Prolonged cyclin-dependent kinase inhibition results in septin perturbations during return to growth and mitosis

Abstract

We investigated how coordinate polarization, budding, and anaphase during a unique developmental program called return to growth (RTG) in which cells in meiosis return to mitosis upon nutrient shift. Cells reentering mitosis from prophase I deviate from the normal cell cycle by budding in G2 instead of G1. We found that cells do not maintain the bipolar budding pattern, a characteristic of diploid cells. Furthermore, strict temporal regulation of M-phase cyclin-dependent kinase (CDK; M-CDK) is important for polarity establishment and morphogenesis. Cells with premature M-CDK activity caused by loss of checkpoint kinase Swe1 failed to polarize and underwent anaphase without budding. Mutants with increased Swe1-dependent M-CDK inhibition showed additional or more penetrant phenotypes in RTG than mitosis, including elongated buds, multiple buds, spindle mispositioning, and septin perturbation. Surprisingly, the enhanced and additional phenotypes were not exclusive to RTG but also occurred with prolonged Swe1-dependent CDK inhibition in mitosis. Our analysis reveals that prolonged activation of the Swe1-dependent checkpoint can be detrimental instead of beneficial.

Growth-Dependent Activation of Protein Kinases Suggests a Mechanism for Measuring Cell Growth

“…Consistent with this, we observed that gin4-AID hsl1-AID caused a failure in cell separation in nearly all cells at the end of the first cell cycle following the addition of auxin (Video 2). Previous studies also found that gin4∆ and hsl1∆ cause defects in spindle positioning (Fraschini et al 2006;Grava et al 2006;Gihana et al 2018). We observed only a few defects in spindle positioning during the first cell division after addition of auxin to gin4-AID hsl1-AID cells.…”

“…However, an alternative model is that aberrant growth caused by loss of Gin4-related kinases is an indirect consequence of mitotic delays. For example, there is evidence that loss of Gin4-related kinases causes defects in positioning the mitotic spindle, which could cause chromosome segregation defects and associated mitotic delays that lead to abnormally prolonged growth (Fraschini et al 2006;Grava et al 2006;Gihana et al 2018). In this case, growth defects would be a secondary consequence of mitotic spindle defects.…”

The 14-3-3 Proteins Bmh1 and Bmh2 Are Key Regulators of Meiotic Commitment

“…Successful completion of meiosis requires that cells remain committed to completing meiosis, even in the presence of a mitosis-inducing signal. Historically, meiotic commitment has been studied in budding yeast due to the ease of providing meiotic cells a mitosis-inducing signal, nutrient-rich medium (Ballew & Lacefield, 2019a, 2019b; Esposito & Esposito, 1974; Gihana, Musser, Thompson, & Lacefield, 2018; Honigberg & Esposito, 1994; Nachman, Regev, & Ramanathan, 2007; Sherman & Roman, 1963; Simchen, 2009; Simchen et al, 1972; Tsuchiya et al, 2014). However, only a few genes have been implicated in meiotic commitment (Ballew & Lacefield, 2019a; Esposito & Esposito, 1974; Honigberg, Conicella, & Espositio, 1992; Honigberg & Esposito, 1994; Tsuchiya et al, 2014).…”

High-throughput genetic screening of meiotic commitment using fluorescence microscopy in Saccharomyces cerevisiae

“…Prior to initiating the protocol, the features of the starting strain for the screen should be carefully considered. To study genes involved in progression through meiosis, meiotic commitment, and return-to-growth, we typically use cells that express three fluorescently-tagged proteins to monitor different stages of meiosis: Zip1-GFP, which marks the synaptonemal complex in prophase I, Spc42-mCherry, a component of spindle pole bodies, and GFP-Tub1, to monitor the spindle morphology at the different stages of meiosis ( Carminati and Stearns, 1997 ; Donaldson and Kilmartin, 1996 ; Gihana et al., 2018 ; Scherthan et al., 2007 ; Tsuchiya et al., 2011 ; Tsuchiya and Lacefield, 2013 ). Although both Zip1 and Tub1 are tagged with GFP, we can easily distinguish between the markers due to differences in temporal regulation and morphology.…”