Carsten Wloka

Biphasic targeting and cleavage furrow ingression directed by the tail of a myosin II

The tail of yeast myosin II is localized to the division site by two distinct molecular pathways and sufficient for promoting actomyosin ring assembly, furrow ingression, and guidance in ECM remodeling.

Mechanisms of cytokinesis in budding yeast.

Cytokinesis is essential for cell proliferation in all domains of life. Because the core components and mechanisms of cytokinesis are conserved from fungi to humans, the budding yeast Saccharomyces cerevisiae has served as an attractive model for studying this fundamental process. Cytokinesis in budding yeast is driven by two interdependent cellular events: actomyosin ring (AMR) constriction and the formation of a chitinous cell wall structure called the primary septum (PS), the functional equivalent of extracellular matrix remodeling during animal cytokinesis. AMR constriction is thought to drive efficient plasma membrane ingression as well as to guide PS formation, whereas PS formation is thought to stabilize the AMR during its constriction. Following the completion of the PS formation, two secondary septa (SS), consisting of glucans and mannoproteins, are synthesized at both sides of the PS. Degradation of the PS and a part of the SS by a chitinase and glucanases then enables cell separation. In this review, we discuss the mechanics of cytokinesis in budding yeast, highlighting its common and unique features as well as the emerging questions.

Architecture and dynamic remodelling of the septin cytoskeleton during the cell cycle.

Septins perform diverse functions through the formation of filaments and higher-order structures. However, the exact architecture of septin structures remains unclear. In the budding yeast Saccharomyces cerevisiae, septins form an “hourglass” at the mother-bud neck before cytokinesis, which is converted into a “double ring” during cytokinesis. Here, using platinum-replica electron microscopy, we find that the early hourglass consists of septin double filaments oriented along the mother-bud axis. In the late hourglass, these double filaments are connected by periodic circumferential single filaments on the membrane-proximal side, and are associated with centrally located, circumferential, myosin-II thick filaments on the membrane-distal side. The double ring consists of exclusively circumferential septin filaments. Live-cell imaging studies indicate that the hourglass-to-double ring transition is accompanied by loss of septin subunits from the hourglass, and reorganization of the remaining subunits into the double ring. This work provides an unparalleled view of septin structures within cells and defines their remodeling dynamics during the cell cycle.

Evidence that a septin diffusion barrier is dispensable for cytokinesis in budding yeast.

Abstract Septins are essential for cytokinesis in Saccharomyces cerevisiae, but their precise roles remain elusive. Currently, it is thought that before cytokinesis, the hourglass-shaped septin structure at the mother-bud neck acts as a scaffold for assembly of the actomyosin ring (AMR) and other cytokinesis factors. At the onset of cytokinesis, the septin hourglass splits to form a double ring that sandwiches the AMR and may function as diffusion barriers to restrict diffusible cytokinesis factors to the division site. Here, we show that in cells lacking the septin Cdc10 or the septin-associated protein Bud4, the septins form a ring-like structure at the mother-bud neck that fails to re-arrange into a double ring early in cytokinesis. Strikingly, AMR assembly and constriction, the localization of membrane-trafficking and extracellular-matrix-remodeling factors, cytokinesis, and cell-wall-septum formation all occur efficiently in cdc10Δ and bud4Δ mutants. Thus, diffusion barriers formed by the septin double ring do not appear to be critical for S. cerevisiae cytokinesis. However, an AMR mutation and a septin mutation have synergistic effects on cytokinesis and the localization of cytokinesis proteins, suggesting that tethering to the AMR and a septin diffusion barrier may function redundantly to localize proteins to the division site.

Mitotic exit kinase Dbf2 directly phosphorylates chitin synthase Chs2 to regulate cytokinesis in budding yeast

In Saccharomyces cerevisiae, the primary septum (PS), a functional equivalent of animal ECM, is synthesized during cytokinesis by the chitin synthase Chs2. The mitotic exit kinase Dbf2 regulates PS formation by direct phosphorylation of Chs2, which triggers its dissociation from the actomyosin ring during the late stage of cytokinesis.

Targeting and functional mechanisms of the cytokinesis‑related F‑BAR protein Hof1 during the cell cycle

Hof1 targets to the division site by interacting with septins and myosin II sequentially during the cell cycle. It plays a role in cytokinesis by coupling actomyosin ring constriction to primary septum formation through interactions with Myo1 and Chs2.

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

Myo1 becomes immobile and acts as a scaffold for proteins involved in septum formation and coordination with the actomyosin ring during yeast cytokinesis.

Hof1 and Chs4 Interact via F-BAR Domain and Sel1-like Repeats to Control Extracellular Matrix Deposition during Cytokinesis

Localized extracellular matrix (ECM) remodeling is thought to stabilize the cleavage furrow and maintain cell shape during cytokinesis [1-14]. This remodeling is spatiotemporally coordinated with a cytoskeletal structure pertaining to a kingdom of life, for example the FtsZ ring in bacteria [15], the phragmoplast in plants [16], and the actomyosin ring in fungi and animals [17, 18]. Although the cytoskeletal structures have been analyzed extensively, the mechanisms of ECM remodeling remain poorly understood. In the budding yeast Saccharomyces cerevisiae, ECM remodeling refers to sequential formations of the primary and secondary septa that are catalyzed by chitin synthase-II (Chs2) and chitin synthase-III (the catalytic subunit Chs3 and its activator Chs4), respectively [18, 19]. Surprisingly, both Chs2 and Chs3 are delivered to the division site at the onset of cytokinesis [6, 20]. What keeps Chs3 inactive until secondary septum formation remains unknown. Here, we show that Hof1 binds to the Sel1-like repeats (SLRs) of Chs4 via its F-BAR domain and inhibits Chs3-mediated chitin synthesis during cytokinesis. In addition, Hof1 is required for rapid accumulation as well as efficient removal of Chs4 at the division site. This study uncovers a mechanism by which Hof1 controls timely activation of Chs3 during cytokinesis and defines a novel interaction and function for the conserved F-BAR domain and SLR that are otherwise known for their abilities to bind membrane lipids [21, 22] and scaffold protein complex formation [23].

Analysis of protein dynamics during cytokinesis in budding yeast

Cytokinesis is essential for development and survival of all organisms by increasing cell number and diversity. It is a highly regulated process that requires spatiotemporal coordination of hundreds of proteins functioning in the assembly, constriction, and disassembly of a contractile actomyosin ring, targeted vesicle fusion, and localized extracellular matrix remodeling. Cytokinesis has been studied in multiple systems with a wide range of technologies to learn the common principles. In this chapter, we describe the analysis of protein dynamics during cytokinesis in the budding yeast Saccharomyces cerevisiae by several live-cell imaging methods. This, in combination with the power of yeast genetics, has yielded novel insights into the mechanism of cytokinesis. Similar approaches are increasingly used to study this fundamental process in more complex systems.