlated with the strain rate along stress fibers but, interestingly, not with the absolute strain magnitude. Moreover, stress fibers are subjected to centripetal flow as well as both contractile and tensile strains along them. Taken together, this novel framework for long-term FRAP analysis allows for unveiling the contribution of overlooked microscopic mechanics to molecular turnover in living cells.

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“…Several candidate mechanisms that could control actomyosin contractile instability have been proposed. One possibility is turnover of myosin and actin ( 24 , 25 ), which presumably tends to homogenize spatial variations ( 12 ). Another suggestion is myosin II diffusivity, which could serve this purpose by smoothing small-scale density variations characteristic of the instability ( 15 , 26 ).…”

mentioning

confidence: 99%

Myosin turnover controls actomyosin contractile instability

Thiyagarajan

Wang

Chew

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Actomyosin contractile force produced by myosin II molecules that bind and pull actin filaments is harnessed for diverse functions, from cell division by the cytokinetic contractile ring to morphogenesis driven by supracellular actomyosin networks during development. However, actomyosin contractility is intrinsically unstable to self-reinforcing spatial variations that may destroy the actomyosin architecture if unopposed. How cells control this threat is not established, and while large myosin fluctuations and punctateness are widely reported, the full course of the instability in cells has not been observed. Here, we observed the instability run its full course in isolated cytokinetic contractile rings in cell ghosts where component turnover processes are absent. Unprotected by turnover, myosin II merged hierarchically into aggregates with increasing amounts of myosin and increasing separation, up to a maximum separation. Molecularly explicit simulations reproduced the hierarchical aggregation which precipitated tension loss and ring fracture and identified the maximum separation as the length of actin filaments mediating mechanical communication between aggregates. In the final simulated dead-end state, aggregates were morphologically quiescent, including asters with polarity-sorted actin, similar to the dead-end state observed in actomyosin systems in vitro. Our results suggest the myosin II turnover time controls actomyosin contractile instability in normal cells, long enough for aggregation to build robust aggregates but sufficiently short to intercept catastrophic hierarchical aggregation and fracture.

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Time-varying mobility and turnover of actomyosin ring components during cytokinesis inSchizosaccharomyces pombe

Abstract: This work investigates the mitotic stage-dependent mobility of fission yeast actomyosin ring proteins in the cytokinetic ring using fluorescence recovery after photobleaching. It reveals a cell cycle–dependent mobility change in the F-BAR protein Cdc15.

Cited by 7 publications

References 44 publications

Multiple polarity kinases inhibit phase separation of F-BAR protein Cdc15 and antagonize cytokinetic ring assembly in fission yeast

Multiple polarity kinases inhibit phase separation of F-BAR protein Cdc15 and antagonize cytokinetic ring assembly in fission yeast

Analysis of chemical and mechanical behaviors in living cells by continuum mechanics-based FRAP

Myosin turnover controls actomyosin contractile instability

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