Septins function in exocytosis via physical interactions with the exocyst complex in fission yeast cytokinesis
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Septins can function as scaffolds for protein recruitment, membrane-bound diffusion barriers, or membrane curvature sensors. Septins are important for cytokinesis, but their exact roles are still obscure. In fission yeast, four septins (Spn1 to Spn4) accumulate at the rim of the division plane as rings. The octameric exocyst complex, which tethers exocytic vesicles to the plasma membrane, exhibits a similar localization and is essential for plasma membrane deposition during cytokinesis. Without septins, the exocyst spreads across the division plane but absent from the rim during septum formation. These results suggest that septins and the exocyst physically interact for proper localization. Indeed, we predicted six pairs of direct interactions between septin and exocyst subunits by AlphaFold2 ColabFold, most of them are confirmed by co-immunoprecipitation and yeast two-hybrid assays. Exocyst mislocalization results in mistargeting of secretory vesicles and their cargos, which leads to cell-separation delay in septin mutants. Our results indicate that septins guide the targeting of exocyst complex on the plasma membrane for vesicle tethering during cytokinesis through direct physical interactions.Keywords:
Exocyst
Septin
Septins are a family of GTP-binding proteins implicated in mammalian cell division. Most studies examining the role of septins in this process have treated the family as a whole, thus neglecting the possibility that individual members may have diverse functions. To address this, we individually depleted each septin family member expressed in HeLa cells by siRNA and assayed for defects in cell division by immunofluorescence and time-lapse microscopy. Depletion of SEPT2, SEPT7, and SEPT11 causes defects in the early stages of cytokinesis, ultimately resulting in binucleation. In sharp contrast, SEPT9 is dispensable for the early stages of cell division, but is critical for the final separation of daughter cells. Rescue experiments indicate that SEPT9 isoforms containing the N-terminal region are sufficient to drive cytokinesis. We demonstrate that SEPT9 mediates the localization of the vesicle-tethering exocyst complex to the midbody, providing mechanistic insight into the role of SEPT9 during abscission.
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Abscission
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Abstract Septins are essential for cytokinesis in Saccharomyces cerevisiae , but their precise roles remain elusive. Currently, it is thought that before cytokinesis, the hourglass-shaped septin structure at the mother-bud neck acts as a scaffold for assembly of the actomyosin ring (AMR) and other cytokinesis factors. At the onset of cytokinesis, the septin hourglass splits to form a double ring that sandwiches the AMR and may function as diffusion barriers to restrict diffusible cytokinesis factors to the division site. Here, we show that in cells lacking the septin Cdc10 or the septin-associated protein Bud4, the septins form a ring-like structure at the mother-bud neck that fails to re-arrange into a double ring early in cytokinesis. Strikingly, AMR assembly and constriction, the localization of membrane-trafficking and extracellular-matrix-remodeling factors, cytokinesis, and cell-wall-septum formation all occur efficiently in cdc10Δ and bud4Δ mutants. Thus, diffusion barriers formed by the septin double ring do not appear to be critical for S. cerevisiae cytokinesis. However, an AMR mutation and a septin mutation have synergistic effects on cytokinesis and the localization of cytokinesis proteins, suggesting that tethering to the AMR and a septin diffusion barrier may function redundantly to localize proteins to the division site.
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In Saccharomyces cerevisiae, five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1/Sep7) form the septin ring at the bud neck during vegetative growth. We show here that disruption of SHS1 caused cold-sensitive growth in the W303 background, with cells arrested in chains, indicative of a cytokinesis defect. Surprisingly, the other four septins appeared to form an apparently normal septin ring in shs1Delta cells grown under the restrictive condition. We found that Myo1 and Iqg1, two components of the actomyosin contractile ring, and Cyk3, a component of the septum formation, were either delocalized or mislocalized in shs1Delta cells, suggesting that Shs1 plays supportive roles in cytokinesis. We also found that deletion of SHS1 enhanced or suppressed the septin defect in cdc10Delta and cdc11Delta cells, respectively, suggesting that Shs1 is involved in septin organization, exerting different effects on septin-ring assembly, depending on the composition of the septin subunits. Furthermore, we constructed an shs1-100c allele that lacks the coding sequence for the C-terminal 32 amino acids. This allele still displayed the genetic interactions with the septin mutants, but did not show cytokinesis defects as described above, suggesting that the roles of Shs1 in septin organization and cytokinesis are separable.
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In the budding yeast Saccharomyces cerevisiae, the Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Sep7p/Shs1p septins assemble early in the cell cycle in a ring that marks the future cytokinetic site.The septins appear to be major structural components of a set of filaments at the mother-bud neck and function as a scaffold for recruiting proteins involved in cytokinesis and other processes.We isolated a novel gene, BNI5, as a dosage suppressor of the cdc12-6 growth defect.Overexpression of BNI5 also suppressed the growth defects of cdc10-1, cdc11-6, and sep7⌬ strains.Loss of BNI5 resulted in a cytokinesis defect, as evidenced by the formation of connected cells with shared cytoplasms, and deletion of BNI5 in a cdc3-6, cdc10-1, cdc11-6, cdc12-6, or sep7⌬ mutant strain resulted in enhanced defects in septin localization and cytokinesis.Bni5p localizes to the mother-bud neck in a septin-dependent manner shortly after bud emergence and disappears from the neck approximately 2 to 3 min before spindle disassembly.Two-hybrid, in vitro binding, and protein-localization studies suggest that Bni5p interacts with the N-terminal domain of Cdc11p, which also appears to be sufficient for the localization of Cdc11p, its interaction with other septins, and other critical aspects of its function.Our data suggest that the Bni5p-septin interaction is important for septin ring stability and function, which is in turn critical for normal cytokinesis.
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Fungal protein
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Cytokinesis mediates the final separation of a mother cell into two daughter cells. Septins are recruited to the cleavage furrow at an early stage. During cytokinetic progression the septin cytoskeleton is constantly rearranged, ultimately leading to a concentration of septins within the intercellular bridge (ICB), and to the formation of two rings adjacent to the midbody that aid ESCRT-dependent abscission. The molecular mechanisms underlying this behavior are poorly understood. Based on observations that septins can associate with actin, microtubules and associated motors, we review here established roles of septins in mammalian cytokinesis, and discuss, how septins may support cytokinetic progression by exerting their functions at particular sites. Finally, we discuss how this might be assisted by phosphoinositide-metabolizing enzymes.
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Cleavage furrow
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Septins are conserved filament-forming proteins that act in diverse cellular processes. They closely associate with membranes and, in some systems, components of the cytoskeleton. It is not well understood how filaments assemble into higher-order structures in vivo or how they are remodeled throughout the cell cycle. In the budding yeast S. cerevisiae, septins are found through most of the cell cycle in an hourglass organization at the mother-bud neck until cytokinesis when the collar splits into two rings that disassemble prior to the next cell cycle. Experiments using polarized fluorescence microscopy have suggested that septins are arranged in ordered, paired filaments in the hourglass and undergo a coordinated 90° reorientation during splitting at cytokinesis. This apparent reorganization could be due to two orthogonal populations of filaments disassembling and reassembling or being preferentially retained at cytokinesis. In support of this idea, we report a decrease in septin concentration at the mother-bud neck during cytokinesis consistent with other reports and the timing of the decrease depends on known septin regulators including the Gin4 kinase. We took a candidate-based approach to examine what factors control reorientation during splitting and used polarized fluorescence microscopy to screen mutant yeast strains deficient in septin interacting proteins. Using this method, we have linked known septin regulators to different aspects of the assembly, stability, and reorganization of septin assemblies. The data support that ring splitting requires Gin4 activity and an anillin-like protein Bud4, and normal accumulation of septins at the ring requires phosphorylation of Shs1. We find distinct regulatory requirements for septin organization in the hourglass compared to split rings. We propose that septin subpopulations can vary in their localization and assembly/disassembly behavior in a cell-cycle dependent manner at cytokinesis.
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Cytokinesis terminates mitosis, resulting in separation of the two sister cells. Septins, a conserved family of GTP-binding cytoskeletal proteins, are an absolute requirement for cytokinesis in budding yeast. We demonstrate that septin-dependence of mammalian cytokinesis differs greatly between cell types: genetic loss of the pivotal septin subunit SEPT7 in vivo reveals that septins are indispensable for cytokinesis in fibroblasts, but expendable in cells of the hematopoietic system. SEPT7-deficient mouse embryos fail to gastrulate, and septin-deficient fibroblasts exhibit pleiotropic defects in the major cytokinetic machinery, including hyperacetylation/stabilization of microtubules and stalled midbody abscission, leading to constitutive multinucleation. We identified the microtubule depolymerizing protein stathmin as a key molecule aiding in septin-independent cytokinesis, demonstrated that stathmin supplementation is sufficient to override cytokinesis failure in SEPT7-null fibroblasts, and that knockdown of stathmin makes proliferation of a hematopoietic cell line sensitive to the septin inhibitor forchlorfenuron. Identification of septin-independent cytokinesis in the hematopoietic system could serve as a key to identify solid tumor-specific molecular targets for inhibition of cell proliferation.
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Cleavage furrow
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Septins are a family of conserved GTP-binding proteins that function in cytokinesis in fungi and animals. In budding yeast, septins form scaffolds for assembly of the actomyosin contractile ring at the cleavage plane, a role that does not appear to be conserved in other organisms. The septins form an hourglass-shaped collar at the mother-bud neck, which splits into two rings flanking the division plane at cytokinesis. A recent study(1) demonstrates that these two septin rings constitute diffusion barriers that create a cytokinetic compartment to retain cortical cytokinetic factors in proximity to the cleavage plane.
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Cleavage furrow
Cleavage (geology)
Budding yeast
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ABSTRACT A Saccharomyces cerevisiae mutant unable to grow in a cdc28-1N background was isolated and shown to be affected in the ELM1 gene. Elm1 is a protein kinase, thought to be a negative regulator of pseudo-hyphal growth. We show that Cdc11, one of the septins, is delocalised in the mutant, indicating that septin localisation is partly controlled by Elm1. Moreover, we show that cytokinesis is delayed in an elm1Δ mutant. Elm1 levels peak at the end of the cell cycle and Elm1 is localised at the bud neck in a septin-dependent fashion from bud emergence until the completion of anaphase, at about the time of cell division. Genetic and biochemical evidence suggest that Elm1 and the three other septin-localised protein kinases, Hsl1, Gin4 and Kcc4, work in parallel pathways to regulate septin behaviour and cytokinesis. In addition, the elm1Δ morphological defects can be suppressed by deletion of the SWE1 gene, but not the cytokinesis defect nor the septin mislocalisation. Our results indicate that cytokinesis in budding yeast is regulated by Elm1.
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Mitotic exit
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