Abstract Morphological complexity and plasticity are hallmarks of polyextremotolerant fungi. Septins are conserved cytoskeletal proteins and key contributors to cell polarity and morphogenesis. They sense membrane curvature, coordinate cell division, and influence diffusion at the plasma membrane. Four septins homologs are conserved from yeasts to humans, the two systems in which septins have been studied most extensively. But there is also a fifth family of septin proteins that remain biochemically mysterious. Members of this family, known as Group 5 septins, appear in the genomes of filamentous fungi, and thus have been understudied due to their absence from ascomycete yeasts.Knufia petricolais an emerging model polyextremotolerant black fungus that can serve as a model system for understudied Group 5 septins. We have recombinantly expressed and biochemically characterizedKpAspE, a Group 5 septin fromK. petricola, demonstrating that this septin––by itselfin vitro–– recapitulates many of the functions of canonical septin hetero-octamers.KpAspE is an active GTPase that forms diverse homo-oligomers, senses membrane curvature, and interacts with the terminal subunit of canonical septin hetero-octamers. These findings raise the possibility that Group 5 septins govern the higher order structures formed by canonical septins, which inK. petricolacells form extended filaments. These findings provide insight into how septin hetero-oligomers evolved from ancient homomers and raise the possibility that Group 5 septins govern the higher order structures formed by canonical septins. Significance StatementSeptins are understudied cytoskeletal proteins. Here, we biochemically characterizedKpAspE, a novel Group 5 septin from a polyextremotolerant black fungus.KpAspE in isolation recapitulates many properties of canonical septin hetero-octamersin vitroand interacts with the Cdc11, the terminal subunit of those octamers.These findings provide insight into how ancient septins may have evolved and diversified from homopolymers and suggest that many of the septin functions were present in the ancestral protein.
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Animal septins contain functional transmembrane domains
Summary Septins, a conserved family of filament-forming proteins, contribute to eukaryotic cell division, polarity, and membrane trafficking. Septins scaffold other proteins to cellular membranes, but it is not fully understood how septins associate with membranes. We identified and characterized an isoform ofCaenorhabditis elegansseptin UNC-61 that was predicted to contain a transmembrane domain (TMD). The TMD isoform is expressed in a subset of tissues where the known septins were known to act, and TMD function was required for tissue integrity of the egg-laying apparatus. We found predicted TMD-containing septins across much of opisthokont phylogeny and demonstrated that the TMD-containing sequence of a primate TMD-septin is sufficient for localization to cellular membranes. Together, our findings reveal a novel mechanism of septin-membrane association with profound implications for septin dynamics and regulation.
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- Award ID(s):
- 2153790
- PAR ID:
- 10577407
- Publisher / Repository:
- bioRxiv
- Date Published:
- Format(s):
- Medium: X
- Institution:
- bioRxiv
- Sponsoring Org:
- National Science Foundation
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Abstract Septin proteins contribute to many eukaryotic processes involving cellular membranes. In the budding yeast Saccharomyces cerevisiae , septin hetero‐oligomers interact with the plasma membrane (PM) almost exclusively at the future site of cytokinesis. While multiple mechanisms of membrane recruitment have been identified, including direct interactions with specific phospholipids and curvature‐sensitive interactions via amphipathic helices, these do not fully explain why yeast septins do not localize all over the inner leaflet of the PM. While engineering an inducible split‐yellow fluorescent protein (YFP) system to measure the kinetics of yeast septin complex assembly, we found that ectopic co‐overexpression of two tagged septins, Cdc3 and Cdc10, resulted in nearly uniform PM localization, as well as perturbation of endogenous septin function. Septin localization and function in gametogenesis were also perturbed. PM localization required the C‐terminal YFP fragment fused to the C terminus of Cdc3, the septin‐associated kinases Cla4 and Gin4, and phosphotidylinositol‐4,5‐bis‐phosphate (PI[4,5]P 2 ), but not the putative PI(4,5)P 2 ‐binding residues in Cdc3. Endogenous Cdc10 was recruited to the PM, likely contributing to the functional interference. PM‐localized septins did not exchange with the cytosolic pool, indicative of stable polymers. These findings provide new clues as to what normally restricts septin localization to specific membranes.more » « less
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Septins are a family of membrane-associated cytoskeletal guanine-nucleotide binding proteins that play crucial roles in various cellular processes, such as cell division, phagocytosis, and organelle fission. Despite their importance, the evolutionary origins and ancestral function of septins remain unclear. In opisthokonts, septins form five distinct groups of orthologs, with subunits from multiple groups assembling into heteropolymers, thus supporting their diverse molecular functions. Recent studies have revealed that septins are also conserved in algae and protists, indicating an ancient origin from the last eukaryotic common ancestor. However, the phylogenetic relationships among septins across eukaryotes remained unclear. Here, we expanded the list of non-opisthokont septins, including previously unrecognized septins from glaucophyte algae. Constructing a rooted phylogenetic tree of 254 total septins, we observed a bifurcation between the major non-opisthokont and opisthokont septin clades. Within the non-opisthokont septins, we identified three major subclades: Group 6 representing chlorophyte green algae (6A mostly for species with single septins, 6B for species with multiple septins), Group 7 representing algae in chlorophytes, heterokonts, haptophytes, chrysophytes, and rhodophytes, and Group 8 representing ciliates. Glaucophyte and some ciliate septins formed orphan lineages in-between all other septins and the outgroup. Combining ancestral-sequence reconstruction and AlphaFold predictions, we tracked the structural evolution of septins across eukaryotes. In the GTPase domain, we identified a conserved GAP-like arginine finger within the G-interface of at least one septin in most algal and ciliate species. This residue is required for homodimerization of the singleChlamydomonasseptin, and its loss coincided with septin duplication events in various lineages. The loss of the arginine finger is often accompanied by the emergence of the α0 helix, a known NC-interface interaction motif, potentially signifying the diversification of septin-septin interaction mechanisms from homo-dimerization to hetero-oligomerization. Lastly, we found amphipathic helices in all septin groups, suggesting that membrane binding is an ancestral trait. Coiled-coil domains were also broadly distributed, while transmembrane domains were found in some septins in Group 6A and 7. In summary, this study advances our understanding of septin distribution and phylogenetic groupings, shedding light on their ancestral features, potential function, and early evolution.more » « less
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ABSTRACT Cells employ cytoskeletal polymers to move, divide, and pass information inside and outside of the cell. Previous work on eukaryotic cytoskeletal elements such as actin, microtubules, and intermediate filaments investigating the mechanisms of polymerization have been critical to understand how cells control the assembly of the cytoskeleton. Most biophysical analyses have considered cooperative versus isodesmic modes of polymerization; this framework is useful for specifying functions of regulatory proteins that control nucleation and understanding how cells regulate elongation in time and space. The septins are considered a fourth component of the eukaryotic cytoskeleton, but they are poorly understood in many ways despite their conserved roles in membrane dynamics, cytokinesis, and cell shape, and in their links to a myriad of human diseases. Because septin function is intimately linked to their assembled state, we set out to investigate the mechanisms by which septin polymers elongate under different conditions. We used simulations,in vitroreconstitution of purified septin complexes, and quantitative microscopy to directly interrogate septin polymerization behaviors in solution and on synthetic lipid bilayers of different geometries. We first used reactive Brownian dynamics simulations to determine if the presence of a membrane induces cooperativity to septin polymerization. We then used fluorescence correlation spectroscopy (FCS) to assess septins’ ability to form filaments in solution at different salt conditions. Finally, we investigated septin membrane adsorption and polymerization on planar and curved supported lipid bilayers. Septins clearly show signs of salt-dependent cooperative assembly in solution, but cooperativity is limited by binding a membrane. Thus, septin assembly is dramatically influenced by extrinsic conditions and substrate properties and can show properties of both isodesmic and cooperative polymers. This versatility in assembly modes may explain the extensive array of assembly types, functions, and subcellular locations in which septins act. SIGNIFICANCEThe septin cytoskeleton plays conserved and essential roles in cell division, membrane remodeling, and intracellular signaling with links to varied human diseases. Unlike actin and microtubules, whose polymerization dynamics have been extensively characterized, the molecular details of septin polymerization remain poorly understood. Here, we investigate the mode of septin polymerization through the lens of isodesmic and cooperative polymer assembly models in solution, on planar and curved supported membranes, and under different ionic conditions. Our findings show that the mechanisms of septin assembly are highly sensitive to ionic conditions, membrane geometry, and protein concentrations. Notably, assembly can show either cooperative or isodesmic properties depending on context, thereby revealing unexpected plasticity.more » « less
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Momany, Michelle (Ed.)Morphological complexity and plasticity are hallmarks of polyextremotolerant fungi. Septins are conserved cytoskeletal proteins and key contributors to cell polarity and morphogenesis. They sense membrane curvature, coordinate cell division, and influence diffusion at the plasma membrane. Four septin homologues are conserved from yeasts to humans, the systems in which septins have been most studied. But there is also a fifth family of opisthokont septins that remain biochemically mysterious. Members of this family, Group 5 septins, appear in the genomes of filamentous fungi, but are understudied due to their absence from ascomycete yeasts. Knufia petricola is an emerging model polyextremotolerant black fungus that can also serve as a model system for Group 5 septins. We have recombinantly expressed and biochemically characterized KpAspE, a Group 5 septin from K. petricola. This septin––by itself in vitro––recapitulates many functions of canonical septin hetero-octamers. KpAspE is an active GTPase that forms diverse homo-oligomers, binds shallow membrane curvatures, and interacts with the terminal subunit of canonical septin hetero-octamers. These findings raise the possibility that Group 5 septins govern the higher-order structures formed by canonical septins, which in K. petricola cells form extended filaments, and provide insight into how septin hetero-oligomers evolved from ancient homomers.more » « less
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