Abstract
SummaryThe position of the division site dictates the size and fate of daughter cells in many organisms. In animal cells, division-site placement involves overlapping mechanisms, including signaling from the central spindle microtubules, astral microtubules, and spindle poles and through polar contractions [1, 2, 3]. In fission yeast, division-site positioning requires overlapping mechanisms involving the anillin-related protein Mid1 and the tip complex (comprising the Kelch-repeat protein Tea1, the Dyrk-kinase Pom1, and the SH3-domain protein Tea4) [4, 5, 6, 7, 8, 9, 10, 11]. In addition to these factors, cell shape has also been shown to participate in the maintenance of the position of the actomyosin ring [12, 13, 14]. The first principles guiding actomyosin ring placement, however, have not been elucidated in any organism. Because actomyosin ring positioning, ring assembly, and cell morphogenesis are genetically separable in fission yeast, we have used it to derive actomyosin ring placement mechanisms from first principles. We report that, during ring assembly in the absence of cytokinetic cues (anillin-related Mid1 and tip-complex proteins), actin bundles follow the path of least curvature and assemble actomyosin rings in an equatorial position in spherical protoplasts and along the long axis in cylindrical cells and compressed protoplasts. The equatorial position of rings is abolished upon treatment of protoplasts with an actin-severing compound or by slowing down actin polymerization. We propose that the physical properties of actin filaments/bundles play key roles in actomyosin ring assembly and positioning, and that key cytokinetic molecules may modulate the length of actin filaments to promote ring assembly along the short axis.
Highlights
To study the ring assembly process, we imaged wild-type spheroplasts expressing LifeAct-EGFP and mCherry-Atb2 in cell suspension using time-lapse microscopy [19]
In spheroplast diameters (Ss). pombe, cell-geometry, cell-wall, and cytokinesis-positioning factors contribute to the determination of the location of the actomyosin ring [4, 12,13,14,15]
To investigate where the actomyosin ring would form in the absence of all these factors, we first generated spherical cells with minimal residual cell wall by enzymatically removing the cell wall in an osmotically stabilized environment
Summary
To study the ring assembly process, we imaged wild-type spheroplasts expressing LifeAct-EGFP (as a proxy for actin filaments) and mCherry-Atb (alpha-tubulin; as a marker of the cellcycle stage) in cell suspension using time-lapse microscopy [19]. We measured and compared the ring diameters (Rs; visualized either by LifeAct-EGFP or Rlc1-GFP) to the spheroplast diameters (Ss). The diameter of the spheroplast (S) is defined as the length of a line through the center of the spheroplast that intersects two points on its circumference. The ratio of these parameters (R/S) was used to express the size of the assembled ring in relation to the diameter of the spheroplast (Figure 1A). The LifeAct-EGFP (Figure S1) and Rlc1-GFP (regulatory light chain of myosin) [20] used did not cause any overt cytokinetic phenotype in cells, and were used in these studies
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