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Micron-Scale Plasma Membrane Curvature Is Recognized by the Septin Cytoskeleton

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Micron-Scale Plasma Membrane Curvature Is Recognized by the Septin Cytoskeleton Dartmouth College Dartmouth Digital Commons Dartmouth Scholarship Faculty Work 4-4-2016 Micron-Scale Plasma Membrane Curvature is Recognized by the Septin Cytoskeleton Andrew A. Bridges Dartmouth College Maximilian S. Jentzsch Dartmouth College Patrick W. Oakes University of Chicago Patricia Occhipinti Dartmouth College Amy S. Gladfelter Dartmouth College Follow this and additional works at: https://digitalcommons.dartmouth.edu/facoa Part of the Biology Commons Dartmouth Digital Commons Citation Bridges, Andrew A.; Jentzsch, Maximilian S.; Oakes, Patrick W.; Occhipinti, Patricia; and Gladfelter, Amy S., "Micron-Scale Plasma Membrane Curvature is Recognized by the Septin Cytoskeleton" (2016). Dartmouth Scholarship. 3661. https://digitalcommons.dartmouth.edu/facoa/3661 This Article is brought to you for free and open access by the Faculty Work at Dartmouth Digital Commons. It has been accepted for inclusion in Dartmouth Scholarship by an authorized administrator of Dartmouth Digital Commons. For more information, please contact [email protected]. JCB: Report Micron-scale plasma membrane curvature is recognized by the septin cytoskeleton Andrew A. Bridges,1,2 Maximilian S. Jentzsch,1 Patrick W. Oakes,3 Patricia Occhipinti,1 and Amy S. Gladfelter1,2 1Department of Biological Sciences, Dartmouth College, Hanover, NH 03755 2The Bell Center, Marine Biological Laboratory, Woods Hole, MA 02543 3Department of Physics, Institute for Biophysical Dynamics and James Franck Institute, University of Chicago, Chicago, IL 60637 Cells change shape in response to diverse environmental and developmental conditions, creating topologies with micron-scale features. Although individual proteins can sense nanometer-scale membrane curvature, it is unclear if a cell could also use nanometer-scale components to sense micron-scale contours, such as the cytokinetic furrow and base of neuronal branches. Septins are filament-forming proteins that serve as signaling platforms and are frequently associated with areas of the plasma membrane where there is micron-scale curvature, including the cytokinetic furrow and the base of cell protrusions. We report here that fungal and human septins are able to distinguish between different degrees of micron-scale curvature in cells. By preparing supported lipid bilayers on beads of different curvature, we reconstitute and measure the intrinsic septin curvature preference. We conclude that micron-scale curvature recognition is a funda- mental property of the septin cytoskeleton that provides the cell with a mechanism to know its local shape. Introduction Micron-scale plasma membrane curvature exists at the base and cancers (Dolat et al., 2014). Despite their importance, little of neuronal outgrowths, cilia, and the cytokinetic furrow. Al- is understood about the molecular function of septins compared though it is well known that proteins can sense and generate with other cytoskeletal proteins. Given their capacity to self- curvature on the nanometer scale (e.g., BAR domain proteins) assemble into rod-shaped complexes that are tens of nanome- via numerous mechanisms, it is unclear whether proteins can ters in length and filaments on the plasma membrane that are directly sense shape on the micron scale, which is the level of microns in length, we hypothesized that septins are capable many cell shape features (Zimmerberg and Kozlov, 2006). Dy- of micron-scale curvature recognition (Sirajuddin et al., 2007; namic changes in cell shape are central to processes as diverse Bertin et al., 2008; Bridges et al., 2014). as blood clotting, neurogenesis, and cancer cell metastasis. Thus, we reasoned that cells may have a capacity to sense local cell shape and use this information to inform behavior. Results and discussion Septins, which are conserved filament-forming, mem- brane-associated proteins, are found at regions of cells which Septins enrich at sites of positive THE JOURNAL OF CELL BIOLOGY are characterized by micron-scale curvature, including the curvature in Ashbya gossypii cytokinetic furrow and the bases of cell branches in neurons, To assess the curvature dependence of septin localization, we fungi, and ciliates (Fares et al., 1995; Helfer and Gladfelter, used Ashbya gossypii, a filamentous fungus that has similar 2006; Tada et al., 2007; Xie et al., 2007; Hu et al., 2010). At genome organization to Saccharomyces cerevisiae (Dietrich et these sites, septins tether organelles, restrict diffusion, rigidify al., 2004). During hyphal growth in A. gossypii, septins assem- the cell cortex, and spatially localize signaling (Longtine et al., ble into three genetically and spatially separable higher-order 2000; Tooley et al., 2009; Gilden and Krummel, 2010; Hu et structures associated with the cell cortex: (1) straight, stable al., 2010; Gilden et al., 2012; Chao et al., 2014). Perturbation bundles or “bars” of filaments; (2) thin, dynamic filaments, of septin genes results in abnormal cell morphology, cytoki- often enriched at sites of cell growth; and (3) dense assemblies nesis defects, and inviability in many organisms (Gladfelter et at the base of lateral branches, reminiscent of septin localiza- al., 2005; Mostowy et al., 2011; Mostowy and Cossart, 2012). tion at the base of outgrowths in neurons (Fig. 1 A, Fig. S1 Humans possess 13 septins which are implicated in numerous A, and Video 1; Helfer and Gladfelter, 2006; DeMay et al., pathologies, including neurodegenerative diseases, infertility, 2009). A. gossypii branches emerge in a variety of orientations Correspondence to Amy S. Gladfelter: [email protected] © 2016 Bridges et al. This article is distributed under the terms of an Attribution– Abbreviations used in this paper: PC, L-α-phosphatidylcholine; PI, L-α phos- Noncommercial–Share Alike–No Mirror Sites license for the first six months after the phatidylinositol; RCF, relative centrifugal force; RhPE, L-α-phosphatidylethanol- publication date (see http​://www​.rupress​.org​/terms). After six months it is available under a amine-N-(lissamine rhodamine B sulfonyl); ROI, region of interest; SUV, small Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, unilamellar vesicle; WT, wild type. as described at http​://creativecommons​.org​/licenses​/by​-nc​-sa​/3​.0​/). The Rockefeller University Press $30.00 J. Cell Biol. Vol. 213 No. 1 23–32 www.jcb.org/cgi/doi/10.1083/jcb.201512029 JCB 23 Figure 1. Septin abundance scales with positive curvature in A. gossypii. (A) Septin higher-order structures in A. gossypii, visualized by Cdc11a-GFP using structured illumination microscopy (SIM). Straight bundles (1), thin filaments (2,) and branch assemblies (3) exist in the same cell. (B) SIM images of Cdc11a-GFP signal at the base of four lateral branches emanating from hyphae at distinct angles, producing different curvatures. (C) Mean curvature heat map produced by imaging Blankophor in the A. gossypii cell wall followed by curvature analysis. For this display, curvature was mapped onto the external surface and values were inverted to represent curvature as viewed from the cell interior. (D) Cdc11a-GFP intensity at the base of branches plotted against positive and negative principal curvatures and mean curvature as viewed from the cell interior. Diagrams illustrate the curvature measured in each plot. (E) Filament orientation at the base of branches visualized by Cdc11a-GFP SIM. (F) Filament orientation in hyphae, away from sites containing a positive curvature component. (G) Filament orientation relative to the hyphal axis was measured compared with a random simulation of filament orientations (solid lines, mean; dotted line, SD; n = 263 filaments in 13 hyphae). (H) Colocalization of Cdc11-mCherry (green) and Hsl7-GFP (magenta) at the base of branches and straight bundles in A. gossypii. creating a situation where different curvatures are present lo- that the bases of branches are saddle shaped, we analyzed septin cally at the base of the same branch. We found that the septin localization versus the two orthogonal principal curvatures and Cdc11a is asymmetrically localized and preferentially enriched mean curvature. For clarity, we report the measured curvature on the side of a branch with the highest curvature, even though of the cytoplasmic face of the cell, which is what septins ex- both surfaces have access to the same local soluble pool of perience. From this perspective, A. gossypii are primarily com- septins (Fig. 1 B). This suggested that septins localize in a cur- posed of a negative principal curvature, which runs around the vature-dependent manner. tube-shaped cells, including at the base of branches. In con- To systematically determine the relationship between trast, a positive principal curvature is only found at the base cell shape and septin localization, we visualized the cell out- of branches, and it runs from the main hyphae into branches, line using a cell wall dye and measured curvature proximal to where the membrane bends inward toward the cytoplasm (Fig. A. gossypii branches (Fig. 1 C and Fig. S2, A–C). Considering S2 A). We found a strong relationship between Cdc11a-GFP 24 JCB • Volume 213 • NumBer 1 • 2016 enrichment and the positive curvature component (r = 0.850) Septin affinity for membranes varies and little correlation with the negative principal curvature (r depending on curvature = 0.145; Fig. 1 D). Consistent with a preference for positive How do septins differentiate between membrane curvatures? We curvature, septin abundance
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