Development of a Fluorescent Assay of Septin Filament Assembly

Assembly at the mother–bud neck of a filamentous collar containing five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) is necessary for proper morphogenesis and cytokinesis. We show that Cdc10 and Cdc12 possess GTPase activity and appropriate mutations in conserved nucleotide-binding residues abrogate GTP binding and/or hydrolysis in vitro. In vivo, mutants unable to bind GTP prevent septin collar formation, whereas mutants that block GTP hydrolysis do not. GTP binding-defective Cdc10 and Cdc12 form soluble heteromeric complexes with other septins both in yeast and in bacteria; yet, unlike wild-type, mutant complexes do not bind GTP and do not assemble into filaments in vitro. Absence of a p21-activated protein kinase (Cla4) perturbs septin collar formation. This defect is greatly exacerbated when combined with GTP binding-defective septins; conversely, the septin collar assembly defect of such mutants is suppressed efficiently by CLA4 overexpression. Cla4 interacts directly with and phosphorylates certain septins in vitro and in vivo. Thus, septin collar formation may correspond to septin filament assembly, and requires both GTP binding and Cla4-mediated phosphorylation of septins.

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Biophysical properties governing septin assembly

10.1101/2021.03.22.436414 ◽

2021 ◽

Author(s):

Benjamin L Woods ◽

Ian L Seim ◽

Jessica Liu ◽

Grace McLaughlin ◽

Kevin S. Cannon ◽

...

Keyword(s):

Physical Model ◽

Yeast Extract ◽

Cell Membranes ◽

Regulatory Proteins ◽

Biophysical Properties ◽

Regulatory Mutants ◽

Filament Assembly ◽

Pure Protein ◽

Crowded Environments ◽

Septin Filament

Septin filaments build structures such as rings, lattices and gauzes that serve as platforms for localizing signaling and organizing cell membranes. How cells control the geometry of septin assemblies in poorly understood. We show here that septins are isodesmic polymers, in contrast to cooperative polymerization exhibited by F-actin and microtubules. We constructed a physical model to analyze and interpret how septin assemblies change in the presence of regulators in yeast extracts. Notably filaments differ in length and curvature in yeast extract compared to pure protein indicating cellular regulators modulate intrinsic biophysical features. Combining analysis of extracts from regulatory mutants with simulations, we found increased filament flexibility and reduced filament fragmentation promote assembly of septin rings, whereas reduced flexibility in crowded environments promotes local filament alignment. This work demonstrates how tuning of intrinsic features of septin filament assembly by regulatory proteins yields a diverse array of structures observed in cells.

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Molecular recognition at septin interfaces: the switches hold the key

10.1101/2020.07.08.161463 ◽

2020 ◽

Author(s):

Higor Vinícius Dias Rosa ◽

Diego Antonio Leonardo ◽

Gabriel Brognara ◽

José Brandão-Neto ◽

Humberto D’Muniz Pereira ◽

...

Keyword(s):

High Resolution ◽

Molecular Recognition ◽

Crystal Structures ◽

Molecular Level ◽

Small Gtpases ◽

Structural Basis ◽

List Type ◽

Filament Assembly ◽

New Interactions ◽

Septin Filament

ABSTRACTThe assembly of a septin filament requires that homologous monomers must distinguish between one another in establishing appropriate interfaces with their neighbours. To understand this phenomenon at the molecular level, we present the first four crystal structures of heterodimeric septin complexes. We describe in detail the two distinct types of G-interface present within the octameric particles which must polymerize to form filaments. These are formed between SEPT2 and SEPT6 and between SEPT7 and SEPT3, and their description permits an understanding of the structural basis for the selectivity necessary for correct filament assembly. By replacing SEPT6 by SEPT8 or SEPT11, it is possible to rationalize Kinoshita’s postulate which predicts the exchangeability of septins from within a subgroup. Switches I and II, which in classical small GTPases provide a mechanism for nucleotide-dependent conformational change, have been repurposed in septins to play a fundamental role in molecular recognition. Specifically, it is switch I which holds the key to discriminating between the two different G-interfaces. Moreover, residues which are characteristic for a given subgroup play subtle, but pivotal, roles in guaranteeing that the correct interfaces are formed.HIGHLIGHTSHigh resolution structures of septin heterodimeric complexes reveal new interactionsSwitches of small GTPases are repurposed in septins to play key roles in interface contactsThe GTP present in catalytically inactive septins participates in molecular recognitionConservation of interface residues allows for subunit exchangeability from within septin subgroupsSpecific residues for each septin subgroup provide selectivity for proper filament assemblyGRAPHICAL ABSTRACT

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Phosphatidylinositol-4,5-bisphosphate Promotes Budding Yeast Septin Filament Assembly and Organization

Journal of Molecular Biology ◽

10.1016/j.jmb.2010.10.002 ◽

2010 ◽

Vol 404 (4) ◽

pp. 711-731 ◽

Cited By ~ 128

Author(s):

Aurélie Bertin ◽

Michael A. McMurray ◽

Luong Thai ◽

Galo Garcia ◽

Violet Votin ◽

...

Keyword(s):

Budding Yeast ◽

Filament Assembly ◽

Septin Filament

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Kinetic partitioning during de novo septin filament assembly creates a critical G1 “window of opportunity” for mutant septin function

Cell Cycle ◽

10.1080/15384101.2016.1196304 ◽

2016 ◽

Vol 15 (18) ◽

pp. 2441-2453 ◽

Cited By ~ 5

Author(s):

Rachel M. Schaefer ◽

Lydia R. Heasley ◽

David J. Odde ◽

Michael A. McMurray

Keyword(s):

De Novo ◽

Window Of Opportunity ◽

Filament Assembly ◽

Septin Filament

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The structure and properties of septin 3: a possible missing link in septin filament formation

Biochemical Journal ◽

10.1042/bj20120851 ◽

2013 ◽

Vol 450 (1) ◽

pp. 95-105 ◽

Cited By ~ 28

Author(s):

Joci N. A. Macedo ◽

Napoleão F. Valadares ◽

Ivo A. Marques ◽

Frederico M. Ferreira ◽

Julio C. P. Damalio ◽

...

Keyword(s):

Crystal Structure ◽

Sequence Similarity ◽

Three Dimensional ◽

Building Blocks ◽

Structural Basis ◽

Filament Assembly ◽

Gtp Binding ◽

Hydrolysis Of ◽

Free Monomer ◽

Septin Filament

The human genome codes for 13 members of a family of filament-forming GTP-binding proteins known as septins. These have been divided into four different subgroups on the basis of sequence similarity. The differences between the subgroups are believed to control their correct assembly into heterofilaments which have specific roles in membrane remodelling events. Many different combinations of the 13 proteins are theoretically possible and it is therefore important to understand the structural basis of specific filament assembly. However, three-dimensional structures are currently available for only three of the four subgroups. In the present study we describe the crystal structure of a construct of human SEPT3 which belongs to the outstanding subgroup. This construct (SEPT3-GC), which includes the GTP-binding and C-terminal domains, purifies as a nucleotide-free monomer, allowing for its characterization in terms of GTP-binding and hydrolysis. In the crystal structure, SEPT3-GC forms foreshortened filaments which employ the same NC and G interfaces observed in the heterotrimeric complex of human septins 2, 6 and 7, reinforcing the notion of ‘promiscuous’ interactions described previously. In the present study we describe these two interfaces and relate the structure to its tendency to form monomers and its efficiency in the hydrolysis of GTP. The relevance of these results is emphasized by the fact that septins from the SEPT3 subgroup may be important determinants of polymerization by occupying the terminal position in octameric units which themselves form the building blocks of at least some heterofilaments.

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Septin 9 has Two Polybasic Domains Critical to Septin Filament Assembly and Golgi Integrity

iScience ◽

10.1016/j.isci.2019.02.015 ◽

2019 ◽

Vol 13 ◽

pp. 138-153 ◽

Cited By ~ 12

Author(s):

Mohyeddine Omrane ◽

Amanda Souza Camara ◽

Cyntia Taveneau ◽

Nassima Benzoubir ◽

Thibault Tubiana ◽

...

Keyword(s):

Septin 9 ◽

Filament Assembly ◽

Polybasic Domains ◽

Septin Filament

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The shared role of the Rsr1 GTPase and Gic1/Gic2 in Cdc42 polarization

Molecular Biology of the Cell ◽

10.1091/mbc.e18-02-0145 ◽

2018 ◽

Vol 29 (20) ◽

pp. 2359-2369 ◽

Cited By ~ 7

Author(s):

Pil Jung Kang ◽

Kristi E. Miller ◽

Julia Guegueniat ◽

Laure Beven ◽

Hay-Oak Park

Keyword(s):

Cell Polarization ◽

G1 Phase ◽

Spatial Cues ◽

Filament Assembly ◽

Bud Emergence ◽

Bud Site ◽

Stepwise Assembly ◽

Redundant Role ◽

Septin Filament

The Cdc42 GTPase plays a central role in polarity development in many species. In budding yeast, Cdc42 is essential for polarized growth at the proper site and also for spontaneous cell polarization in the absence of spatial cues. Cdc42 polarization is critical for multiple events in the G1 phase prior to bud emergence, including bud-site assembly, polarization of the actin cytoskeleton, and septin filament assembly to form a ring at the new bud site. Yet the mechanism by which Cdc42 polarizes is not fully understood. Here we report that biphasic Cdc42 polarization in the G1 phase is coupled to stepwise assembly of the septin ring for bud emergence. We show that the Rsr1 GTPase shares a partially redundant role with Gic1 and Gic2, two related Cdc42 effectors, in the first phase of Cdc42 polarization in haploid cells. We propose that the first phase of Cdc42 polarization is mediated by positive feedback loops that function in parallel—one involving Rsr1 via local activation of Cdc42 in response to spatial cues and another involving Gic1 or Gic2 via reduction of diffusion of active Cdc42.

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