Abstract
Actin-based protrusions are reinforced through positive feedback, but it is unclear what restricts their size, or limits positive signals when they retract or split. We identify an evolutionarily conserved regulator of actin-based protrusion: CYRI (CYFIP-related Rac interactor) also known as Fam49 (family of unknown function 49). CYRI binds activated Rac1 via a domain of unknown function (DUF1394) shared with CYFIP, defining DUF1394 as a Rac1-binding module. CYRI-depleted cells have broad lamellipodia enriched in Scar/WAVE, but reduced protrusion–retraction dynamics. Pseudopods induced by optogenetic Rac1 activation in CYRI-depleted cells are larger and longer lived. Conversely, CYRI overexpression suppresses recruitment of active Scar/WAVE to the cell edge, resulting in short-lived, unproductive protrusions. CYRI thus focuses protrusion signals and regulates pseudopod complexity by inhibiting Scar/WAVE-induced actin polymerization. It thus behaves like a ‘local inhibitor’ as predicted in widely accepted mathematical models, but not previously identified in cells. CYRI therefore regulates chemotaxis, cell migration and epithelial polarization by controlling the polarity and plasticity of protrusions.
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Data availability
Mass spectrometry data have been deposited in ProteomeXchange with the primary accession code PXD 010460. Source data for Figs. 1–7 and Supplementary Figs 2–6 have been provided as Supplementary Table 6. All other data supporting the findings of this study are available from the corresponding authors on reasonable request.
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Acknowledgements
We thank M. O’Prey and the BAIR imaging facility for help with microscopy, C. Tesniere for careful work with CYRI overexpression plasmids, M. Mcilwraith for help with protein purification, B. Tyrell for isolation of the inducible Rac1fl/fl fibroblasts, K. Rottners and M. Schaks for technical advice and discussion, and R. Wedlich-Söldner for the GFP-LifeAct construct. We thank CRUK for core funding to L.M.M. (grant A15673), R.H.I. (grant A15672) and S.Z. (C596/A12935), BBSRC for funding to L.H.C. and N.C.O.T. (BB/L022087/1), and NIH for funding to G.S.M. (NIH RO1 EY025205). Fundação para a Ciência e a Tecnologia, Portugal awarded a PhD scholarship (reference SFRH/BD/69003/2010) to J.M.B.
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Contributions
R.H.I. and J.M.B. conceived and carried out the initial screen and recognized the similarity of CYRI to CYFIP. L.F. designed and carried out the majority of the experiments on mammalian CYRI-B. L.M.M., R.H.I. and L.F. conceived the study and wrote the paper. P.A.T. designed and constructed the mitochondrial relocalization tools and carried out the Dictyostelium experiments in Figs. 2 and 6. K.J.M. and K.I.A. designed the Raichu FRET probe and, along with L.F., carried out the FRET experiments. P.B. and L.F. carried out the SPR experiments. J.G., N.C.O.T. and L.H.C. synthesized probes for, advised on and carried out the myristoylation experiments with L.F. S.L. and S.Z. carried out and analysed the mass spectrometry with L.F. and J.M.B. P.A.T., G.S.M., J.A.W., H.J.S., L.T. and S.I. provided essential advice, carried out experiments and analysed the data. M.P.N. and R.H.I. constructed the model and advised on its use.
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P.T. works with Hybrigenics, which performs yeast two-hybrid screening for commercial purposes.
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Supplementary Figure 1 CYRI proteins are evolutionarily conserved and contain a putative Rac1-binding site similar to CYFIP.
a - Co-evolution of Arp2/3 (green), Scar/WAVE (brown) and CYRI (orange) across the 4 main superfamilies of the eukaryotic tree 1. An incomplete circle corresponds to the absence of some subunits from the complex as in 2. Each supergroup was assigned to a specific background colour: Unikonts (yellow), Chromalveolates (pink), Excavates (blue) and Plants (green). Branch length does not reflect evolutionary distances. For C. elegans and R. allomycis, * and red highlight around circle denotes lack of putative N-terminal myristoylation site on CYRI (see below). Different coloured branches in Unikonts represent amoebazoa (yellow branches), metazoan (purple branches), fungi (brown branches) and Rozella allomycis, the earliest diverged of the fungi (blue branch). b - Alignment of D. discoideum and H. sapiens CYFIP and CYRI sequences. UniProt accession numbers are reported. Colour code represents the number of entries with identical amino acid at this position. c - HMM logo from the highlighted region in (b). Logo was generated by feeding the full Pfam dataset (915 sequences) of the DUF1394 domain to Skylign. Letter stack represents the conservation of the residue at this position across the dataset. Occupancy score is also mentioned below each letter (light blue) and is a readout of the presence of a letter across the dataset, ranging from 0 (no present) to 1 (present).
Supplementary Figure 2 CYRI-B interacts directly with active Rac1.
a – Summary of yeast 2-hybrid derived interacting regions between CYRI-B and constitutively active Rac1G12V. Confidence score A = best to D = low (see Methods). b – Schematic of clones from yeast two-hybrid screen (a). Overlaps define core Rac1-binding domain (RBD) of CYRI-B aa30-236. c - Schematic of GFP and GST constructs. eCFP-CRIB-PBD represents the CRIB region of PAK1, used as a positive control. d-f – MBP trap beads were loaded with recombinant WT or mutant maltose binding protein MBP-Rac1 protein and incubated with recombinant GST-CRIB-PBD, GST-CYRI-B RBDWT and GST-CYRI-B RBDR161D. Ponceau staining shows MBP-Rac1 (d). Binding (arrowhead) represents ratio relative to Rac1WT (e-f). (n = 3 independent experiments). See Supplementary Fig. 7. g-h - GST-pulldowns showing specific binding of CYRI-B RBD to Rac1. Lysate from CHL-1 cells expressing GFP (negative control), eCFP-CRIB-PBD, GFP-Rhotekin (reporter for Rac1 or RhoA activity respectively) or GFP-RBDWT mixed with immbolized GST, GST-Rac1WT, GST-Rac1Q61L, GST-RhoAQ63L, GST-Cdc42Q61L. Probed for GFP and GST (g). Binding (arrowhead) was assessed relative to GST (h). (n = 3 independent pull-down). See Supplementary Fig. 7. i - Control PLA of COS-7 cells co-expressing CYRI-BWT -HA and MYC-Rac1Q61L using anti-HA or anti- MYC antibodies. PLA signal (yellow), F-actin (magenta) and nuclei (blue). Scale bar = 50 μm. j - Cell area for each field of view (fov); average cell area was similar between conditions. (Myc-WT/WT-HA n = 34 fov; Myc-WT/R160-161D-HA n = 35 fov; Myc-T17N/WT-HA n = 34 fov; Myc-T17N/ R160-161D-HA n = 55 fov; Myc-Q61L/WT-HA n = 39 fov; Myc-Q61L/ R160-161D-HA n = 36 fov). k-l - Proximity ligation assay COS-7 cells on laminin and co-expressing pLIX-mVenus-CYRI-B-HA (WT or mutant R160/161D) and MYC-tagged Rac1 as indicated. (k). Randomized fields of view across 2 independent experiments. PLA signal was thresholded using Fiji and compared with cell area (see Methods) (l). One-way ANOVA with Dunn’s post-test between CYRI-BWT and MYC-Rac1 as indicated. Two-tailed Mann-Whitney test between CYRI-BWT and CYRI-BR160/161D for each MYC-Rac1 construct. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. (anti-HA n = 34 ; anti-Myc n = 33 ; Myc-WT/WT-HA n = 40 ; Myc-WT/R160/161D-HA n = 30 ; Myc-T17N/WT-HA n = 63 ; Myc-T17N/ R160/161D-HA n = 34 ; Myc-Q61L/WT-HA n = 22 ; Myc-Q61L/R160/161D-HA n = 40). Scale bar = 50 μm. Bar and scatter plots show data points with mean and s.e.m.
Supplementary Figure 3 CYRI proteins oppose Rac1-dependent recruitment of Scar/WAVE complex.
a-b - Western blot of CYRI-B siRNA in COS-7 cells. (a). Densitometry normalized to scrambled (control) siRNA (b). c-d – Western blot of two independent cyri-b knockout CHL-1 lines with independent guide RNA (sequences #1 and #2). (a-d represent n = 3 independent blots). e-g - Immunofluorescence of control (Vector Ctr) or independent CrispR cyri-b knockout CHL-1 cell lines (#1 and #2) on collagen-I for 4h showing WAVE2 (green), nuclei (blue) and F-actin (magenta). Scale bar = 50 μm. Box insets show zoom. Scale bar = 10 μm. (e). The ratio of perimeter positive for WAVE2 staining (yellow dotted line) vs the total perimeter (f) . Cell area (g). One-way ANOVA with Dunn’s post-test. *** p ≤ 0.001. (e-f: Ctr n = 89 cells, #1 n = 109 cells, #2 n = 97 cells – g: Ctr n = 286 cells, #1 n = 257 cells, #2 n = 245 cells). h - Western blot of Scar/WAVE subunits in control or cyri-b CrispR knockout (#1 and #2) CHL-1 cells. i-k - Western blot of siCYRI-B-treated COS-7 cells expressing pLIX-mVenus empty vector (EV) or containing siRNA-resistant CYRI-B (WT or R160/161D). Probed as indicated after 48h (i). densitometry in (j) (n = 3 independent blots). Representative immunofluorescence (k) shown Figure 3 e-f. Scale bar = 50 μm. l-m - Western blot confirming re-expression of siRNA-resistant constructs as in i-k, but with CYRI-B sequence (WT or G2A) and probed as indicated. Two exposures of the same blot for GFP captures varying expression levels (l). densitometry in (m) (n = 3 independent blots). n - Still pictures from time-lapse of WT and cyri KO Ax3 Dictyostelium, expressing GFP-HSPC300 and chemotaxing under agarose. Yellow dotted lines- GFP-HSCP300. Scale bar = 5 μm. See also Supplementary Movie 1. Polar kymograph shows Scar/WAVE complex intensity at cell periphery. o - Western blot of ROSA26::Cre-ERT2+; p16Ink4a-/-; Rac1fl/fl mouse tail fibroblasts after 7d DMSO (control) or OHT to induce Rac1 recombination. Cells transfected with scrambled (control) or Cyri-B siRNA (#1 and #2). p-q - FLIM/FRET of (Vector Ctr) or cyri-b knockout (#1 and #2) CHL-1 cells on collagen expressing Raichu- Rac1 probe. Jet2 colour code shows probe average lifetime 1-4 ns (blue to red) (p). FRET efficiency (q). One-way ANOVA with Dunn’s post-test ** p ≤ 0.01, *** p ≤ 0.001. (Ctr n = 61 cells, #1 n = 63 cells, #2 n = 56 cells). Scale bar = 50 μm. (N = 3 independent assays). For a-a, N = 3 biologically independent experiments. For a, c, h, I,o see also Supplementary Fig. 7. Bar and scatter plots data points, mean and s.e.m. Whisker plots 10-90 percentile, median (bar) and mean (cross).
Supplementary Figure 4 Cells containing CYRI-B inducible vector but treated with the vehicle solution show similar phenotype to the control cells.
a-b - Western blot of COS-7 cells transiently transfected with a mVenus-TetON inducible CYRI-B construct probed with anti-GFP (detects mVenus co-expression). Cells were collected and analysed 48h after vehicle or doxycycline treatment (a). Percentage of overexpression relative to the EV (standardized at 100% - Red dotted line) (b) (n = 3 independent western blot). See also Supplementary Fig. 7. c-f - Immunofluorescence of vehicle-treated negative control (EV) or CYRI-B COS-7 cells, fixed after 4h of spreading and stained for WAVE2 (magenta), nuclei (blue) and GFP (green). Scale bar = 50 μm. Inset panels are a magnified view of the white dashed field. Scale bar = 10 μm (c). WAVE2 ratio and circularity were measured and reported in (d) and (e) respectively. Cell area quantification was based on the phalloidin staining (f). Two-tailed Mann-Whitney test was applied. n.s. p > 0.05. (Veh/EV n = 81 cells ; Veh/CYRI-B n = 78 cells). All data presented are representative of at least 3 biologically independent experiments. Bar and scatter plots show data points with mean and s.e.m.
Supplementary Figure 5 CYRI regulates cell shape and motility and acts as a Meinhardt local inhibitor.
a - Still phase contrast pictures from a random migration assay of control (Vector Ctr) or cyri-b CrispR knockout (#1 and #2) CHL-1 cells plated on collagen-I-coated glass-bottom dishes. Yellow arrowheads denote C-shaped cells. Scale bar = 100 μm (Movie S4). b - Correlation between cell shape and average speed in control (Vector Ctr) or cyri-b CrispR knockout (#1 and #2) CHL-1 cells. Pearson coefficient and R2 value are shown for each condition. (Ctr n = 45 cells, #1 n = 53 cells, #2 n = 42 cells). c - Random migration assay movies were analysed and the percentage of cells presenting a C shape during the time of the experiment is reported. Cochran-Mantel-Haenszel test was performed. ** p ≤ 0.001; *** p ≤ 0.0005. (n = 3 independent assay representing >75 cells). d-e - Western blot analysis of CYRI-B expression in parental (Par.), CrispR control (Vector Ctr) or four independent cyri-b CrispR knockout WM852 melanoma cells (CYRI-B #1-4). Membrane was blotted for CYRI-B and α-Tubulin (d). Bar graph (e) represents quantification of CYRI-B expression relative to the Vector Ctr cell line. (n = 3 independent western blot except for Parental cell line n = 2, see also Supplementary Fig. 7). [Please remove the statistics when n = 2.]. f - Establishment of cyri knockout and CYRI rescued Ax3 D. discoideum cells was confirmed by western blot analysis. Membrane was blotted for D. discoideum CYRI (arrowhead) and MCC1 as a loading control. See also Supplementary Fig. 7. g - Representative phase contrast pictures from an under-agarose chemotaxis assay of Ax3 (WT) and Ax3-derived cell lines migrating towards a folate gradient, as illustrated (Movie S7). Scale bar = 25 μm. h - Cells chemotaxing in a folate under-agarose experiment were tracked automatically using an ImageJ plugin (see material and methods). To monitor the persistence of cell movement, the angle between each step was calculated from x and y positions at each time point and is plotted on the right-hand side graph (Mean and S.D. are shown). One-way ANOVA with Dunn’s post-test was performed. **** p ≤ 0.0001. (WT n = 424 cells, cyri KO n = 581 cells, cyri KO + CYRIWT = 727 cells). i - Pictures extracted from the modelling of CYRI proteins as local inhibitor (Supp. movie 9). In each panel, top graph shows the outline of a simulated cell, with red areas of the periphery having more activator than local inhibitor and blue having more local inhibitor than activator. The x- and y-axes correspond to arbitrary units representing distance. The simulation was run from time t = 0 and panels are taken from times shown (arbitrary units of time). Bottom graph shows the concentrations of the activator (red) and the local inhibitor (blue), where the x-axis represents the scaled arc-length around the perimeter of the cell and the y-axis represents the concentration in arbitrary units. Panel 1 (t = 4) shows the generation of a pseudopod, where the local inhibitor causes the edges of the activated region to sharpen. Panel 2 (t = 8) shows a split in the activator profile (and, consequently, a split in the associated pseudopod), which results from the higher relative concentration of the local inhibitor near the centre of the activated region. Panel 3 (t = 14) shows the cell with two smaller pseudopods, which compete for dominance and determine the direction of migration. Panel 4 (t = 18) shows the winning pseudopod starting the cycle over again and the local inhibitor rising in response to the rise in activator. All data presented are representative of at least 3 biologically independent experiments except if mentioned otherwise. Bar and scatter plots show data points with mean and s.e.m.
Supplementary Figure 6 CYRI-B regulates mitotic angle and Rac1-dependent lumenogenesis.
a-b - Western blot analysis of parental (Par.), shRNA Control (Vector Ctr) or four independent shCYRI-B MDCK cell lines. Membranes were blotted against CYRI-B and α-Tubulin (a). Bar graph represents quantification of CYRI-B expression relative to the control cell line. (b) (n = 3 independent western blot). See also Supplementary Fig. 7. c-e - Immunofluorescence of control (Vector Ctr) or cyri-b knockdown MDCK cysts grown in 3D. Cysts were fixed after 5 days and stained for Pdx1 (green), Tubulin (red) and nuclei (blue). Scale bar = 25 μm (c). Mitotic angle (formed between mitotic spindle axis and apico-basal axis) was quantified in (d). One-way ANOVA with Dunn’s post-test was performed. *** p ≤ 0.001. Angles were divided as greater (normal) or lower (abnormal) than 45 degrees and plotted as a bar graph (e). Two-tailed Chi-square test (95% confidence) was applied for each condition compared with the control cells. *** p ≤ 0.001. (d-e: Ctr n = 43 cysts, #2 n = 64 cysts, #4 n = 53 cysts). f - Number of lumina from control (Vector Ctr) or cyri-b knockdown cysts treated or not with 100 μM NSC23766. One-Way ANOVA with Dunn’s post-test was applied between vehicle-treated knockdown cysts and vehicle-treated control cysts. Unpaired two-tailed t-test was applied to untreated versus treated cysts. n.s. p > 0.05, *** p ≤ 0.001. (Ctr/Veh n = 248 cysts ; Ctr/NSC n = 248 cysts; #2/Veh n = 242 cysts; #2/NSC n = 243 cysts; #4/Veh n = 243 cysts; #4/NSC n = 239 cysts). All data presented are representative of at least 3 biologically independent experiments. Bar and scatter plots show data points with mean and s.e.m. Whisker plots show 10-90 percentile and mean (cross).
Supplementary Figure 7 Unprocessed western blots.
Pictures of individual blots shown throughout this study
Supplementary information
Supplementary Information
Supplementary Figures 1–7, legends for Supplementary Videos 1–9 and Supplementary Tables 1–6, and Supplementary Notes 1 and 2
Supplementary Video 1
cyri KO Ax3 cells show a broader recruitment of the Scar–WAVE complex.Under-agarose chemotaxis assay toward folate of wild type or cyri KO Ax3 cells transfected with HSPC300-GFP. 1 frame per 6 sec. Scale bar, 10 μm. Experiment was repeated independently 3 times with similar results
Supplementary Video 2
Automatic segmentation of the cell edge Segmentation of CRISPR control (Vector Ctr) or cyri-b knockout (cyri-b #1) CHL-1 cells, transfected with GFP-LifeAct. Cell periphery is shown in magenta.1 frame per sec. Scale bar, 25 μm
Supplementary Video 3
cyri-b KO MEFs show a defect in the restriction of Rac1-induced protrusions.Time-lapse movie of control (DMSO - top) and CYRI-B -/- (OHT - bottom) MEF cells, transfected with the photo-activable mCherry Rac1 probe. Phase and mCherry channels are shown as a merge. Probe is locally photo-activated (blue circle area) after 1 min (top left label switches for “DARK” to “458 23nm”). 1 frame/2 sec. Scale bar, 25 μm. Experiment was repeated independently 4 times with similar results
Supplementary Video 4
Loss of CYRI-B promotes a more migratory phenotype in CHL-1 cells. Random migration assay of control (Vector Ctr) or cyri-b CRISPR knockout (#1 and #2) CHL-1 cells. Movies are representative of an 8h timelapse experiment. Scale bar, 100 μm. 1 frame per 10 min. Experiment was repeated independently 3 times with similar results
Supplementary Video 5
cyri-b KO WM852 cells lose their ability to chemotax toward FBS. Chemotaxis assay using control (Vector Ctr), or cyri-b CRISPR knockout (#1 and #2) WM852 cells. Cells are migrating from a serum-free environment (Left side of each movie) to a 10% FBS supplemented media (right side of each movie). Tracks are displayed for this movie. Scale bar, 50 μm. 1 frame per 15 min. Experiments was repeated independently 4 times with similar results
Supplementary Video 6
Expression of CYRIWT but not the Rac1-binding defective mutant rescues the shape phenotype associated with the loss of CYRI in D. discoideum DIC movie from an under agarose chemotaxis assay of WT, cyri KO, or the REMI rescue strains (cyri KO+ CYRIWT and cyri KO + CYRIR155/156D) migrating toward folate. 1 frame per 2 sec. Scale bar, 25 μm. Experiment was repeated independently 3 times with similar results
Supplementary Video 7
Expression of CYRIWT but not the Rac1-binding defective mutant rescues the migratory phenotype associated with the loss of CYRI in D. discoideum Phase contrast movie from an under agarose chemotaxis assay toward folate. Scale bar, 100 μm. 1 frame per 30 sec. Experiment was repeated independently 3 times with similar results
Supplementary Video 8
cyri KO Ax3 cells have a defect to reorientate their pseudopod toward a sharp gradient of cAMP. Needle assay using WT or cyri knockout Ax3 cells, responding to cAMP. 1 frame per 5 sec. Scale bar, 20 μm. Experiment was repeated independently 4 times with similar results
Supplementary Video 9
Mathematical model of a cell protrusion and motility. Cell protrusions are generated following an increase of an activator (Red: Rac1 or Scar–WAVE complex), cross-talking to a local inhibitor (Blue: CYRI) that restricts pseudopod width. Accumulation of the local inhibitor causes pseudopod spitting. See text for more details
Supplementary Table 1
Gene and protein nomenclature Gene and protein nomenclature based on HGNC guidelines
Supplementary Table 2
Significant candidates obtained from NAP1 pull down after reversible crosslink treatment in D. discoideum. Uniprot accession numbers are listed for each putative interactor. Welch’s t test and further analysis were performed using MaxQuant software
Supplementary Table 3
List of Antibodies Information regarding provider, catalogue number and working dilution of antibodies and fluorescent probes/stains used in this study
Supplementary Table 4
List of constructs Information regarding the plasmids used in this study
Supplementary Table 5
List of siRNA, shRNA, gRNA and primer sequences corresponding to the oligos used in this study
Supplementary Table 6
Statistics Source Data. Individual data points presented in this study
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Fort, L., Batista, J.M., Thomason, P.A. et al. Fam49/CYRI interacts with Rac1 and locally suppresses protrusions. Nat Cell Biol 20, 1159–1171 (2018). https://doi.org/10.1038/s41556-018-0198-9
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DOI: https://doi.org/10.1038/s41556-018-0198-9
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