Decision letter: The role of Cdc42 and Gic1 in the regulation of septin filament formation and dissociation

Septins belong to a family of polymerizing GTP-binding proteins that are important for cytokinesis and other processes that involve spatial organization of the cell cortex. We reconstituted a recombinant Drosophila septin complex and compared activities of the wild-type and several mutant septin complex variants both in vitro and in vivo. We show that Drosophila septin complex functions depend on the intact GTP-binding and/or hydrolysis domains of Pnut, Sep1, and Sep2. The presence of the functional C-terminal domain of septins is required for the integrity of the complex. Drosophila Orc6 protein, the smallest subunit of the origin recognition complex (ORC), directly binds to septin complex and facilitates septin filament formation. Orc6 forms dimers through the interactions of its N-terminal, TFIIB-like domains. This ability of the protein suggests a direct bridging role for Orc6 in stimulating septin polymerization in Drosophila. Studies reported here provide a functional dissection of a Drosophila septin complex and highlight the basic conserved and divergent features among metazoan septin complexes.

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The role of Cdc42 and Gic1 in the regulation of septin filament formation and dissociation

eLife ◽

10.7554/elife.01085 ◽

2013 ◽

Vol 2 ◽

Cited By ~ 43

Author(s):

Yashar Sadian ◽

Christos Gatsogiannis ◽

Csilla Patasi ◽

Oliver Hofnagel ◽

Roger S Goody ◽

...

Keyword(s):

Electron Tomography ◽

Scaffolding Protein ◽

Structural Basis ◽

Dual Function ◽

Filament Formation ◽

Guanine Nucleotide Binding ◽

Nucleotide Binding Proteins ◽

Guanine Nucleotide Binding Proteins ◽

Septin Filament

Septins are guanine nucleotide-binding proteins that polymerize into filamentous and higher-order structures. Cdc42 and its effector Gic1 are involved in septin recruitment, ring formation and dissociation. The regulatory mechanisms behind these processes are not well understood. Here, we have used electron microscopy and cryo electron tomography to elucidate the structural basis of the Gic1-septin and Gic1-Cdc42-septin interaction. We show that Gic1 acts as a scaffolding protein for septin filaments forming long and flexible filament cables. Cdc42 in its GTP-form binds to Gic1, which ultimately leads to the dissociation of Gic1 from the filament cables. Surprisingly, Cdc42-GDP is not inactive, but in the absence of Gic1 directly interacts with septin filaments resulting in their disassembly. We suggest that this unanticipated dual function of Cdc42 is crucial for the cell cycle. Based on our results we propose a novel regulatory mechanism for septin filament formation and dissociation.

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Author response: The role of Cdc42 and Gic1 in the regulation of septin filament formation and dissociation

10.7554/elife.01085.030 ◽

2013 ◽

Author(s):

Yashar Sadian ◽

Christos Gatsogiannis ◽

Csilla Patasi ◽

Oliver Hofnagel ◽

Roger S Goody ◽

...

Keyword(s):

Author Response ◽

Filament Formation ◽

Septin Filament

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The Role of Cdc42 and Gic1 in the Regulation of Septin Filament Formation and Dissociation

Biophysical Journal ◽

10.1016/j.bpj.2013.11.956 ◽

2014 ◽

Vol 106 (2) ◽

pp. 168a

Author(s):

Yashar Sadian ◽

Christos Gatsogiannis ◽

Csilla Patasi ◽

Oliver Hofnagel ◽

Roger S. Goody ◽

...

Keyword(s):

Filament Formation ◽

Septin Filament

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Faculty Opinions recommendation of Septin9 is involved in septin filament formation and cellular stability.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.13370097.14740252 ◽

2011 ◽

Author(s):

Elias Spiliotis

Keyword(s):

Filament Formation ◽

Septin Filament

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The epigenetic regulator LSH maintains fork protection and genomic stability via MacroH2A deposition and RAD51 filament formation

Nature Communications ◽

10.1038/s41467-021-23809-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Xiaoping Xu ◽

Kai Ni ◽

Yafeng He ◽

Jianke Ren ◽

Chongkui Sun ◽

...

Keyword(s):

Chromatin Remodeling ◽

Critical Role ◽

Genomic Stability ◽

Dna Degradation ◽

Replication Forks ◽

Filament Formation ◽

Epigenetic Regulator ◽

Centromeric Instability ◽

Stalled Replication Forks

AbstractThe Immunodeficiency Centromeric Instability Facial Anomalies (ICF) 4 syndrome is caused by mutations in LSH/HELLS, a chromatin remodeler promoting incorporation of histone variant macroH2A. Here, we demonstrate that LSH depletion results in degradation of nascent DNA at stalled replication forks and the generation of genomic instability. The protection of stalled forks is mediated by macroH2A, whose knockdown mimics LSH depletion and whose overexpression rescues nascent DNA degradation. LSH or macroH2A deficiency leads to an impairment of RAD51 loading, a factor that prevents MRE11 and EXO1 mediated nascent DNA degradation. The defect in RAD51 loading is linked to a disbalance of BRCA1 and 53BP1 accumulation at stalled forks. This is associated with perturbed histone modifications, including abnormal H4K20 methylation that is critical for BRCA1 enrichment and 53BP1 exclusion. Altogether, our results illuminate the mechanism underlying a human syndrome and reveal a critical role of LSH mediated chromatin remodeling in genomic stability.

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The role of VPS4 in ESCRT-III polymer remodeling

Biochemical Society Transactions ◽

10.1042/bst20180026 ◽

2019 ◽

Vol 47 (1) ◽

pp. 441-448 ◽

Cited By ~ 8

Author(s):

Christophe Caillat ◽

Sourav Maity ◽

Nolwenn Miguet ◽

Wouter H. Roos ◽

Winfried Weissenhorn

Keyword(s):

Active Role ◽

Mechanistic Models ◽

Membrane Remodeling ◽

Filament Formation ◽

Enveloped Viruses ◽

Endosomal Sorting ◽

Membrane Fission ◽

Dynamic Assembly ◽

Inside Out

Abstract The endosomal sorting complex required for transport-III (ESCRT-III) and VPS4 catalyze a variety of membrane-remodeling processes in eukaryotes and archaea. Common to these processes is the dynamic recruitment of ESCRT-III proteins from the cytosol to the inner face of a membrane neck structure, their activation and filament formation inside or at the membrane neck and the subsequent or concomitant recruitment of the AAA-type ATPase VPS4. The dynamic assembly of ESCRT-III filaments and VPS4 on cellular membranes induces constriction of membrane necks with large diameters such as the cytokinetic midbody and necks with small diameters such as those of intraluminal vesicles or enveloped viruses. The two processes seem to use different sets of ESCRT-III filaments. Constriction is then thought to set the stage for membrane fission. Here, we review recent progress in understanding the structural transitions of ESCRT-III proteins required for filament formation, the functional role of VPS4 in dynamic ESCRT-III assembly and its active role in filament constriction. The recent data will be discussed in the context of different mechanistic models for inside-out membrane fission.

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Multiple Roles of Flagellar Export Chaperones for Efficient and Robust Flagellar Filament Formation in Salmonella

Frontiers in Microbiology ◽

10.3389/fmicb.2021.756044 ◽

2021 ◽

Vol 12 ◽

Author(s):

Tohru Minamino ◽

Yusuke V. Morimoto ◽

Miki Kinoshita ◽

Keiichi Namba

Keyword(s):

Type Iii Secretion ◽

Culture Media ◽

Multiple Roles ◽

Filament Formation ◽

Dynamic Interactions ◽

Filament Assembly ◽

Suppressor Mutations ◽

Folded Structure ◽

Flagellar Filament

FlgN, FliS, and FliT are flagellar export chaperones specific for FlgK/FlgL, FliC, and FliD, respectively, which are essential component proteins for filament formation. These chaperones facilitate the docking of their cognate substrates to a transmembrane export gate protein, FlhA, to facilitate their subsequent unfolding and export by the flagellar type III secretion system (fT3SS). Dynamic interactions of the chaperones with FlhA are thought to determine the substrate export order. To clarify the role of flagellar chaperones in filament assembly, we constructed cells lacking FlgN, FliS, and/or FliT. Removal of either FlgN, FliS, or FliT resulted in leakage of a large amount of unassembled FliC monomers into the culture media, indicating that these chaperones contribute to robust and efficient filament formation. The ∆flgN ∆fliS ∆fliT (∆NST) cells produced short filaments similarly to the ∆fliS mutant. Suppressor mutations of the ∆NST cells, which lengthened the filament, were all found in FliC and destabilized the folded structure of FliC monomer. Deletion of FliS inhibited FliC export and filament elongation only after FliC synthesis was complete. We propose that FliS is not involved in the transport of FliC upon onset of filament formation, but FliS-assisted unfolding of FliC by the fT3SS becomes essential for its rapid and efficient export to form a long filament when FliC becomes fully expressed in the cytoplasm.

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Filament formation by metabolic enzymes is a specific adaptation to an advanced state of cellular starvation

10.1101/003277 ◽

2014 ◽

Cited By ~ 2

Author(s):

Ivana Petrovska ◽

Elisabeth Nüske ◽

Matthias C Munder ◽

Gayathrie Kulasegaran ◽

Liliana Malinovska ◽

...

Keyword(s):

Metabolic Enzymes ◽

Low Ph ◽

General Mechanism ◽

Filament Formation ◽

Ph Change ◽

Cooperative Process ◽

A Cell ◽

Enzymatic Inactivation ◽

Physical Organization

One of the key questions in biology is how the metabolism of a cell responds to changes in the environment. In budding yeast, starvation causes a drop in intracellular pH, but the functional role of this pH change is not well understood. Here, we show that the enzyme glutamine synthetase (Gln1) forms filaments at low pH and that filament formation leads to enzymatic inactivation. Filament formation by Gln1 is a highly cooperative process, strongly dependent on macromolecular crowding, and involves back-to-back stacking of cylindrical homo-decamers into filaments that associate laterally to form higher order fibrils. Other metabolic enzymes also assemble into filaments at low pH. Hence, we propose that filament formation is a general mechanism to inactivate and store key metabolic enzymes during a state of advanced cellular starvation. These findings have broad implications for understanding the interplay between nutritional stress, the metabolism and the physical organization of a cell.

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DSS1 and ssDNA regulate oligomerization of BRCA2

Nucleic Acids Research ◽

10.1093/nar/gkaa555 ◽

2020 ◽

Vol 48 (14) ◽

pp. 7818-7833 ◽

Cited By ~ 1

Author(s):

Hang Phuong Le ◽

Xiaoyan Ma ◽

Jorge Vaquero ◽

Megan Brinkmeyer ◽

Fei Guo ◽

...

Keyword(s):

Replication Fork ◽

Microscopic Analysis ◽

Filament Formation ◽

Electron Microscopic ◽

Microscopic Imaging ◽

Cellular Events ◽

Self Association ◽

Oligomeric Forms

Abstract The tumor suppressor BRCA2 plays a key role in initiating homologous recombination by facilitating RAD51 filament formation on single-stranded DNA. The small acidic protein DSS1 is a crucial partner to BRCA2 in this process. In vitro and in cells (1,2), BRCA2 associates into oligomeric complexes besides also existing as monomers. A dimeric structure was further characterized by electron microscopic analysis (3), but the functional significance of the different BRCA2 assemblies remains to be determined. Here, we used biochemistry and electron microscopic imaging to demonstrate that the multimerization of BRCA2 is counteracted by DSS1 and ssDNA. When validating the findings, we identified three self-interacting regions and two types of self-association, the N-to-C terminal and the N-to-N terminal interactions. The N-to-C terminal self-interaction of BRCA2 is sensitive to DSS1 and ssDNA. The N-to-N terminal self-interaction is modulated by ssDNA. Our results define a novel role of DSS1 to regulate BRCA2 in an RPA-independent fashion. Since DSS1 is required for BRCA2 function in recombination, we speculate that the monomeric and oligomeric forms of BRCA2 might be active for different cellular events in recombinational DNA repair and replication fork stabilization.

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