Black arrowheads, aberrant PSMs. that directed PSM extension fails in many septin-mutant cells, and, for those that do succeed, walls are abnormal, leading to increased susceptibility to heating, freezing, and digestion by theDrosophilagut. Septin mutants mislocalize the leading-edge protein (LEP) complex required for normal PSM and wall biogenesis, and ectopic expression of the LEP protein Ssp1 perturbs mitotic septin localization and function, suggesting a functional interaction. Strikingly, extra copies of septinCDC10rescue sporulation and LEP localization in cells lacking Sma1, a phospholipase Dassociated protein dispensable for initiation of PSM assembly and PSM curvature but required for PSM extension. These findings point to key septin functions in directing efficient membrane and cell wall synthesis during budding yeast gametogenesis. == INTRODUCTION == When deprived of nutrients, diploid yeast cells exit the mitotic cell cycle and undergo sporulation (Neiman, 2011). Unlike cytokinesis in vegetative (mitotically dividing) cells, in which a single plasma membrane is divided in two, sporulating cells in meiosis II initiate de novo synthesis of four new membranes, each of which encapsulates a haploid nucleus and essential cellular components. As the four haploid lobes of the nucleus separate, the process of spore morphogenesis begins at the spindle pole bodies (SPBs) implanted in the nuclear envelope (Lynn and Magee, 1970; Neiman, 1998; Knop and Strasser, 2000). Prospore membrane (PSM) development proceeds through a series of transitions, beginning as SPB-proximal disks and forming spheres upon closure (Neiman, 2011). During subsequent spore wall synthesis, mannan and -1, 3-glucan layers are deposited in the lumen formed by the double-bilayer PSM (Smitset al., 2001). The outermost two layers, rich in chitosan (Coluccioet al., 2004) and dityrosine (Brizaet al., 1990b), form after breakdown of the outer PSM leaflet and endow spores with resistance to environmental assaults (Neiman, 2011). Septins were Angptl2 first identified inSaccharomyces cerevisiaeand are found throughout nonplant eukaryotes (Panet al., 2007; Nishihamaet al., 2011). Five septin proteins expressed in vegetative cells (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) assemble into hetero-octamers that interact with the plasma membrane and anneal into a filamentous network (Bertinet al., 2008, 2010, 2012; Garciaet al., 2011; Onget al., 2014). Filamentous septin rings at the site of vegetative cytokinesis, the motherbud neck, act as scaffolds to recruit chitin synthases (Larsonet al., 2008) and components of the actomyosin ring (Rohet al., 2002; Ohet al., 2013; Fenget al., 2015; Finniganet al., 2015). Septin rings are also associated with a BMS-927711 diffusion barrier function that compartmentalizes regions of the cortex (Dobbelaere and Barral, 2004). By contrast, roles for septins in sporulation are poorly understood. Expression ofCDC3, CDC10, and the two sporulation-specific septinsSPR3andSPR28is highly induced during sporulation via the transcription factor Ndt80 (Kaback and Feldberg, 1985; Chu and Herskowitz, 1998; Pierceet al., 2003). The products of these genes colocalize with Cdc11 in BMS-927711 PSM-associated higher-order structures (Ozsaracet BMS-927711 al., 1995; De Virgilioet al., 1996; Fareset al., 1996; McMurray and Thorner, 2008; Pablo-Hernandoet al., 2008), whereas Cdc12 and Shs1 are largely evicted from existing complexes and appear to be replaced by Spr3 and Spr28, respectively (McMurray and Thorner, 2008). Septin assemblies probably interact with the inner PSM leaflet, because vegetatively synthesized Cdc10 molecules are inherited by mature spores and are recycled for use upon spore germination (Joseph-Strausset al., 2007; McMurray and Thorner, 2008); were they on the outer leaflet, they would be left behind in the ascal cytoplasm. Others previously found only mild sporulation defects in septin mutants, leading to the idea that septins are unimportant for this process (Neiman, 2011). Here we carefully analyze PSM dynamics and spore wall composition in mutant cells to identify specific functional requirements for septins during budding yeast sporulation. == RESULTS AND DISCUSSION == In previous studies, the magnitude of effects of septin mutations on sporulation varied with strain background (De Virgilioet al., 1996; Fareset al., 1996). Overall sporulation efficiency was calculated as the frequency of asci per total number of cells. We constructed homozygous deletions ofSPR3orSPR28in two nearly isogenic strain backgrounds with very different sporulation efficiencies. BY4743 sporulates with relatively low BMS-927711 efficiency.