Septin ring size scaling and dynamics require the coiled-coil region of Shs1p

Meseroll, Rebecca A.; Howard, Louisa; Gladfelter, Amy S.

doi:10.1091/mbc.e12-03-0207

Cited by 28 publications

(50 citation statements)

References 51 publications

(56 reference statements)

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“…For this reason, we revisited the ability of AgShs1 to substitute for ScShs1 using our well-defined Shs1-dependent genetic backgrounds. We found, first, and contrary to the other study (Meseroll et al 2012), that AgShs1 expressed from the chromosomal SHS1 locus is not functional in S. cerevisiae because, unlike authentic ScShs1 expressed in the identical manner, it did not support the growth of either cdc10D cells at 25°on Gal medium ( Figure S6A, top) or the growth of Cdc11(DCTE)-mC cells at 30°on glucose medium ( Figure S6A, middle). Yet, AgShs1 was stably expressed and was clearly able to associate with Cdc12 as judged by two crteria, its ability to kill cdc11D cells ( Figure S6A, bottom) and its efficient incorporation at the bud neck as seen by fluorescence microscopy ( Figure S6B).…”

Section: The Ctes Of Cdc11 and Shs1 Can Function In Transcontrasting

confidence: 93%

“…Titration of Shs1-capped hetero-octamers with increasing amounts of Cdc11-capped hetero-octamers reduces the thickness and increases the diameter of the rings formed in vitro . Consistent with a role for Shs1-capped rods in modulating the plasticity of septin-based structures, the Shs1 ortholog in Candida albicans contributes to septin ring dynamics (Gonzalez-Novo et al 2008) and the Shs1 ortholog in Ashbya gossypii has been implicated in scaling the size of the septin ring in vivo (Meseroll et al 2012). Moreover, in keeping with a primarily regulatory role, Shs1 undergoes extensive posttranslational modification during passage through the cell cycle, including SUMOylation (Johnson and Blobel 1999) and phosphorylation (Mortensen et al 2002;Dobbelaere et al 2003;Smolka et al 2006;Egelhofer et al 2008).…”

mentioning

confidence: 84%

“…Thus, cycles of phosphorylation and dephosphorylation of the CTE can have no significant role in regulating the function of this septin. Similarly, in the filamentous fungus A. gossypii, its Shs1 ortholog is also multiply phosphorylated within its CTE near its predicted coiled coil domain, and two protein kinases (the orthologs of Elm1 and Gin4) have been implicated in septin ring assembly in this organism; yet, unlike loss of the kinases, a shs1-9A allele did not display any marked defect in septin ring assembly or dynamics (Meseroll et al 2012).…”

Section: Phosphorylation Of Shs1 Within Its Cte Is Not Required For Fmentioning

confidence: 97%

“…Finally, it has been reported that, when expressed from a plasmid as a GFP-tagged protein, the Shs1 ortholog of A. gossypii is efficiently incorporated into the septin collar at the bud neck in both WT and shs1D S. cerevisiae cells, and that expression of WT AgShs1 in either WT or shs1D S. cerevisiae is inocuous, whereas AgShs1(DCTE)-GFP, and much less so AgShs1(DCC)-GFP, caused shs1D cells (but not WT cells) to assume a markedly aberrant cell morphology (Meseroll et al 2012). These findings are at odds with our demonstration here that the CC element in the Shs1 CTE is the segment that mediates its physiological function.…”

Section: The Ctes Of Cdc11 and Shs1 Can Function In Transmentioning

confidence: 99%

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Comprehensive Genetic Analysis of Paralogous Terminal Septin Subunits Shs1 and Cdc11 inSaccharomyces cerevisiae

et al. 2015

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Septins are a family of GTP-binding proteins considered to be cytoskeletal elements because they self-assemble into filaments and other higher-order structures in vivo. In budding yeast, septins establish a diffusion barrier at the bud neck between a mother and daughter cell, promote membrane curvature there, and serve as a scaffold to recruit other proteins to the site of cytokinesis. However, the mechanism by which any septin engages a partner protein has been unclear. The two most related and recently evolved subunits appear to be Cdc11 and Shs1, and the basic building blocks for assembling septin structures are hetero-octameric rods (Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11 and Shs1-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Shs1). Loss of Cdc11 is not normally tolerated, whereas cells lacking Shs1 do not appear grossly abnormal. We established several different sensitized genetic backgrounds wherein Shs1 is indispensable, which allowed us to carry out the first comprehensive and detailed genetic analysis of Shs1 in vivo. Our analysis revealed several novel insights, including: (i) the sole portion of Shs1 essential for its function is a predicted coiled-coil-forming segment in its C-terminal extension (CTE); (ii) the CTE of Cdc11 shares this function; (iii) this role for the CTEs of Cdc11 and Shs1 is quite distinct from that of the CTEs of Cdc3 and Cdc12; and (iv) heterotypic Cdc11 and Shs1 junctions likely occur in vivo. KEYWORDS yeast; cytoskeleton; complexes; filaments; mutants S EPTINS are GTP-binding proteins conserved across Eukarya (except higher plants) (Pan et al. 2007;Nishihama et al. 2011). This protein family was first identified because the corresponding loci were among the temperature-sensitive (ts) cell division cycle (cdc) mutations isolated in Saccharomyces cerevisiae (Hartwell 1978). At the restrictive temperature, cdc3, cdc10, cdc11, and cdc12 mutants continued to bud, replicate, and segregate their chromosomes, yet failed to execute cell division, resulting in the formation of chains of multinucleate cells (Hartwell 1971). Shifting a ts allele of any of these four homologous gene products to restrictive temperature prevented formation of what appeared to be rings of highly ordered membrane-associated filaments at the bud neck (Byers and Goetsch 1976). Antibody decoration (Haarer and Pringle 1987;Ford and Pringle 1991;Kim et al. 1991) and, later, use of fusions to green fluorescent protein (GFP) (Cid et al. 1998) demonstrated that these four proteins colocalized with and were likely constituents of these filaments. Because loss of the function of these proteins prevents cytokinesis and cell septation, and they seemed to be integral components of the filamentous structures erected at the bud neck, they were dubbed septins (Sanders and Field 1994;Pringle 2008). Indeed, subsequent purification of these proteins from yeast (Frazier et al. 1998), and their production as recombinant proteins in bacteria Versele and Thorner 2004;Farkasovsky et al. 2005), demonstrated that Cdc3, Cdc10, Cdc11...

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Section: The Ctes Of Cdc11 and Shs1 Can Function In Transcontrasting

confidence: 93%

mentioning

confidence: 84%

Section: Phosphorylation Of Shs1 Within Its Cte Is Not Required For Fmentioning

confidence: 97%

Section: The Ctes Of Cdc11 and Shs1 Can Function In Transmentioning

confidence: 99%

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Comprehensive Genetic Analysis of Paralogous Terminal Septin Subunits Shs1 and Cdc11 inSaccharomyces cerevisiae

et al. 2015

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“…First, as measured by FCS, septins oligomerize into small complexes that are not filaments in the cytoplasm. These complexes are likely to be rods that are heterooctamers or decamers in fungi based on previous work with purified and recombinant proteins in high-salt conditions (17,18,31,32). Subsequently, when rods contact the plasma membrane, they are concentrated, diffuse, and merge to grow into short filaments through annealing.…”

Section: Discussionmentioning

confidence: 99%

Septin assemblies form by diffusion-driven annealing on membranes

Bridges

Zhang

Mehta

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Septins assemble into filaments and higher-order structures that act as scaffolds for diverse cell functions including cytokinesis, cell polarity, and membrane remodeling. Despite their conserved role in cell organization, little is known about how septin filaments elongate and are knitted together into higher-order assemblies. Using fluorescence correlation spectroscopy, we determined that cytosolic septins are in small complexes, suggesting that septin filaments are not formed in the cytosol. When the plasma membrane of live cells is monitored by total internal reflection fluorescence microscopy, we see that septin complexes of variable size diffuse in two dimensions. Diffusing septin complexes collide and make end-on associations to form elongated filaments and higher-order structures, an assembly process we call annealing. Septin assembly by annealing can be reconstituted in vitro on supported lipid bilayers with purified septin complexes. Using the reconstitution assay, we show that septin filaments are highly flexible, grow only from free filament ends, and do not exchange subunits in the middle of filaments. This work shows that annealing is a previously unidentified intrinsic property of septins in the presence of membranes and demonstrates that cells exploit this mechanism to build large septin assemblies.cytoskeleton | biophysics S eptin filaments form rings, bars, and gauzes that serve as a scaffold at cell division sites; act to retract blebbed regions of membrane; and restrict diffusion between cell compartments (1-4). Septin function is required for cell division and viability in many eukaryotes whereas misregulation is associated with cancers and neurodegenerative disorders (5-8). Furthermore, septins mediate entry of both bacterial and fungal pathogens into host cells (9-11). In vivo, septin assembly is restricted both in time and in space through local activation of small GTPases such as Cdc42. Localized signaling leads to higher-order septin structures forming closely apposed to the plasma membrane at the plane of division, sites of polarity, and curved membranes (10,(12)(13)(14). Notably, eukaryotic cells of different geometries build higher-order septin assemblies of various shapes, sizes, and functions (4, 15, 16). Although septins are critical for spatial organization of cell plasma membranes, their assembly and disassembly dynamics are not understood (15).Electron microscopy (EM) studies of recombinant and immunoprecipitated Saccharomyces cerevisiae septins have shown that septins form nonpolar hetero-octameric rod-shaped complexes in high-salt buffers (>300 mM) and elongated filaments when dialyzed into low-salt buffers (<100 mM) (17, 18). Structural analyses of worm and mammalian septins have revealed that the heteromeric, rod-shaped complex is conserved (19-21). Thus, septin rods characterized to date contain two copies of each septin subunit assembled into a nonpolar, heteromeric complex (Fig. S1). Association of purified septin proteins with phosphoinositide-containing membrane monola...

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