Septins buffer actomyosin forces to protect the nucleus from genotoxic mechanical stress

Extended Data, Fig. 1:. Perinuclear septin cables consist of SEPT9_i1 and SEPT7, and are spatially distinct from the nuclear vimentin basket. a, Sagittal view of SEPT9_i1 (green) and the nucleus (DAPI, blue) in a 3D volume region of a U2OS cell confined in an 8 mm-wide pore. Scale bar, 2 μm. b, Top-down and sagittal side views of a confined U2OS subcellular region after staining for SEPT7 (green), SEPT9_i1 (magenta) and the nucleus (DAPI, blue). Images were 3D reconstructed, and volume rendered. Scale bars, 1 μm (top-down view) and 2 μm (sideview). c-d, Single optical sections of a confined U2OS cell region taken from medial (c) and basal (d) depths after staining for SEPT7 (green) and SEPT9_i1 (magenta). Scale bar, 1 μm. e, Sagittal sideviews of the nucleus (DAPI, blue), F-actin (phalloidin, grayscale), SEPT9_i1 (green) and vimentin (magenta) in a 3D volume region of a U2OS cell confined in an 8 mm-wide pore. The view shown in the top row of images is rotated 180 degrees in the bottom row. The perinuclear vimentin basket is outlined with a yellow dashed line. Scale bars, 2 μm. All U2OS cell images were acquired with super-resolution SoRa SDCM 12 h after plating on 8 μm-wide pores.

Extended Data, Fig. 2:. Spatiotemporal dynamics of perinuclear actin and septin cables in confined regions of cell migration. a, Top-down (top) and oblique angle (middle, bottom) views of still frames from time-lapse 3D SoRa SDCM imaging of LAP2b-AcGFP1 (green) and SPY555_FastAct_X (magenta) in the U2OS cell region confined in a Matrigel/laminin-filled 8 μm-wide pore. Images were rendered and reconstructed in 3D for each time point. Angled side views are more zoomed in on the nuclear envelope, which appears to surround the pore. Nuclear envelope creases and folds (arrows) are seen in the 60 and 120 minute time frames. Note that the nuclear envelope preserves its continuity in the 120 minute timeframe, but the front side becomes dim due to higher signal intensity in the creased regions of the 3D-rendered image. Scale bars, 2 μm. b, Time-lapse frames of a single optical slice from SoRa SDCM imaging of a U2OS cell region confined in a Matrigel/laminin-filled 8 μm-wide pore. U2OS cells were transfected with GFP-SEPT9_i1 (green) and labeled live with SPY650_FastAct_X (magents). White arrows point to actin that colocalizes with GFP-SEPT9_i1, and enlarges overtime. Blue arrows point to actoseptin that appears…

Extended Data, Fig. 3:. Septin expression in genome edited U2OS cells, and zyxin increase in confined SEPT9_i1-depleted U2OS cells. a, Western blots of lysates of parental U2OS and cell clones after selection for stable expression of SEPT9_i1 targeting sgRNA relative. Cell lysates were blotted with antibodies against the SEPT9_i1 isoform and SEPT7. b, Wide-field microscopy images of U2OS cells that stably express control and SEPT9_i1 sgRNAs after staining for F-actin (phalloidin, magenta) and the SEPT9_i1 isoform (green). Insets show outlined regions in higher magnification. Scale bars, 20 μm. c-e, Sagittal side views of 3D rendered images of the confined regions of control and SEPT9_i1-depleted U2OS cells in 8 μm (c), 5 μm (d) and 3 μm (e) wide pores. Cells were stained with phalloidin (F-actin, magenta) and antibody against zyxin (grayscale). Nuclear areas are outlined with dashed yellow line. Outlined regions with zyxin-decorated actin cables are shown in higher magnification. Scale bars, 2 μm (c, d) and 1 μm (e).

Extended Data, Fig. 4:. SEPT9 expression and localization in Sept9+/+ and Sept9−/− MEFs. a, Wide-field microscopy images of Sept9+/+ MEFs on collagen type I ECM after staining for F-actin (phalloidin, magenta) and endogenous Sept9 with an antibody against all isoforms. Bottom panels show in higher magnification the outlined region with Sept9 filaments colocalizing with actin stress fibers. Scale bars, 10 μm. b, Wide-field microscopy images of Sept9−/− MEFs on collagen type I ECM after staining for F-actin (phalloidin, magenta) and endogenous Sept9 (green) with an antibody against all isoforms. Bottom panels show in higher magnification an outlined region with actin stress fibers which lack Sept9 filaments. Absence of Sept9 expression is evident by the faint spotty background-level fluorescence signal. Scale bars, 10 μm.

Extended Data, Fig. 5:. SEPT9 expression and localization in MDA-MB-231 cells during confined migration, and effects of SEPT9_i1 depletion on genomic integrity and cell viability. a, Sagittal 3D volume view of an MDA-MB-231 cell region confined in a Matrigel-filled 3 μm wide pore after staining for nucleus (DAPI, blue), F-actin (phalloidin, magenta) and SEPT9 (green). Images were acquired with 3D SoRa SDCM and rendered in 3D. Outlined regions is shown in higher magnification. Scale bars, 1 μm. b, Images show single SoRa SDCM optical slices taken from the indicated depths of the 3 μm wide pore (see lines in a). Arrowheads point to F-actin and SEPT9 colocalization. Scale bars, 1 μm. c, Sagittal 3D volume view of an MDA-MB-231 cell region confined in a Matrigel-filled 3 μm wide pore after staining for DAPI (blue), F-actin (phalloidin, magenta). Images were acquired with 3D SoRa SDCM and rendered in 3D. Scale bars, 1 μm. d, Images show single SoRa SDCM optical slices taken from the indicated depths of the 3 μm wide pore (see lines in c), and stained for nucleus (DAPI, blue), F-actin (phalloidin, magenta), pMLC2 (green) and SEPT9 (grayscale). Arrowheads point to F-actin, SEPT9 and pMLC2 colocalization. Scale…

Extended Data, Fig. 6.. SEPT9 gene copy amplification associates with thresholds of lower genomic alteration in aggressive tumors a, Stacked bar plots showing the proportions of each indicated tumor type harboring SEPT9 copy-number amplification (magenta), gain (cyan), shallow (olive) and deep depletion (pink), or copy-neutral status (green). Data are shown for pan-cancer primary tumors from TCGA (n = 11,245). b, Kernel density plots showing the distribution of FGA values in normal breast tissue (n = 36), luminal A breast cancer (n = 496), luminal B breast cancer (n = 197), basal/triple negative breast cancer (n = 171) and all breast tumors harboring SEPT9 copy-number gain or amplification (n = 403).

Fig. 1:. Septins align with perinuclear contractile actomyosin cables during confined cell migration a, Schematic of the experimental configuration for imaging confined cell migration in 3D. Transwell polycarbonate membranes with pores of variable diameter were coated with ECM, and U2OS cells migrated through the pores by chemotaxis along an EGF gradient. Cellular regions within the pore were imaged by super-resolution SoRa SDCM. b, Maximum intensity projections of cross-sectional images from ~1 μm-thick regions at apical, apical-medial, medial, basal-medial, and basal depths of the pore, indicated by the yellow band in the schematic below each fluorescence image. Insets show magnified views of phalloidin-stained F-actin clusters (groups of vertical filaments; magenta) surrounding the confined nuclear mass (DAPI, blue) with semi-regular spacing along the pore circumference. The basal cross-section shows cortical actin at the cell membrane extending from the pore (dotted circle) to the basal surface of the porous membrane. c, Side view of a 3D volume showing F-actin (phalloidin, magenta) relative to the confined nucleus (DAPI, blue) within the pore and the plasma membrane extending onto…

Fig. 2. Septins reduce mechanical stress on the actomyosin cytoskeleton during migration through increasingly narrow pores a-c, Maximum intensity projections of cross-sectional images from ~1 μm-thick regions at apical, medial and basal depths of 8 μm (a), 5 μm (b) and 3 μm (c) wide pores. The confined regions of genome-edited U2OS cells (parental control and SEPT9_i1 CRISPRi) are shown after staining for F-actin (phalloidin, magenta) and zyxin (grayscale). Scale bars, 1 μm. d, Sagittal view of a subcellular 3D volume region of a SEPT9_i1-depleted U2OS cell confined in a 5 μm wide pore. Arrows point to zyxin (grayscale) localization along the length of actomyosin cables (phalloidin, magenta). Scale bar, 0.5 μm. e, Violin plots show percentage of zyxin decorated actin cables in genome-edited U2OS cell (parental control and SEPT9_i1 CRISPRi) regions confined in 3 μm (n = 96–102 cables; 10 cells), 5 μm (n = 185–190 cables; 10 cells) and 8 μm (n = 381–390 cables; 10 cells) pores. Statistical analysis between the indicated pair groups was done with the Welch’s t-test. f, Violin plot shows pMLC2-decorated actin cables with SEPT9 per cell regions confined in 3 μm (n = 40) and 8 μm (n = 63) wide…

Fig. 3. Septins relieve actin stress fibers from tensile stress induced by myosin II hyperactivation. a-b, Wide-field microscopy images show F-actin (phalloidin, green) and paxillin (magenta) in mouse embryonic fibroblasts (MEFs) from Sept9+/+ (a) and Sept9−/− (b) mice, which were plated on collagen type I and treated with calyculin A (10 nM) for 30 minutes. Regions in dashed rectangles are shown in higher magnification below. Arrows and arrowheads point to breaks and buckles in actin stress fibers, respectively. Scale bars, 5 μm. c, Violin plots show percentage of actin stress fibers with breaks per cell after 0 (n = 230–289 stress fibers; 10 cells), 15 (n = 235–285 stress fibers; 10 cells) and 30 (n = 239–255 stress fibers; 10 cells) minute treatment of Sept9+/+ and SEPT9−/− MEFs with calyculin A (10 nM). Statistical comparisons between the indicated groups were done with Brown-Forsythe and Welch one-way ANOVA test with Dunnett T3 multiple comparisons test. ***, p<0.001; ****, p<0.0001. d, Violin plots show percentage of actin stress fibers with buckles per cell after 0 (n = 230–289 stress fibers; 10 cells), 15 (n = 235–285 stress fibers; 10 cells) and 30 (n = 239–255 stress fibers; 10…

Fig. 4. Septins protect the nuclear envelope from the mechanical stress of actomyosin contraction. a, Still frames of SDCM images of live Sept9+/+ and SEPT9−/− MEFs expressing LAP2b-AcGFP1 (green) and treated with calyculin A (10 nM) which was administered 5 minutes prior to start of time-lapse imaging. Arrow points to latten region of the circular nuclear envelope. Arrowheads point to nuclear envelope dimples. Scale bars, 5 μm. b, SDCM images show in higher magnification regions of the nuclear envelope outlined by the dashed lines in still frames of the time series above (a). Scale bars, 1 μm. c, Wide field microscopy images of Sept9+/+ and Sept9−/− immunostained for nucleoporins after 0 and 30 min of treatment with calyculin A (10 nM). Numbered regions outlined by dashed lines are shown in higher magnification. Arrows point to sites of nuclear envelope rupture. Scale bars, 10 μm. d, Violin plots show nuclear surface areas in Sept9+/+ MEFs after 0 minutes (n = 55 cells) and 30 minutes (n = 38 cells) of calyculin A treatment, and in SEPT9−/− MEFs after 0 minutes (n = 50 cells) and 30 minutes (n = 50 cells) of calyculin A treatment. Statistical comparisons between the indicated groups…

Fig. 5:. SEPT9_i1 protects nuclei from mechanical damage during confined migration, suppressing DNA damage, nuclear defects associated with genomic instability, and cell death. a, SoRa SDCM images of nuclei (DAPI, blue), gH2AX (green) and actin (phalloidin, magenta) in parental control and SEPT9_i1-depleted MDA-MB-231 cells, taken from the basal side of polycarbonate membranes with 3 μm wide pores after 36 h of apicobasal transmigration. Arrow points to a large nuclear region with gH2AX accumulation. Inset shows a gH2AX-enriched micronucleus in higher magnification. Scale bars, 5 μm (1 μm in inset). b, SDCM images of nuclei (DAPI, blue), nucleoporins (green) and actin (phalloidin, magenta) in parental control and SEPT9_i1-depleted MDA-MB-231 cells, taken from the basal side of polycarbonate membranes with 3 μm wide pores after 36 h of apicobasal transmigration. Inset shows in higher magnification a region of nuclear blebbing, where nuclear DNA protrudes from a site of nuclear envelope rupture. Scale bars, 5 μm (1 μm in inset). c, Violin plots show percentage of parental control and SEPT9_i1-depleted MDA-MB-231 cells with large gH2AX foci on basal membrane regions 3 μm (n = 26 regions;…

Fig. 6:. SEPT9 amplification associates with suppression of excessive genomic instability and lower patient survival a, Kaplan-Maier plot of survival in a pan-cancer TCGA cohort stratified by SEPT9 copy number status. Patients with copy-neutral (diploid) SEPT9 tumors (blue; n = 6,912) are compared with patients whose tumors harbor SEPT9 copy-number gain or amplification (red; n = 2,873). Survival curves were compared using the log rank test (p < 0.0001; n = 3,135 events). b, Volcano plot showing log2 odds ratios for SEPT9 amplification (positive values) and deletion (negative values) across tumor types, plotted against statistical significance (−log10 FDR). Tumor types with FDR < 0.05 are highlighted. Breast cancer shows the strongest enrichment for SEPT9 amplification, while more modest enrichment is observed in hepatobiliary cancer. Several tumor types including (blue squares) glioblastoma, head and neck cancer, thyroid cancer, and renal clear cell carcinoma, exhibit significant depletion of SEPT9 copy number. Data were derived from TCGA. c, SEPT9 mRNA expression (log2 (value+1)) is shown for breast tumors stratified by SEPT9 GISTIC copy-number state: deep (n = 2) and shallow deletion…