Equatorial assembly of the cell-division actomyosin ring in the absence of cytokinetic spatial cues

The 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

Studies of cytokinesis in fission yeast protoplast

In many organisms, cytokinesis is facilitated by an actomyosin-based contractile ring. Positioning of the ring requires close coordination of positive and negative-signaling cues, coupled with cell geometry and nuclear position. The fission yeast S. pombe relies on these spatial cues to accomplish stable ring assembly. After proper positioning of the actomyosin ring, the ring contracts to drive membrane invagination, cell wall assembly, and to complete cell separation into two daughter cells. Although this model organism has its specificities, knowledge of the basic mechanisms and roles of actomyosin ring could be useful to understand similar mechanisms in other organisms. We were curious about how a fission yeast maintain a robust cell division machinery, if majority of cell wall is absent. In cylindrical fission yeast cells, coordination of actomyosin ring positioning is facilitated by a positive spatial cue, mid1 and a negative spatial cue, the tip-complex. In spherical protoplast of fission yeast, we observed mislocalized spatial cues, while the actomyosin ring consistently assemble at the equatorial region. Although removal of mid1 and the tip-complex in cylindrical cells caused the actomyosin ring to assemble along the long axis, it did not hinder the equatorial assembly of actomyosin ring in the spherical protoplasts. We found that actin filaments played a role as major determinant of positioning actomyosin ring in the absence of spatial cues. Given the spheroplasts are capable of forming an equatorial ring, we then asked whether the actomyosin ring is capable of contracting and bring about cytofission. The cps1 mutant, a cell wall mutant lacking b-glucan synthase function is unable to overcome its turgor pressure and undergo actomyosin ring contraction. We found that by generating protoplasts, cytofission (which we define as a process that separates the cytosol into two membrane-bound entities) took place and the protoplast was divided into two entities. We report that this event does not require a-glucan (another component of cell wall), exoglucanases to facilitate breakdown residual septum, and ESCRT proteins. The actomyosin ring is however essential for cytofission

Inhibition of cell membrane ingression at the division site by cell wall in fission yeast

Eukaryotic cells assemble an actomyosin ring during cytokinesis to function as a force-generating machine to drive membrane invagination, and to counteract the intracellular pressure and the cell surface tension. It is unclear whether additional factors such as the extracellular matrix (cell wall in yeasts and fungi) affect the actomyosin ring contraction. While studying the fission yeast β-glucan synthase mutant cps1-191, which is defective in division septum synthesis and actomyosin ring contraction, we found that significantly weakening of the extracellular glycan matrix caused the spheroplasts to divide at the non-permissive condition. This division was dependent on a functional actomyosin ring and vesicular trafficking, but independent of normal septum synthesis. cps1-191 cells with weakened extracellular glycan matrix divide non-medially with a much slower ring contraction rate compared to wild type cells under similar conditions, which we term as cytofission. Interestingly, the high turgor pressure appears to play minimal roles in inhibiting ring contraction in cps1-191 mutants as decreasing the turgor pressure alone does not enable cytofission. We propose that during cytokinesis, the extracellular glycan matrix restricts actomyosin ring contraction and membrane ingression, and remodeling of the extracellular components through division septum synthesis relieves the inhibition and facilitates actomyosin ring contraction

mNG-tagged fusion proteins and nanobodies to visualize tropomyosins in yeast and mammalian cells

Tropomyosins are structurally conserved α-helical coiled-coil proteins that bind along the length of filamentous actin (F-actin) in fungi and animals. Tropomyosins play essential roles in the stability of actin filaments and in regulating myosin II contractility. Despite the crucial role of tropomyosin in actin cytoskeletal regulation, in vivo investigations of tropomyosin are limited, mainly due to the suboptimal live-cell imaging tools currently available. Here, we report on an mNeonGreen (mNG)-tagged tropomyosin, with native promoter and linker length configuration, that clearly reports tropomyosin dynamics in Schizosaccharomyces pombe (Cdc8), Schizosaccharomyces japonicus (Cdc8) and Saccharomyces cerevisiae (Tpm1 and Tpm2). We also describe a fluorescent probe to visualize mammalian tropomyosin (TPM2 isoform). Finally, we generated a camelid nanobody against S. pombe Cdc8, which mimics the localization of mNG–Cdc8 in vivo. Using these tools, we report the presence of tropomyosin in previously unappreciated patch-like structures in fission and budding yeasts, show flow of tropomyosin (F-actin) cables to the cytokinetic actomyosin ring and identify rearrangements of the actin cytoskeleton during mating. These powerful tools and strategies will aid better analyses of tropomyosin and F-actin cables in vivo