No abstract

Eisosomes generate spatial domains in the plasma membrane of yeast cells. The core eisosome protein Pil1 is shown to form filaments in vitro and in cells. Pil1 filaments are stable at the cell cortex, and cytoplasmic Pil1 filament rods appear upon overexpression. This shows a role for self-assembly in organizing cortical domains.

Summary Cell size control requires mechanisms that integrate cell growth and division. Key to this integration in fission yeast is the SAD family kinase Cdr2, which organizes a set of cortical nodes in the cell middle to promote mitotic entry through Wee1 and Cdk1 [1-3]. Cdr2 is inhibited by a spatial gradient of the DYRK kinase Pom1 emanating from cell tips in a cell size-dependent manner [2, 3], but how the Pom1 gradient inhibits Cdr2 activity during cell growth is unknown. Here, we show that Pom1 acts to prevent activation of Cdr2 kinase activity by the CaMKK Ssp1. We found that Ssp1 activates Cdr2 through phosphorylation of a conserved threonine residue (Thr166) in the activation loop of the Cdr2 N-terminal kinase domain both in vitro and in cells. The levels of this activating phosphorylation increased with cell cycle progression, and genetic epistasis demonstrated that Ssp1 promotes mitotic entry through Cdr2. Intriguingly, Pom1 phosophorylated the C-terminal domain (CTD) of Cdr2, and this modification reduced Cdr2-T166 phosphorylation by Ssp1. These findings show how activation of the conserved mitotic inducer Cdr2 is integrated with an inhibitory spatial gradient to ensure proper cell size control at mitosis.

c Spatial control of cytokinesis is essential for proper cell division. The molecular mechanisms that anchor the dynamic assembly and constriction of the cytokinetic ring at the plasma membrane remain unclear. In the fission yeast Schizosaccharomyces pombe, the cytokinetic ring is assembled in the cell middle from cortical node precursors that are positioned by the anillin-like protein Mid1. During mitotic entry, cortical nodes mature and then compact into a contractile ring positioned in the cell middle. The molecular link between Mid1 and medial cortical nodes remains poorly defined. Here we show that Blt1, a previously enigmatic cortical node protein, promotes the robust association of Mid1 with cortical nodes. Blt1 interacts with Mid1 through the RhoGEF Gef2 to stabilize nodes at the cell cortex during the early stages of contractile ring assembly. The Blt1 N terminus is required for localization and function, while the Blt1 C terminus promotes cortical localization by interacting with phospholipids. In cells lacking membrane binding by both Mid1 and Blt1, nodes detach from the cell cortex and generate aberrant cytokinetic rings. We conclude that Blt1 acts as a scaffolding protein for precursors of the cytokinetic ring and that Blt1 and Mid1 provide overlapping membrane anchors for proper division plane positioning. Cell division requires the spatial and temporal coordination of many cellular activities. During cytokinesis, the final act of the cell cycle, a contractile actomyosin ring constricts to separate the two daughter cells. The contractile ring must be properly assembled and positioned to ensure equal segregation of cellular materials to each daughter cell. The contracting cytokinetic ring maintains association with the cell cortex, which undergoes dramatic remodeling and membrane bending during this process. The mechanisms that position and anchor components of the cytokinetic ring in the plasma membrane have been the subject of intense study, as defects in this process can lead to a range of cellular defects and disease states (1-4).Many insights into eukaryotic cytokinesis have come from work on the fission yeast Schizosaccharomyces pombe (1, 5). These rod-shaped cells grow in a linear manner at the cell ends and then position the contractile ring precisely in the cell middle at division. This positioning occurs through the combination of inhibitory signals emanating from the cell ends and positive cues from the nucleus in the cell middle. These positional cues act largely through the protein Mid1, which is similar to anillin in metazoans (6, 7). During interphase, Mid1 localizes to the nucleus and to a band of cortical nodes that are positioned in the cell middle. These interphase nodes are organized by the protein kinase Cdr2 and are spatially restricted to the cell middle by inhibitory cues from the cell tips (8-12). During interphase, these cortical nodes also contain a cell cycle regulatory network that couples mitotic entry with cell size (11, 13). Proteomic studies have revealed additional ...