Cell cycle control of septin ring dynamics in the budding yeast

Abstract

Septins constitute a cytoskeletal structure that is conserved in eukaryotes. In Saccharomyces cerevisiae, the Cdc3, Cdc10, Cdc11, Cdc12 and Shs1/Sep7 septins assemble as a ring that marks the cytokinetic plane throughout the budding cycle. This structure participates in different aspects of morphogenesis, such as selection of cell polarity, localization of chitin synthesis, the switch from hyperpolar to isotropic bud growth after bud emergence and the spatial regulation of septation. The septin cytoskeleton assembles at the pre-bud site before bud emergence, remains there during bud growth and duplicates at late mitosis eventually disappearing after cell separation. Using a septin-GFP fusion and time-lapse confocal microscopy, we have determined that septin dynamics are maintained in budding zygotes and during unipolar synchronous growth in pseudohyphae. By means of specific cell cycle arrests and deregulation of cell cycle controls we show that septin assembly is dependent on G1 cyclin/Cdc28-mediated cell cycle signals and that the small GTPase Cdc42, but not Rho1, are essential for this event. However, during bud growth, the septin ring shapes a bud-neck-spanning structure that is unaffected by failures in the regulation of mitosis, such as activation of the DNA repair or spindle assembly checkpoints or inactivation of the anaphase-promoting complex (APC). At the end of the cell cycle, the splitting of the ring into two independent structures depends on the function of the mitotic exit network in which the protein phosphatase Cdc14 participates. Our data support a role of cell cycle control mechanisms in the regulation of septin dynamics to accurately coordinate morphogenesis throughout the budding process in yeast.

The Role of Cdc42p GTPase-activating Proteins in Assembly of the Septin Ring in Yeast

“…Ford and Cid et al, 2001; C. Caruso and J. Pringle, unpublished results). These observations suggest that the transition from a low-to a high-stability state as seen by FRAP may correspond to the development of the higher-order structure that results in the apparent filaments seen in the EM.…”

“…Note that the longer bud of cell 1 (A) is out of the plane of focus in B. Ford and Pringle, 1991;Cid et al, 2001;C. Caruso and J. Pringle, unpublished results).…”

“…Because the initial recruitment step appears to depend on both Cdc42p and Cdc24p Cid et al, 2001;Gladfelter et al, 2002;H.B. Kim, B.K.…”

“…The Rho-family GTPase Cdc42p and its activating factor (GEF) Cdc24p are required for the polarized assembly of both the actin cytoskeleton and the septins at the beginning of the cell cycle Johnson, 1999;Pruyne and Bretscher, 2000;Cid et al, 2001;Gladfelter et al, 2001a). However, actin and the septins appear to be independent of each other for their organization at the presumptive bud site (Adams and Pringle, 1984;Ford and Pringle, 1991;Ayscough et al, 1997;Harkins et al, 2001).…”

Inhibition of Cdc42-dependent signalling in Saccharomyces cerevisiae by phosphatase-dead SigD/SopB from Salmonella typhimurium

“…In all cases, fidelity of the amplified DNA was verified by DNA sequencing. Other plasmids used in this work were pLA10H (Cid et al, 2001a), used to express GFP-tagged septin, and GAL-GST-CDC24, kindly provided by Daniel Lew (Duke University, GST pull-down assays. Cultures were grown overnight in raffinose-based medium to exponential phase, and expression of the GST or GFP fusions was induced with galactose for 8 h. Lysates were obtained from these cultures in ice-cold lysis buffer (10 %, v/v, glycerol, 50 mM Tris/HCl pH 7?5, 0?1 % NP40, 150 mM NaCl, 5 mM EDTA pH 8, 50 mM NaF, 5 mM sodium pyrophosphate, 50 mM b-glycerol phosphate, 1 mM sodium vanadate), clarified by centrifugation at 13 000 r.p.m.…”