Abstract
Most human cells require anchorage for survival. Cell–substrate adhesion activates diverse signalling pathways, without which cells undergo anoikis—a form of programmed cell death1. Acquisition of anoikis resistance is a pivotal step in cancer disease progression, as metastasizing cells often lose firm attachment to surrounding tissue2,3. In these poorly attached states, cells adopt rounded morphologies and form small hemispherical plasma membrane protrusions called blebs4,5,6,7,8,9,10,11. Bleb function has been thoroughly investigated in the context of amoeboid migration, but it has been examined far less in other scenarios12. Here we show by three-dimensional imaging and manipulation of cell morphological states that blebbing triggers the formation of plasma membrane-proximal signalling hubs that confer anoikis resistance. Specifically, in melanoma cells, blebbing generates plasma membrane contours that recruit curvature-sensing septin proteins as scaffolds for constitutively active mutant NRAS and effectors. These signalling hubs activate ERK and PI3K—well-established promoters of pro-survival pathways. Inhibition of blebs or septins has little effect on the survival of well-adhered cells, but in detached cells it causes NRAS mislocalization, reduced MAPK and PI3K activity, and ultimately, death. This unveils a morphological requirement for mutant NRAS to operate as an effective oncoprotein. Furthermore, whereas some BRAF-mutated melanoma cells do not rely on this survival pathway in a basal state, inhibition of BRAF and MEK strongly sensitizes them to both bleb and septin inhibition. Moreover, fibroblasts engineered to sustain blebbing acquire the same anoikis resistance as cancer cells even without harbouring oncogenic mutations. Thus, blebs are potent signalling organelles capable of integrating myriad cellular information flows into concerted cellular responses, in this case granting robust anoikis resistance.
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Data availability
The proteomic datasets generated during this study are available in the MassIVE repository at https://doi.org/10.25345/C5HQ3S35T (corresponding to Supplementary Table 1) and https://doi.org/10.25345/C5NG4GW9J (corresponding to Supplementary Table 2). All additional data that support the findings of this study are available in a Zenodo repository (https://doi.org/10.5281/zenodo.7416301). Source data are provided with this paper.
Code availability
All code, algorithms and software central to the findings of this study are available from the corresponding author upon reasonable request. Most can be found at https://github.com/DanuserLab, with code specific to this paper at https://github.com/DanuserLab/Weems-Septin-Paper. This code is also available in a Zenodo repository (https://doi.org/10.5281/zenodo.7435279).
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Acknowledgements
We thank J. Huang of TheWell Bioscience for providing information about the physical properties of VitroGel, D. Segal for discussions about septin inhibition, X. Jiang for conversations about optimal transport, P. Roudot for conversations on 3D image analysis, T. Isogai for advice on proximity proteomics, B. Nanes for assistance in data blinding and M. McMurray for feedback on the manuscript. We thank the UT Southwestern BioHPC team for providing necessary computing infrastructure. Funding for this work in the Danuser lab has been provided through grants R35 GM136428 (NIH) and I-1840-20200401 (Welch Foundation), and in the Fiolka lab through R33 CA235254 (NIH) and R35 GM133522 (NIH). M.D. was supported by the fellowship K99 GM123221 (NIH). A.D.W.s is a fellow of the Jane Coffin Childs Memorial Fund.
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Authors and Affiliations
Contributions
Study conceived by A.D.W. and developed by A.D.W., E.S.W., M.K.D. and G.D. Experimental design by A.D.W., E.S.W., M.K.D., G.M.G., V.S.M. and G.D. Experiments carried out by A.D.W., E.S.W., B.G.W., G.M.G., V.S.M. and D.R. Data analysed by A.D.W., M.K.D., H.M.-F. and F.Z. Cloning and cell line creation carried out by J.C. Statistical analysis carried out by A.D.W., M.K.D. and F.Z. Data visualization and figure preparation carried out by A.D.W. Figure layouts designed by A.D.W. and G.D. Software designed by M.K.D. (u-shape3D), H.M.-F. (EMD analysis) and F.Z. (time series analysis). B.-J.C., K.M.D. and R.F. designed, built and maintained 3D light-sheet microscopes. A.D.W. and G.D. wrote the paper, which was edited and approved by all authors. Experiments in Fig. 1a carried out by A.D.W. and E.S.W. and analysed by A.D.W. All other experiments in Fig. 1 carried out and analysed by A.D.W. Experiments in Fig. 2d carried out by E.S.W. and analysed by M.K.D. Experiment in Fig. 2j carried out by A.D.W. and E.S.W. Analysis in Fig. 2l carried out by F.Z. All other experiments and analysis in Fig. 2 carried out by A.D.W. Experiments in Fig. 3b carried out by A.D.W. and E.S.W. and analysed by A.D.W. All other experiments in Fig. 3 carried out by A.D.W. Analysis in Fig. 4b carried out by H.M.-F. All other experiments and analysis in Fig. 4 carried out by A.D.W. All experiments and analysis in Fig. 5 carried out by A.D.W. Experiments in Extended Data Fig. 1A carried out by E.S.W. and analysed by M.K.D. Experiments in Extended Data Fig. 1B–D carried out by A.D.W. and E.S.W. All other analysis in Extended Data Fig. 1 carried out by A.D.W. Experiments and analysis in Extended Data Fig. 2 carried out by A.D.W. Experiments in Extended Data Fig. 3 carried out by V.S.M., D.R. and A.D.W. and analysed by A.D.W. Experiments and analysis in Extended Data Fig. 4 carried out by A.D.W. Experiments in Extended Data Fig. 5 carried out by A.D.W. and analysed by F.Z. Experiments and analysis in Extended Data Fig. 6 carried out by A.D.W. Experiments in Extended Data Fig. 7 carried out by G.M.G. and analysed by G.M.G. and A.D.W. Experiments and analysis in Extended Data Figs. 8–10 carried out by A.D.W. Experiments in Extended Data Table 2 carried out by B.G.W and analysed by A.D.W. Experiments and analysis in Supplementary Videos 1–4 carried out by A.D.W.
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The Board of Regents of the University of Texas System has filed for a patent (US patent 63/334,029, filed 22 April 2022) covering therapeutic targeting of septins and blebbing, which was based in part on data from this article. A.D.W., G.D., E.S.W. and M.K.D. are named as inventors. The authors declare no other competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Replicates of bleb inhibition viability experiments.
(A) Fraction of cell surface comprised of blebs for MV3 cells treated with different concentrations of WGA. Dashed lines separate quartiles. Dots represent individual cells. (B) Individual bleb inhibition viability experiments, as summarized in Fig. 1a. Each pairing of adhered/detached cell experiments was performed on the same day and seeded with cells from the same diluted cell suspensions. (C) Morphology of cells in “adhered” conditions, related to Fig. 1a. Cells grown without perturbation for 24 h on adherent chamber slides. Representative of either 688 (MV3), 405 (M498), or 576 (A375) cells observed. (D) Paired mean differences between control and experimental condition shown in Gardner-Altman estimation plots for the data presented in Figs. 1d, 3b, 4d, 4e, and 4f. Gray distributions represent 5000 bootstrapped samples, black bars represent 95% confidence intervals, black dots represent mean difference.
Extended Data Fig. 2 SEPT6-GFP localization and the effects of septin inhibition.
(A) Mouse SEPT6-GFP probe localization in MV3 cells embedded in soft bovine collagen. Maximum intensity projections (left) and single z-slices of 0.16 micron thickness (right) shown for representative cells in two columns. (B) Maximum intensity projections of mouse SEPT6-GFP probe localization in MV3 cells with and without inhibition by FCF or SEPT2(33–306) expression. Cells embedded in soft bovine collagen. (Below) Fraction of cell surface comprised of blebs for MV3 cells with and without SEPT6-GFP expression, FCF treatment, and SEPT2(33–306) expression. Dashed lines separate quartiles. Dots represent individual cells. (C) Mouse SEPT6-GFP probe localization in MV3 cells embedded in soft bovine collagen. Maximum intensity projections and single z-slices of 0.16 micron thickness shown for representative cells in two columns. (Above) Cells expressing the SEPT2(33–306) mutant. (Below) Cells expressing cytosolic mCherry (not shown) using the same pLVX-IRES-Hyg vector as the SEPT2(33–306) construct. Included as a negative control demonstrating that the septin mislocalization in SEPT2(33–306)-expressing cells is not due to effects arising from the protein expression construct.
Extended Data Fig. 3 Endogenous septin localization via immunofluorescence.
(A) MV3 cells adhered to fibronectin-coated glass slides showing either the mouse SEPT6-GFP probe or anti-SEPT2 immunofluorescence localization. While the well-established septin localization to actin stress fibers (https://doi.org/10.1016/S1534-5807(02)00366-0) is apparent in both samples, the anti-SEPT2 signal is irregular, punctate, and possesses low signal-to-noise ratio (SNR) compared to SEPT6-GFP. Scale bars are 10 µm. (B) Anti-SEPT2 signal in a rounded blebbing MV3 cell embedded in soft bovine collagen, with maximum intensity projections (top) and single z-slices of 0.16 micron thickness (below). Phalloidin signal from the same cell shown on right to visualize the cell surface. Anti-SEPT2 signal possesses low SNR, as seen in adhered cells. Despite this, the signal is enriched at the cell surface in blebby regions of the cell just as seen in cells expressing the SEPT6-GFP probe (see Extended Data Fig. 2a). (C) Cell surface distribution of anti-SEPT2 signal for the cell shown in Extended Data Fig. 3b. (D) Local anti-SEPT2 or SEPT6-GFP intensity and intracellular mean curvature as a function of distance from bleb edges. Mean intensity is comprised only of the brightest 50% of the cell surface to account for punctate and discontinuous immunofluorescent signal. (E) Fraction of cortical voxels (within 0.96 µm of surface) with anti-SEPT2 intensity higher than cytoplasmic mean intensity in MV3 cells with and without expression of SEPT6-GFP. Dashed lines separate quartiles and dots represent individual cells.
Extended Data Fig. 4 A mutation that disrupts curvature sensing blocks septin localization to the cell surface.
Top, cartoon illustrating the mechanism underlying the altered function of the SEPT6(ΔAH)-GFP mutant. Bottom left, mean local SEPT6(ΔAH)-GFP intensity and intracellular mean curvature as a function of distance from bleb edges, calculated from 630 blebs across 5 MV3 cells. Error bands indicate 95% confidence intervals. Bottom right, difference in mean local septin intensity compared to local maxima as a function of distance from bleb edge for both SEPT6-GFP (as seen in Fig. 2e) and SEPT6(ΔAH)-GFP.
Extended Data Fig. 5 Analysis of high-speed timelapse SEPT6-GFP data in MV3.
(A) Autocorrelation curves for the local mean curvature and SEPT6-GFP data presented in Fig. 2l; the fluorescence signal was normalized to account for photobleaching as described in Methods. (B) Temporal cross-correlation functions as shown in Fig. 2l, here with extended range of time lags. (C) Mean rates of absolute change in SEPT6-GFP intensity for positive and negative intracellular mean curvature contigs, expressed as a function of the dynamicity of the contigs they occur within. Dynamicity is defined as the total number of timepoints showing intensity increases and decreases that occur within a contig, divided by the total number of timepoints within that contig. Derived from the same dataset used in Fig. 2l. Vertical dashed line represents the threshold for low and high dynamic groups as shown in Figs. 2l and S7B.
Extended Data Fig. 6 A mutation that disrupts inter-oligomeric polymerization blocks septin localization to the cell surface.
Top, a cartoon illustrating the mechanism underlying the altered function of the SEPT2(33–306) mutant. Bottom left above, mean local SEPT6-GFP intensity and intracellular mean curvature as a function of distance from bleb edges in cells expressing SEPT2(33–306), calculated from 1293 blebs across 8 MV3 cells. Error bands indicate 95% confidence intervals. Bottom left below, difference in mean local SEPT6-GFP intensity compared to local maxima as a function of distance from bleb edge for cells expressing SEPT2(33–306) and control cells (as seen in Fig. 2e). Bottom right, Western blot of SEPT2 in MV3 cells with and without SEPT2(33–306) expression. Calculated mass of SEPT2 is 41.5kD and SEPT2(33–306) is 31.7 kD. Annotations show positions of the indicated ladder bands.
Extended Data Fig. 7 Changes in septin expression upon MAPKi treatment of A375 cells.
Top, Western blots of SEPT2, SEPT6, SEPT7, and SEPT9 abundance (the 4 septins with highest expression in A375 cells, as measured by mass spectrometry, see Extended Data Table 2) upon combined treatment with Dabrafenib and Trametinib at doses indicated. Three independent experiments were performed, with data from all replicates shown alongside vinculin loading controls. Bottom, densitometry analysis of Western blots showing fold change in septin expression as dose response curves. Dashed lines indicate individual experiments (color-coded according to the color bars indicating Western blot replicates); solid red lines indicate means of all replicates.
Extended Data Fig. 8 Viability effects of SEPT2(33–306) expression.
Viability in adhered and detached melanoma cells expressing SEPT2(33–306). Cells grown for 24 h and assayed for cell death using ethidium homodimer staining. All treatment groups grown and assayed in simultaneous paired experiments. Dots represent individual experiments. Cell counts for all replicates (see Extended Data Table 1 for individual counts): MV3 (951, 872), A375 (653, 1302). MV3 and A375 datasets were tested using one-sided Paired T Tests, p = 1.02x10−3 and p = 0.388.
Extended Data Fig. 9 Effect of SEPT6 expression on NRAS Localization.
(A) Observed NRAS-GFP % enrichment at the cortex (voxels within 0.96 µm of surface) of individual unperturbed and septin-inhibited MV3 cells. Basal/FCF tested with one-sided Welch’s T-test (p = 4.27x10−5), normality tested with Shapiro-Wilk (p = 0.219 & 0.219), variance tested with two-tailed F test (p = 0.0054). Basal/SEPT2(33–306) tested with two sample one-sided t-test using pooled variance (p = 0.0101), normality tested with Shapiro-Wilk (p = 0.219 & 0.724), variance tested with two-tailed F test (p = 0.768). Dashed lines separate quartiles. (B) Observed NRAS-GFP percent enrichment at the cortex (voxels within 0.96 µm of surface) of individual MV3 cells with and without expression of SEPT6-HALO using the same pLVX-IRES-Hyg vector as the SEPT2(33–306) construct. Included as a negative control demonstrating that the NRAS mislocalization in SEPT2(33–306)-expressing cells is not due to effects arising from the protein expression construct. Significance tested with two sample one-sided T-test with pooled variance (p = 0.738), normality tested with Shapiro-Wilk (p = 0.315 & 0.426), variance tested with two-tailed F test (p = 0.181). Dashed lines separate quartiles. (C) Cell death upon expression of dominant negative NRAS(S17N) for adhered and detached MV3 cells. Cells grown as in Fig. 1a for 24 h. Data were normalized by subtracting paired negative control values from each treatment group. Dots represent individual experiments. Cell counts for all replicates, with control counts in parentheses (see Extended Data Table 1 for individual counts): Adh. 1105(1039), Det. 799(734).
Extended Data Fig. 10 Anoikis resistance in non-malignant cells.
(A) SEPT6-GFP localization in a recently detached HEK cell, embedded in soft bovine collagen. Top, maximum intensity projection (MIP); bottom, individual 0.16 µm z-slice from the same cell. (B) Additional fraction of MV3 cells showing caspase activation after 4 h of treatment with 10 µg/mL WGA (compared to matched, same-day negative controls) for adhered and detached cells. Total cell counts with control in parenthesis: Replicate 1: Adh 143(220), Det 310(544); Replicate 2: Adh 182(152), Det 256(215); Replicate 3: Adh 134(157), Det 89(267). (C) The same data shown in Fig. 5c, without normalization. Solid, dashed, and dotted lines represent paired, same-day experiments. Total cell counts for each condition, with adhered group in parenthesis: Replicate 1: Con 49(89), DYN2(K44A) 38(55), DYN2(K44A)+WGA 32(25), DYN2(K44A)+FCF 27(33); Replicate 2: Con 23(25), DYN2(K44A) 28(39), DYN2(K44A)+WGA 27(43), DYN2(K44A)+FCF 28(34); Replicate 3: Con 92(54), DYN2(K44A) 121(84), DYN2(K44A)+WGA 101(115), DYN2(K44A)+FCF 141(70).
Extended Data Fig. 11 Graphical Abstract.
Summary illustration of the information flow via bleb signaling as well as the mechanisms through which pro-survival bleb signaling hubs are constructed.
Supplementary information
Supplementary Fig. 1
Uncropped images of western blots shown in this study.
Supplementary Table 1
Summary of mass spectrometry results from MV3 lysate and SEPT6 pulldown experiments. Proteins that were identified with only 1 PSM are highlighted in orange, proteins that were identified but not quantified are highlighted in blue, and potential contaminants from experimenter are highlighted in yellow. Details of column titles are as follows—Protein FDR confidence: High (1% false discovery rate), Medium (5% false discovery rate) or Low (>5% false discovery rate); Master: if more than one protein in a group has the same score, and an equal number of PSMs, and an equal number of peptides, the protein with the longest sequence is designated as the master protein; Accession: protein accession number (from UniProtKB); Description: description taken from UniProt; Coverage (%): percentage of the protein sequence that was covered by the peptides identified for the protein; #PSMs: number of peptide spectrum matches, or the number of spectra assigned to peptides that contributed to the inference of the protein; MW [kDa]: molecular weight of the protein based on the sequence from Uniprot; Abundance: the sum of the peak intensities for each peptide identified for that protein (these values can be used to compare relative abundance of a protein between samples).
Supplementary Table 2
Summary of mass spectrometry results from SEPT6 BioID proximity labelling experiment. Proteins that were identified with only 1 PSM are highlighted in orange, proteins that were identified but not quantified are highlighted in blue, and potential contaminants from experimenter are highlighted in yellow. Details of column titles are as follows—Protein FDR confidence: High (1% false discovery rate), Medium (5% false discovery rate) or low (>5% false discovery rate); Master: if more than one protein in a group has the same score, and an equal number of PSMs, and an equal number of peptides, the protein with the longest sequence is designated as the master protein; Accession: protein accession number (from UniProtKB); Description: description taken from UniProt; Coverage (%): percentage of the protein sequence that was covered by the peptides identified for the protein; #PSMs: number of peptide spectrum matches, or the number of spectra assigned to peptides that contributed to the inference of the protein; MW [kDa]: molecular weight of the protein based on the sequence from Uniprot; Abundance: the sum of the peak intensities for each peptide identified for that protein (these values can be used to compare relative abundance of a protein between samples).
Supplementary Video 1
Mammalian cells become rounded and blebby after detachment. Top: three common cell lines (MEF cells, human epithelial kidney 293 cells and human bronchial epithelial cells) grown under standard adherent conditions in plastic Petri dishes. Bottom: Cells from the same cultures 3 min after treatment with trypsin and gentle pipetting to cause detachment. All time lapses played at ≈15× speed.
Supplementary Video 2
Conversion of raw light-sheet microscope data to 3D surfaces using u-Shape3D. Poorly adhered MV3 cell embedded in soft bovine collagen. Septins visualized with mouse SEPT6–GFP probe.
Supplementary Video 3
Septins ‘pulse’ at the curvy bases of blebs. Poorly adhered MV3 cell embedded in soft bovine collagen. Septins visualized with mouse SEPT6–GFP probe. Five-min time lapse acquired at ≈0.83 Hz played twice, the second time with arrowheads and insets highlighting septin pulses.
Supplementary Video 4
Iterative blebbing produces stable septin structures. Poorly adhered MV3 cell embedded in soft bovine collagen. Septins visualized with mouse SEPT6–GFP probe. Five-min time lapse acquired at ≈0.83 Hz. Three orthogonal views show tightly spaced iterative blebbing producing septin pulses that combine to form a bright stable structure that maintains signal intensity independent of local blebbing.
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Weems, A.D., Welf, E.S., Driscoll, M.K. et al. Blebs promote cell survival by assembling oncogenic signalling hubs. Nature 615, 517–525 (2023). https://doi.org/10.1038/s41586-023-05758-6
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DOI: https://doi.org/10.1038/s41586-023-05758-6
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