Crutchley
John M.
Crutchley
John M.
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ArticleInterplay of septin amphipathic helices in sensing membrane-curvature and filament bundling(American Society for Cell Biology, 2021-09-09) Woods, Benjamin L. ; Cannon, Kevin S. ; Vogt, Ellysa J. D. ; Crutchley, John M. ; Gladfelter, Amy S.The curvature of the membrane defines cell shape. Septins are GTP-binding proteins that assemble into heteromeric complexes and polymerize into filaments at areas of micron-scale membrane curvature. An amphipathic helix (AH) domain within the septin complex is necessary and sufficient for septins to preferentially assemble onto micron-scale curvature. Here we report that the nonessential fungal septin, Shs1, also has an AH domain capable of recognizing membrane curvature. In a septin mutant strain lacking a fully functional Cdc12 AH domain (cdc12-6), the C-terminal extension of Shs1, containing an AH domain, becomes essential. Additionally, we find that the Cdc12 AH domain is important for regulating septin filament bundling, suggesting septin AH domains have multiple, distinct functions and that bundling and membrane binding may be coordinately controlled.
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ArticleFXR1 splicing is important for muscle development and biomolecular condensates in muscle cells(Rockefeller University Press, 2020-03-13) Smith, Jean A. ; Curry, Ennessa G. ; Blue, R. Eric ; Roden, Christine ; Dundon, Samantha E.R. ; Rodríguez-Vargas, Anthony ; Jordan, Danielle C. ; Chen, Xiaomin ; Lyons, Shawn M. ; Crutchley, John M. ; Anderson, Paul ; Horb, Marko E. ; Gladfelter, Amy S. ; Giudice, JimenaFragile-X mental retardation autosomal homologue-1 (FXR1) is a muscle-enriched RNA-binding protein. FXR1 depletion is perinatally lethal in mice, Xenopus, and zebrafish; however, the mechanisms driving these phenotypes remain unclear. The FXR1 gene undergoes alternative splicing, producing multiple protein isoforms and mis-splicing has been implicated in disease. Furthermore, mutations that cause frameshifts in muscle-specific isoforms result in congenital multi-minicore myopathy. We observed that FXR1 alternative splicing is pronounced in the serine- and arginine-rich intrinsically disordered domain; these domains are known to promote biomolecular condensation. Here, we show that tissue-specific splicing of fxr1 is required for Xenopus development and alters the disordered domain of FXR1. FXR1 isoforms vary in the formation of RNA-dependent biomolecular condensates in cells and in vitro. This work shows that regulation of tissue-specific splicing can influence FXR1 condensates in muscle development and how mis-splicing promotes disease.
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PreprintmRNA structure determines specificity of a polyQ-driven phase separation( 2018-04) Langdon, Erin M. ; Qiu, Yupeng ; Ghanbari Niaki, Amirhossein ; McLaughlin, Grace A. ; Weidmann, Chase ; Gerbich, Therese M. ; Smith, Jean A. ; Crutchley, John M. ; Termini, Christina M. ; Weeks, Kevin M. ; Myong, Sua ; Gladfelter, Amy S.RNA promotes liquid-liquid phase separation (LLPS) to build membrane-less compartments in cells. How distinct molecular compositions are established and maintained in these liquid compartments is unknown. Here we report that secondary structure allows mRNAs to self-associate and determines if an mRNA is recruited to or excluded from liquid compartments. The polyQ-protein Whi3 induces conformational changes in RNA structure and generates distinct molecular fluctuations depending on the RNA sequence. These data support a model in which structure-based, RNA-RNA interactions promote assembly of distinct droplets and protein-driven, conformational dynamics of the RNA maintain this identity. Thus, the shape of RNA can promote the formation and coexistence of the diverse array of RNA-rich liquid compartments found in a single cell.
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ArticleSpatial heterogeneity of the cytosol revealed by machine learning-based 3D particle tracking(American Society for Cell Biology, 2020-06-29) McLaughlin, Grace A. ; Langdon, Erin M. ; Crutchley, John M. ; Holt, Liam J. ; Forest, M. Gregory ; Newby, Jay M. ; Gladfelter, Amy S.The spatial structure and physical properties of the cytosol are not well understood. Measurements of the material state of the cytosol are challenging due to its spatial and temporal heterogeneity. Recent development of genetically encoded multimeric nanoparticles (GEMs) has opened up study of the cytosol at the length scales of multiprotein complexes (20-60 nm). We developed an image analysis pipeline for 3D imaging of GEMs in the context of large, multinucleate fungi where there is evidence of functional compartmentalization of the cytosol for both the nuclear division cycle and branching. We applied a neural network to track particles in 3D and then created quantitative visualizations of spatially varying diffusivity. Using this pipeline to analyze spatial diffusivity patterns, we found that there is substantial variability in the properties of the cytosol. We detected zones where GEMs display especially low diffusivity at hyphal tips and near some nuclei, showing that the physical state of the cytosol varies spatially within a single cell. Additionally, we observed significant cell-to-cell variability in the average diffusivity of GEMs. Thus, the physical properties of the cytosol vary substantially in time and space and can be a source of heterogeneity within individual cells and across populations.
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ArticleAn amphipathic helix enables septins to sense micrometer-scale membrane curvature(Rockefeller University Press, 2019-01-18) Cannon, Kevin S. ; Woods, Benjamin L. ; Crutchley, John M. ; Gladfelter, Amy S.Cell shape is well described by membrane curvature. Septins are filament-forming, GTP-binding proteins that assemble on positive, micrometer-scale curvatures. Here, we examine the molecular basis of curvature sensing by septins. We show that differences in affinity and the number of binding sites drive curvature-specific adsorption of septins. Moreover, we find septin assembly onto curved membranes is cooperative and show that geometry influences higher-order arrangement of septin filaments. Although septins must form polymers to stay associated with membranes, septin filaments do not have to span micrometers in length to sense curvature, as we find that single-septin complexes have curvature-dependent association rates. We trace this ability to an amphipathic helix (AH) located on the C-terminus of Cdc12. The AH domain is necessary and sufficient for curvature sensing both in vitro and in vivo. These data show that curvature sensing by septins operates at much smaller length scales than the micrometer curvatures being detected.