Septins are a family of conserved GTP-binding proteins that assemble into hetero-oligomeric complexes and polymerize into filaments and other supramolecular arrangements. Septin structures associate with the plasma membrane by interacting with a specific phosphoinositide, and are involved in cell compartmentation, in cell division, and other membrane remodeling events. In budding yeast, septins establish a diffusion barrier at the neck between a mother and daughter cell, promote membrane curvature, and act as a scaffold to recruit other proteins to the site of cytokinesis. In humans, septin structures serve very similar functions; they are localized at the cleavage furrow, at the root of the primary cilium, at the base of every dendritic spine, and within the annulus in spermatozoa. Marked alterations of septin gene expression are found in solid tumors of certain tissues and septin gene translocations in mixed lineage leukemias. SEPT9 mutations are one apparent cause of hereditary neuralgic amyotrophy. Substitution mutations preventing GTP binding to SEPT12 disrupt the sperm annulus and cause male infertiity. In-depth structural and functional analysis of septins is necessary to understand the molecular mechanisms governing septin organization and function and obtain new insights to illuminate their pathophysiology. This project aims to apply novel tools and the experimental advantages of yeast to interrogate septin-based structures and eludicate fundamental properties of their organization, regulation and function that should be applicable to the highly homologous septin-based structures in human cells.
Our specific aims i nclude experimental tests of the following hypotheses. (1) Post-translational modifications (PTMs) of septins and septin-associated proteins drive the dramatic changes in septin structural organization that occur during passage through the cell division cycle. Therefore, comprehensive analysis by mass spectrometry of PTMs (especially phosphorylation and SUMOylation) that septins undergo during cell cycle progression, and subsequent genetic analysis of the physiological importance of those modifications by site-directed mutagenesis in vivo and a FRET-based method in vitro, will be conducted. (2) Sequential recruitment of septin-binding proteins exert and order the spatio-temporal changes in septin organization and function that impose proper cell morphology. Hence, comprehensive analysis of the in vivo septin interactome using a tripartite split-GFP method will be carried out. In addition, a novel clonable tag for in vivo labeling and protein localization at the ultrastructural level, which will be applied to dynamic analysis of the septin interactome using correlated light (fluorescence) and electron microscopy (CLEM), will be developed; and, (3) Association with PtdIns4,5P2 is obligatory for septin recruitment to the plasma membrane. Thus, to gain unprecedented insight as to how septins associate with a PtdIns4,5P2-containing lipid surface and how specific septin-binding proteins influence that interaction, the newest instrumentation for cryo-EM will be used to visualize these structures at near-atomic resolution.

Public Health Relevance

Septins, first discovered in yeast, are proteins conserved in all eukaryotes (except higher plants), are encoded by a gene family, and are considered components of the intracellular cytoskeleton because they associate into hetero-oligomeric complexes that polymerize into filaments and other higher-order structures. Septin-based structures associate with the plasma membrane and are required for cell morphogenesis and compartmentation? from cell division to cell differentiation. Pronounced up-regulation of certain splice variants of human septin SEPT9 is a hallmark of breast, ovarian and other tumor types, and expression changes in septins appear to be causal in certain neuropathological conditions, indicating that further investigation of septin assembly and function could provide new insights about how septin dysfunction contributes to disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM101314-05A1
Application #
9445749
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Flicker, Paula F
Project Start
2013-01-01
Project End
2021-12-31
Budget Start
2018-01-01
Budget End
2018-12-31
Support Year
5
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of California Berkeley
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Booth, E A; Thorner, J (2016) A FRET-based method for monitoring septin polymerization and binding of septin-associated proteins. Methods Cell Biol 136:35-56
Finnigan, Gregory C; Thorner, Jeremy (2016) mCAL: A New Approach for Versatile Multiplex Action of Cas9 Using One sgRNA and Loci Flanked by a Programmed Target Sequence. G3 (Bethesda) 6:2147-56
Perez, Adam M; Finnigan, Gregory C; Roelants, Françoise M et al. (2016) Septin-Associated Protein Kinases in the Yeast Saccharomyces cerevisiae. Front Cell Dev Biol 4:119
Finnigan, Gregory C; Duvalyan, Angela; Liao, Elizabeth N et al. (2016) Detection of protein-protein interactions at the septin collar in Saccharomyces cerevisiae using a tripartite split-GFP system. Mol Biol Cell 27:2708-25
Booth, Elizabeth A; Sterling, Sarah M; Dovala, Dustin et al. (2016) Effects of Bni5 Binding on Septin Filament Organization. J Mol Biol 428:4962-4980
Bertin, A; Nogales, E (2016) Preparing recombinant yeast septins and their analysis by electron microscopy. Methods Cell Biol 136:21-34
Finnigan, Gregory C; Sterling, Sarah M; Duvalyan, Angela et al. (2016) Coordinate action of distinct sequence elements localizes checkpoint kinase Hsl1 to the septin collar at the bud neck in Saccharomyces cerevisiae. Mol Biol Cell 27:2213-33
Garcia 3rd, Galo; Finnigan, Gregory C; Heasley, Lydia R et al. (2016) Assembly, molecular organization, and membrane-binding properties of development-specific septins. J Cell Biol 212:515-29
Bertin, Aurélie; Nogales, Eva (2016) Characterization of Septin Ultrastructure in Budding Yeast Using Electron Tomography. Methods Mol Biol 1369:113-23
Finnigan, Gregory C; Booth, Elizabeth A; Duvalyan, Angela et al. (2015) The Carboxy-Terminal Tails of Septins Cdc11 and Shs1 Recruit Myosin-II Binding Factor Bni5 to the Bud Neck in Saccharomyces cerevisiae. Genetics 200:843-62

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