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Endoplasmic reticulum chaperone Gp96 controls actomyosin dynamics and protects against pore‐forming toxins

2016; Springer Nature; Volume: 18; Issue: 2 Linguagem: Inglês

10.15252/embr.201642833

1469-3178

Francisco S. Mesquita, Cláudia Brito, María J. Mazón, Jorge Pinheiro, Serge Mostowy, Didier Cabanes, Sandra Sousa,

Microbial Inactivation Methods

Article30 December 2016free access Transparent process Endoplasmic reticulum chaperone Gp96 controls actomyosin dynamics and protects against pore-forming toxins Francisco Sarmento Mesquita Francisco Sarmento Mesquita I3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal Group of Molecular Microbiology, IBMC, Universidade do Porto, Porto, Portugal Search for more papers by this author Cláudia Brito Cláudia Brito I3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal Group of Molecular Microbiology, IBMC, Universidade do Porto, Porto, Portugal Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Porto, Portugal Search for more papers by this author Maria J Mazon Moya Maria J Mazon Moya Section of Microbiology, MRC Centre for Molecular Bacteriology and Infection (CMBI), Imperial College London, London, UK Search for more papers by this author Jorge Campos Pinheiro Jorge Campos Pinheiro I3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal Group of Molecular Microbiology, IBMC, Universidade do Porto, Porto, Portugal Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Porto, Portugal Search for more papers by this author Serge Mostowy Serge Mostowy orcid.org/0000-0002-7286-6503 Section of Microbiology, MRC Centre for Molecular Bacteriology and Infection (CMBI), Imperial College London, London, UK Search for more papers by this author Didier Cabanes Corresponding Author Didier Cabanes [email protected] I3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal Group of Molecular Microbiology, IBMC, Universidade do Porto, Porto, Portugal Search for more papers by this author Sandra Sousa Corresponding Author Sandra Sousa [email protected] orcid.org/0000-0001-8578-0461 I3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal Group of Molecular Microbiology, IBMC, Universidade do Porto, Porto, Portugal Search for more papers by this author Francisco Sarmento Mesquita Francisco Sarmento Mesquita I3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal Group of Molecular Microbiology, IBMC, Universidade do Porto, Porto, Portugal Search for more papers by this author Cláudia Brito Cláudia Brito I3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal Group of Molecular Microbiology, IBMC, Universidade do Porto, Porto, Portugal Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Porto, Portugal Search for more papers by this author Maria J Mazon Moya Maria J Mazon Moya Section of Microbiology, MRC Centre for Molecular Bacteriology and Infection (CMBI), Imperial College London, London, UK Search for more papers by this author Jorge Campos Pinheiro Jorge Campos Pinheiro I3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal Group of Molecular Microbiology, IBMC, Universidade do Porto, Porto, Portugal Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Porto, Portugal Search for more papers by this author Serge Mostowy Serge Mostowy orcid.org/0000-0002-7286-6503 Section of Microbiology, MRC Centre for Molecular Bacteriology and Infection (CMBI), Imperial College London, London, UK Search for more papers by this author Didier Cabanes Corresponding Author Didier Cabanes [email protected] I3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal Group of Molecular Microbiology, IBMC, Universidade do Porto, Porto, Portugal Search for more papers by this author Sandra Sousa Corresponding Author Sandra Sousa [email protected] orcid.org/0000-0001-8578-0461 I3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal Group of Molecular Microbiology, IBMC, Universidade do Porto, Porto, Portugal Search for more papers by this author Author Information Francisco Sarmento Mesquita1,2, Cláudia Brito1,2,3, Maria J Mazon Moya4, Jorge Campos Pinheiro1,2,3, Serge Mostowy4, Didier Cabanes *,1,2 and Sandra Sousa *,1,2 1I3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal 2Group of Molecular Microbiology, IBMC, Universidade do Porto, Porto, Portugal 3Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Porto, Portugal 4Section of Microbiology, MRC Centre for Molecular Bacteriology and Infection (CMBI), Imperial College London, London, UK *Corresponding author. Tel: +351 220 408 800; E-mail: [email protected] *Corresponding author. Tel: +351 220 408 800; E-mail: [email protected] EMBO Reports (2017)18:303-318https://doi.org/10.15252/embr.201642833 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract During infection, plasma membrane (PM) blebs protect host cells against bacterial pore-forming toxins (PFTs), but were also proposed to promote pathogen dissemination. However, the details and impact of blebbing regulation during infection remained unclear. Here, we identify the endoplasmic reticulum chaperone Gp96 as a novel regulator of PFT-induced blebbing. Gp96 interacts with non-muscle myosin heavy chain IIA (NMHCIIA) and controls its activity and remodelling, which is required for appropriate coordination of bleb formation and retraction. This mechanism involves NMHCIIA–Gp96 interaction and their recruitment to PM blebs and strongly resembles retraction of uropod-like structures from polarized migrating cells, a process that also promotes NMHCIIA–Gp96 association. Consistently, Gp96 and NMHCIIA not only protect the PM integrity from listeriolysin O (LLO) during infection by Listeria monocytogenes but also affect cytoskeletal organization and cell migration. Finally, we validate the association between Gp96 and NMHCIIA in vivo and show that Gp96 is required to protect hosts from LLO-dependent killing. Synopsis Gp96 plays a role in actomyosin remodelling and coordination of plasma membrane blebbing triggered by pore-forming toxins. This process protects membrane integrity and host survival during infection and has implications in cytoskeletal organization. Gp96 controls NMHCIIA network and NMII activity during PFT-induced PM blebbing. Gp96 and NMHCIIA protect PM integrity against bacterial PFT-mediated damage. Gp96–NMHCIIA interplay during PFT responses resembles polarized cell migration process. Gp96 promotes host survival during in vivo Listeria infection. Introduction Plasma membrane (PM) blebs are dynamic cell protrusions, which depend on non-muscle myosin II (NMII) activity and have been associated with multiple processes such as apoptosis, cytokinesis and cell migration 12. Regarding bacterial infections, blebs preserve PM integrity upon damage caused by bacterial pore-forming toxins (PFTs) 3; allow the establishment of an intracellular replicative niche for Pseudomonas aeruginosa 4; and are released by infected macrophages promoting Mycobacterium tuberculosis killing by neighbouring macrophages (efferocytosis) 5 or favouring cell-to-cell spreading of Listeria monocytogenes 6. Listeria monocytogenes (Lm) is a facultative intracellular human foodborne pathogen that causes severe infection in susceptible hosts. Virulence mainly depends on the activity of its secreted PFT, the cholesterol-dependent cytolysin listeriolysin O (LLO) 78. LLO targets phagosomal membranes allowing escape of Lm to the cytosol and has numerous other roles during infection 7. In particular, LLO promotes pathogen dissemination by inducing controlled necrosis 9 and release of bacterial-containing blebs 6. Endoplasmic reticulum (ER) stress pathways are central for host survival against PFTs, including LLO, and ER distribution is often altered during intoxication 1011. Concurrently, we have shown that Lm infection redistributes the ER chaperone Gp96 to the PM through an uncharacterized mechanism 12. Gp96 is an ER-resident HSP90 paralogue, which controls the expression and folding of proteins assigned to the secretory pathway and has crucial roles in cellular homoeostasis, host development and immunity 131415. Gp96 translocates to the PM where it interacts with different bacteria, modulating adherence, internalization 161718, survival 19 and endothelial permeability 20. Cascades involved in these processes may rely on calcium signalling, protein kinase C (PKC) activation and nitric oxide production. However, the molecular outcomes underlying such events remain poorly defined. Given that PFTs alter ER distribution, we evaluated whether LLO affects Gp96 distribution and function, thereby modulating host responses to Lm. We found that LLO triggers the interaction between Gp96 and NMHCIIA and the concomitant assembly of unique NMHCIIA cortical bundles, which coordinate the formation and retraction of PM blebs and preserve PM integrity during Lm cellular infection. Strikingly, this process resembles the formation of uropod-like structures required for tail retraction during polarized cell migration 2122 and is controlled by Gp96 that regulates NMII activity and general cytoskeleton-driven cell properties. Importantly, Gp96 interacts with NMHCIIA during in vivo Lm infection and promotes host survival. Our data establish a novel ER–cytoskeletal interplay crucial for host protection against PFT-mediated bacterial infection. Results Listeria monocytogenes causes LLO-dependent ER redistribution and promotes the interaction between Gp96 and NMHCIIA Given that Lm infection leads to increased levels of PM-associated Gp96 and to ER expansion and that different PFTs were reported to alter ER distribution, we studied the effect of LLO on ER morphology and Gp96 surface levels. HeLa cells infected with wild-type (wt) Lm, but not with LLO-deficient bacteria (Δhly), displayed increased Gp96 PM levels and contained distinct ER vacuoles/structures harbouring proteins with ER retention signal (KDEL), including Gp96 (Fig EV1A and B). Purified LLO was sufficient to induce a dose-dependent ER-Gp96 redistribution with ER vacuoles/structures expanding close to the PM (Fig EV1C–E). Concurrently, surface-exposed Gp96 was also increased upon LLO treatment (Fig EV1F). Click here to expand this figure. Figure EV1. Listeria monocytogenes infection induces LLO-dependent ER redistribution and NMHCIIA–Gp96 interaction Flow cytometry analysis of surface-exposed Gp96 levels in HeLa cells left uninfected (U) or infected with wt or ∆hly Lm for 1 h. Values are mean ± SEM (n ≥ 3), and P-values were calculated using one-way ANOVA with Tukey's post hoc analyses, **P < 0.01 Confocal microscopy images of HeLa cells left uninfected or infected with wt or ∆hly Lm for 1 h, fixed and immunolabelled for ER-KDEL (green) and Gp96 (red) and stained with DAPI (blue). Insets show ER-Gp96 vacuoles. Arrows indicate ER-KDEL vacuoles. Scale bars, 10 μm. Confocal microscopy images of HeLa cells left untreated or treated with the indicated concentrations of LLO for 15 min, fixed and immunolabelled for ER-KDEL (green) and stained with DAPI. Arrows indicate ER-KDEL vacuoles. Scale bars, 10 μm. Confocal microscopy images of HeLa cells left untreated or treated with 0.5 nM LLO for 15 min and immunolabelled for Gp96 (red) and ER-KDEL (green). Arrows indicate ER-Gp96 vacuoles shown enlarged in the insets 1 and 2. Scale bars, 10 μm. TEM images of HeLa cells left untreated or treated with 0.5 nM LLO for 15 min. Arrowheads indicate normal ER cisternae, and arrows show ER vacuoles at the proximity of PM. Flow cytometry analysis of surface-exposed Gp96 levels in HeLa cells left untreated (U) or treated with 0.5 nM LLO for 15 min. Values are mean ± SEM (n ≥ 3), and P-values were calculated using two-tailed unpaired Student's t-test, *P < 0.05 Amino acid sequence of the human NMHCIIA with functional domains indicated. Peptide sequences recovered from MS/MS are highlighted in red. Download figure Download PowerPoint We tested whether the LLO-mediated ER redistribution could alter Gp96 interacting partners. Gp96 immunoprecipitation (IP) fractions from untreated or LLO-treated HeLa cells were resolved by SDS–PAGE. A specific band from LLO-treated sample was identified, by tandem mass spectrometry (MS) analysis, as non-muscle myosin heavy chain IIA or NMHCIIA (P < 0.05). Peptide coverage spanned 23% of the NMHCIIA sequence, covering all functional domains (Fig EV1G). Immunoblot analysis of Gp96 IP confirmed that LLO triggers NMHCIIA–Gp96 co-IP in a dose-responsive manner (Fig 1A), yet the interaction decreased upon high concentrations of LLO possibly due to cytotoxicity. LLO increased NMHCIIA–Gp96 co-IP also during Lm infection (Figs 1B and EV2A–D). The specificity of the IPs was confirmed using isotype antibodies (Fig EV2A and B). Moreover, the NMHCIIA–Gp96 interaction was amplified throughout the infection time (Fig 1C) and could also be detected in colon epithelial (Caco-2) and macrophage-like (RAW264.7) cells (Fig EV2C and D), showing that Lm infection promotes LLO-dependent NMHCIIA–Gp96 co-IP in various cell types. Figure 1. Listeria monocytogenes causes LLO-dependent ER and NMHCIIA redistribution and promotes the interaction between Gp96 and NMHCIIA A–C. Immunoblots of Gp96 and NMHCIIA levels from whole-cell lysates (WCL) and Gp96 IP fractions (IP Gp96) of HeLa cells: (A) left untreated or treated with increasing concentrations of LLO for 15 min; (B) left uninfected (U) or infected with wt or Δhly Lm for 1 h; or (C) infected with wt Lm for the indicated time points. (A, C) Quantifications of NMHCIIA in IP Gp96 are the mean ± SEM (n ≥ 3) (a.u., arbitrary units). D. Confocal microscopy images of HeLa cells left untreated or treated with LLO (0.5 nM, 15 min), immunolabelled for NMHCIIA (green) and Gp96 (red) and stained with DAPI (blue). Arrows point to various NMHCIIA–Gp96-positive cortical bundles in different cells. Insets show high-magnification image. E. Quantification of Pearson's correlation coefficient (PCC) within uninfected cells (U), LLO-treated cells, cortical NMHCIIA bundles or equivalent-size control cellular ROI (Ctrl ROI). Data are mean ± SEM (n = 6); P-values were calculated using one-way ANOVA with Tukey's post hoc analyses, *P < 0.05, **P < 0.01, ***P < 0.001. F. Confocal microscopy images of HeLa cells infected with wt or Δhly Lm for 6 h, immunolabelled for NMHCIIA (green), Gp96 (red) and Lm (blue) and stained with DAPI (white). Arrows indicate NMHCIIA–Gp96-positive cortical bundles at cortical sites close to wt Lm. Data information: Scale bars, 10 μm. See also Figs EV1 and EV2. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. NMHCIIA–Gp96 interaction and association at cortical structures in HeLa, Caco-2 and RAW 264.7 cells infected with Listeria monocytogenes or treated with LLO A–D. Immunoblots of NMHCIIA and Gp96 levels from whole-cell lysates (WCL) and (A) NMHCIIA IP or (B–D) Gp96 IP fractions from (A, B) HeLa cells, (C) Caco-2 cells or (D) RAW264.7 macrophages left uninfected (U) or infected with wt Lm for 1 h, probed with anti-Gp96 and anti-NMHCIIA antibodies. (A, B) Control IP was carried out using unspecific isotype antibodies (IgGiso). E. Confocal microscopy images of Caco-2 cells left untreated or treated with 0.5 nM LLO for 15 min, immunolabelled for NMHCIIA (green) and Gp96 (red) and stained with DAPI (blue). Arrows indicate Gp96-positive NMHCIIA cortical structures. Scale bars, 10 μm. Download figure Download PowerPoint Analysis of NMHCIIA and Gp96 cellular distribution revealed that, besides NMHCIIA–Gp96 co-IP, LLO induced the formation of distinct NMHCIIA cortical clusters or bundles which associated with cortical Gp96 in HeLa (Fig 1D) and Caco-2 (Fig EV2E). Quantification of NMHCIIA–Gp96 co-localization confirmed that both proteins co-localized within cortical bundles when compared to other cellular regions (control region of interest, ROI) or to the overall NMHCIIA–Gp96 correlation in the entire cell in both untreated and LLO-treated cells (Fig 1E). Infection with wt but not Δhly Lm promoted the formation of similar cortical structures, often associated with intracellular bacteria (Fig 1F). Thus, through LLO, Lm redistributes the ER, Gp96 and NMHCIIA into unique cortical bundles and triggers novel association between the ER and the host cytoskeleton. NMHCIIA-ER/Gp96 bundles are hallmarks of PFT-induced blebbing NMII activity is required for PFT-induced PM blebbing 3. We thus analysed the dynamics of LLO-induced NMHCIIA remodelling in HeLa cells ectopically expressing GFPNMHCIIA and/or mcherryKDEL (mcherry fused to KDEL signature). LLO induced a profound reorganization of the NMHCIIA network into cortical bundles at sites of PM blebbing (Fig 2A and Movie EV1). Cortical ER structures and vesicles associated with NMHCIIA bundles and localized within PM blebs (Fig 2B and Movie EV2). PM labelling with fluorescently conjugated wheat germ agglutinin (FITCWGA) showed NMHCIIA bundles at PM blebs, which occasionally appeared detached from the cell body (Fig 2C). Phosphorylated ezrin (p-ezrin), which connects the PM to cortical actin and is recruited to PM blebs 23, was present at NMHCIIA bundles (Appendix Fig S1A), further validating the link between bundles and PM blebs. Figure 2. NMHCIIA–Gp96 bundles are hallmarks of PFT-induced PM blebbing A, B. Sequential frames of time-lapse confocal microscopy sequence of LLO-treated HeLa cells expressing (A) GFPNMHCIIA or (B) simultaneously GFPNMHCIIA and mcherryKDEL. LLO was added to culture medium 10 s before t0. DIC, differential interference contrast. (A) Arrows indicate NMHCIIA bundles at PM blebbing sites. (B) Highlights depicting ER structures within NMHCIIA bundles and PM blebs. Arrows indicate cortical ER surrounding NMHCIIA accumulations, arrowheads indicate contact points between ER vesicles and NMHCIIA cables, and asterisks point to ER vacuoles within PM blebs. C. Confocal microscopy images of HeLa cells left untreated (control) or treated with LLO (0.5 nM, 15 min). Cells were stained with FITCWGA (plasma membrane, PM;red) and immunolabelled for NMHCIIA (green). Insets show PM blebs and arrows indicate recruitment of NMHCIIA bundles to PM blebs associated (1) or detached (2) from the cell body. D–F. Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated (U) or treated with LLO (0.5 nM, 15 min) (LLO) and (D) LLO pre-incubated with cholesterol (LLOCHT); (E) LLO in medium supplemented with 140 mM K+ (HighK+) and LLO in Ca2+-free medium (Ca2+free); (F) LLO in the presence of 25 μM blebbistatin (BB). G. Confocal microscopy images of HeLa cells treated as indicated and immunolabelled for ER-KDEL (blue), NMHCIIA (green) and Gp96 (red). Arrows indicate NMHCIIA bundles. H. Confocal microscopy images of HeLa cells treated with aerolysin and immunolabelled for NMHCIIA (green) and Gp96 (red). Insets show NMHCIIA bundles and arrows indicate association between Gp96 and NMHCIIA. I. Immunoblots of NMHCIIA and Gp96 levels from Gp96 IP of HeLa cells left untreated (U) or treated with LLO (0.5 nM, 15 min), SLO (1.5 μg/ml, 30 min) (SLO) or aerolysin (0.2 nM, 40 min) (AL). Data information: All scale bars are 10 μm. See also Appendix Fig S1. Download figure Download PowerPoint Pore-forming toxins-induced PM blebbing depends on Ca2+ influx from the extracellular milieu caused by PM damage 3. Accordingly, treatment of HeLa cells with LLO pre-incubated with cholesterol, which blocks its pore-forming activity but allows cell surface binding and signalling 24, did not enhance NMHCIIA–Gp96 interaction or affect their cellular distribution (Fig 2D and G). LLO treatment carried out in Ca2+-depleted medium disrupted normal NMHCIIA-ER/Gp96 distribution but failed to trigger formation of distinct NMHCIIA bundles and reduced NMHCIIA–Gp96 co-IP (Fig 2E and G). In contrast, increasing the extracellular concentration of K+, which prevents the K+ efflux responsible for numerous host responses to PFTs 25, did not affect NMHCIIA bundle formation or NMHCIIA–Gp96 co-IP (Fig 2E and G). Using blebbistatin, which inhibits the NMII contractile force required for PM blebbing 26, we showed that NMII activity is also required for both NMHCIIA–Gp96 co-IP and NMHCIIA bundle formation (Fig 2F and G). We then tested other PFTs inducing PM blebbing and showed that aerolysin (AL) from Aeromonas hydrophila induced formation of NMHCIIA–Gp96 cortical bundles, and AL and streptolysin O (SLO) from Streptococcus pneumonia promoted NMHCIIA–Gp96 co-IP (Fig 2H and I). Finally, in accordance with the transient nature of PM blebbing, host cells recovered normal NMHCIIA and ER distribution by 8 h after LLO washout (Appendix Fig S1B–D). NMHCIIA–Gp96 association and cortical bundling appear thus as hallmarks of PFT-induced PM blebbing that rely on NMII activity and Ca2+ influx upon PFT-induced PM damage. Gp96 and NMHCIIA regulate PM blebbing through modulation of NMII activity Next, we evaluated the frequency of cells harbouring NMHCIIA bundles following incubation with LLO, AL or Lm infection of HeLa cells expressing control oligonucleotides (shControl) or oligonucleotides targeting the expression of Gp96 or NMHCIIA (shGp96 and shNMHCIIA) (Appendix Fig S2). For all conditions, Gp96 depletion significantly reduced the frequency of cells with NMHCIIA bundles when compared to controls (Fig 3A–D). Such effect was observed in response to different LLO concentrations (Fig EV3A). LLO-treated shGp96 cells displayed less bundles per cell (Fig EV3B) and showed a disrupted cortical actomyosin network, which appeared unable to bundle. Expectedly, actin co-localized with NMHCIIA bundles (Fig EV3C) and could also hallmark PFT-induced cortical cytoskeletal bundles. Similar to blebbistatin treatment, depletion of NMHCIIA impaired bundling (Figs 3A–C and EV3C). Figure 3. Gp96 controls NMHCIIA remodelling and PM blebbing in response to PFTs A. Confocal microscopy images of shCtrl, shGp96 or shNMHCIIA HeLa cells treated with LLO (0.5 nM, 15 min) and immunolabelled for KDEL proteins (blue), NMHCIIA (green) and Gp96 (red). Insets show compact NMHCIIA bundles in shCtrl cells and dispersed NMHCIIA bundles in shGp96 cells. Scale bar, 10 μm. B–D. Quantification of the percentage of cells harbouring NMHCIIA bundles after incubation with (B) LLO (0.5 nM, 15 min), (C) aerolysin (0.2 nM, 40 min) or (D) wt Listeria monocytogenes. Values are the mean ± SEM (n ≥ 3); P-values were calculated using (B, C) one-way ANOVA with Dunnett's post hoc analyses and (D) two-tailed unpaired Student's t-test, *P < 0.5, ***P < 0.001. For shNMHCIIA, bundles were detected following actin staining (Fig EV3C). E, F. Number of (E) blebs per cell or (F) retracting blebs per cell evaluated by time-lapse microscopy analysis of LLO-treated shCtrl or shGp96 cells. shCtrl n = 32 cells and shGp96 n = 40 cells; P-values were calculated using two-tailed unpaired Student's t-test, *P < 0.5. G. Sequential frames of time-lapse microscopy analysis of LLO-treated HeLa cells expressing GFPNMHCIIA [shCtrl, shGp96 and shCtrl with 25 μM blebbistatin (shCtrl-BB)]. LLO was added to culture medium 10 s before t0. Scale bar, 10 μm. H. Immunoblots of Ser19-phosphorylated MRLC (pMRLC), MRLC and actin levels from shCtrl or shGp96 cells left untreated (U) or treated with 0.1 nM LLO for the indicated time points. Quantification of pMRLC levels corresponds to the mean ± SEM (n ≥ 3); P-values were calculated using one-way ANOVA with Tukey's post hoc analyses, **P < 0.01, ***P < 0.001. Data information: See also Appendix Fig S2 and Fig EV3. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Gp96 and NMHCIIA control actomyosin remodelling during LLO treatment Quantification of the percentage of shControl and shGp96 HeLa cells harbouring NMHCIIA bundles in response to increasing concentrations of LLO. Quantification of the percentage of shCtrl and shGp96 HeLa cells harbouring at least 1–5 or > 5 NMHCIIA bundles per cell in response to 0.5 nM LLO (15 min). Confocal images of shCtrl, shGp96 or shNMHCIIA HeLa cells treated with 0.5 nM LLO for 15 min and immunolabelled for NMHCIIA (green) and stained with phalloidin (actin, red) and DAPI (blue). Arrow indicates compact actomyosin cortical bundle. Scale bars, 10 μm. Data information: For all quantifications, values are mean ± SEM (n ≥ 3) and P-values were calculated using one-way ANOVA with Tukey's post hoc analyses, *P < 0.05, **P < 0.01. Download figure Download PowerPoint Subsequently, we studied GFPNMHCIIA dynamics during LLO intoxication in control or Gp96-depleted cells. Upon LLO treatment, shControl cells assembled NMHCIIA bundles and displayed organized PM blebbing with PM blebs expanding and retracting (Fig 3G and Movie EV3). In contrast, shGp96 cells failed to stabilize their NMHCIIA network into cortical bundles and displayed uncontrolled PM blebbing, with more blebs per cell and less bleb retraction throughout the experiment (Fig 3E–G and Movie EV4). Blebbistatin inhibited NMHCIIA rearrangements, causing disruption of cell morphology during LLO treatment (Fig 3G and Movie EV5). These cells showed passive blebbing with limited expansion, which agrees with the requirement of NMII activity for PM blebbing 1323. NMII activity and actomyosin filaments turnover are regulated by phosphorylation of the myosin II regulatory light chain (MRLC) 2728. In resting conditions, when compared to shControl, shGp96 cells showed higher levels of MRLC phosphorylation (pMRLC) (Fig 3H). Moreover, whereas MRLC was rapidly dephosphorylated in shControl cells upon LLO treatment, its phosphorylation was sustained longer in shGp96 cells (Fig 3H). These data demonstrate that Gp96 modulates NMHCIIA activity and dynamics required for actomyosin network remodelling during PM blebbing. Coordination of PM blebbing resembles control of cell polarity and trailing edge retraction during polarized cell migration To our knowledge, ER–cytoskeletal interactions during PM blebbing have never been described. However, the ER was associated with the assembly of trailing edge uropod-like Wnt-receptor–actin–myosin polarity (WRAMP) structures during polarized migration 22. We thus surveyed PFT-induced NMHCIIA bundles for proteins found in WRAMPs. Our co-localization analysis demonstrated that NMHCIIA co-localized with WRAMP markers such as NMHCIIB, lysosomal-associated glycoprotein 1 (LAMP1), tubulin, ERK and filamin A within the cortical cytoskeletal bundles. In addition, actin and calpain 2 also co-localized at cortical bundles (Fig 4A). Notably, filamin A, an actin-crosslinking protein required for uropod retraction 29, also co-immunoprecipitated with Gp96 upon LLO treatment (Fig 4B). Moreover, promoting WRAMP/uropod assembly with Wnt5a 22 induced NMHCIIA–Gp96 co-IP and polarized distribution of NMHCIIA-ER/Gp96 (Fig 4C and D). Figure 4. NMHCIIA bundles resemble tail-retraction structures and Gp96 regulates cytoskeleton remodelling A. Confocal microscopy images (i) of LLO-treated HeLa cells immunolabelled for the indicated proteins. Insets show co-localization between NMHCIIA and WRAMP components at cortical bundles. (ii) Quantification of Pearson's correlation coefficient (PCC) between NMHCIIA and the indicated proteins within entire LLO-treated cells, cortical bundles or equivalent-size control cellular ROI (Ctrl ROI). For calpain 2, cortical bundles were defined by actin staining. Data are representative of one experiment repeated three independent times with similar results. The boxes extends from the 25th to 75th percentiles. The whiskers are min to max and the horizontal line is plotted at the median. B. Immunoblots of NMHCIIA, Gp96 and filamin A levels from WCL and Gp96 IP of HeLa cells left untreated (U) or treated with LLO (0.5 nM, 15 min) (LLO). C. Immunoblots of NMHCIIA and Gp96 levels from WCL and Gp96 IP of HeLa cells left untreated (U) or treated with 25 μg/ml of Wnt5a for the indicated time. D. Confocal microscopy images of HeLa cells untreated (Mock) or treated with 25 μg/ml of Wnt5a for 30 min. Cells were immunolabelled for ER-KDEL (blue), Gp96 (red) and NMHCIIA (green). Arrows indicate polarized localization of NMHCIIA, ER-KDEL and Gp96 in Wnt5a-treated cells. E, F. Confocal microscopy images of shCtrl, shGp96 or shNMHCIIA HeLa cells stained for actin (red) and DAPI (blue) and immunolabelled for (E) NMHCIIA (green) or (F) focal adhesion kinase (FAK) (green). Insets in (F) show sites with focal adhesion points indicated by arrows. G, H. Quantification of the percentage of cells with (G) stress fibres or (H) focal adhesion points labelled by FAK. I, J. Wound closure assay. (I) Sequential frames of time-lapse microscopy of HeLa cells grown to confluence separated by a stopper. Stopper was removed at t0 and cellular migration was imaged for the indicated times. (J) Quantification of the percentage of wound area occupied by migrating cells over time, stopper removal (upper panel), and respective rate of closure (lower panel). Data information: Scale bars, 10 μm. Data in (G, H and J) are the mean ± SEM (n ≥ 3). P-values were calculated using one-way ANOVA with Tukey's post hoc analyses (A) or Dunnett's post hoc analyses (G, H and J), *P < 0.05, **P < 0.01, ***P < 0.001, ns, non-significant. See also Fig EV4. Download figur