Cells Respond to Mechanical Stress by Rapid Disassembly of Caveolae Bidisha Sinha,1,2,14 Darius Koster,1,2,14 �� Richard Ruez,3,4 Pauline Gonnord,3,4 Michele Bastiani,8,9 Daniel Abankwa,8,9 Radu V. Stan,10 Gillian Butler-Browne,11 Benoit Vedie,12 Ludger Johannes,3,4 Nobuhiro Morone,13 Robert G. Parton,8,9 Grac��a Raposo,3,5,6 Pierre Sens,7 Christophe Lamaze,3,4,15,* and Pierre Nassoy1,2,15,* 1Universite �� P. et M. Curie/CNRS UMR168 2Institut Curie, Centre de Recherche, Laboratoire Physico-Chimie 3CNRS UMR144 4Institut Curie, Centre de Recherche, Laboratoire Trafic, Signalisation et Ciblage Intracellulaires 5PICT IBiSA Institut Curie 6Centre de Recherche, Laboratoire Structure et Compartiments Membranaires, Institut Curie 26 rue d���Ulm, 75248 Paris Cedex 05, France 7ESPCI, CNRS-UMR 7083, Physico-Chimie Theorique, �� 10 rue Vauquelin, 75231 Paris Cedex 05, France 8The University of Queensland, Institute for Molecular Bioscience 9Center for Microscopy and Microanalysis Brisbane, Queensland 4072, Australia 10Dartmouth Medical School, Borwell 502W, HB7600, One Medical Center Drive, 03756 Lebanon, NH, USA 11Institut de Myologie, Hopital �� Pitie-Salpetriere, �� �� ` UM76 UPMC, U974 Inserm, UMR7215, CNRS-AIM, 47, bld de l���hopital, �� 75651 Paris Cedex 13, France 12Laboratoire de Biochimie, Hopital �� Europeen �� Georges Pompidou, 20 rue Leblanc, 75015 Paris, France 13National Center of Neurology and Psychiatry, National Institute of Neuroscience, Department of Ultrastructural Research, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan 14These authors contributed equally to this work 15These authors contributed equally to this work *Correspondence: christophe.lamaze@curie.fr (C.L.), pierre.nassoy@curie.fr (P.N.) DOI 10.1016/j.cell.2010.12.031 SUMMARY The functions of caveolae, the characteristic plasma membrane invaginations, remain debated. Their abundance in cells experiencing mechanical stress led us to investigate their role in membrane-mediated mechanical response. Acute mechanical stress induced by osmotic swelling or by uniaxial stretching results in a rapid disappearance of caveolae, in a reduced caveolin/Cavin1 interaction, and in an increase of free caveolins at the plasma membrane. Tether-pulling force measurements in cells and in plasmamembranespheresdemonstratethatcaveola flattening and disassembly is the primary actin- and ATP-independent cell response that buffers mem- brane tension surges during mechanical stress. Conversely, stress release leads to complete caveola reassembly in an actin- and ATP-dependent process. The absence of a functional caveola reservoir in myo- tubes from muscular dystrophic patients enhanced membrane fragility under mechanical stress. Our findings support a new role for caveolae as a physio- logical membrane reservoir that quickly accommo- dates sudden and acute mechanical stresses. INTRODUCTION Caveolae were first described in the early 1950s through the seminal electron microscopy studies of Palade and Yamada (Palade, 1953 Yamada, 1955). These characteristic 60���80 nm cup-shaped uncoated invaginations are highly enriched in cholesterol and sphingolipids (Richter et al., 2008). Present at the plasma membrane of many cells with the exception of neurons and lymphocytes, they are particularly abundant in muscle cells, adipocytes, and endothelial cells. The identification of caveolin-1 (Cav1) (Rothberg et al., 1992 Kurzchalia et al., 1992) and caveolin-2 (Scherer et al., 1996) as the main constitu- ents of the caveolar structure was instrumental to gain insight into the cell biology, structural, and genetic features of caveolae (Stan, 2005). They have been associated with endocytosis, cell signaling, lipid metabolism, and other functions in physiological as well as in pathological conditions. Nevertheless, the role of these specialized membrane domains remains debated, and little is known about the molecular mechanisms involved in their formation and proposed functions (Parton and Simons, 2007). Recent studies have suggested that the distribution of Cav1 and caveolae-mediated signaling can be affected by external mechanical cues. In endothelial cells, chronic shear exposure activates the ERK pathway in a caveolae-dependent manner (Boyd et al., 2003 Park et al., 2000 Rizzo et al., 2003). In smooth-muscle cells, cyclic stretch can cause association of 402 Cell 144, 402���413, February 4, 2011 ��2011 Elsevier Inc.

some kinases with Cav1 (Sedding et al., 2005). To date, the role of Cav1/caveolae in mechanotransduction is mainly viewed as a downstream signaling platform, whereas their function in primary mechanosensing has not been directly addressed. A recent theoretical study has proposed that budded membrane domains like caveolae could play the role of membrane-medi- ated sensors and regulators of the plasma membrane tension (Sens and Turner, 2006). Endowed with a high membrane and lipid storage capacity, owing to the invaginated structure and high lipid packing, caveolae are well equipped to play such a role. We have challenged the homeostasis of the plasma mem- brane tension with different types of controlled mechanical stresses and analyzed the role of caveolae in the cell short- term response. We show in endothelial cells and muscle cells that functional caveolae are required to buffer the variations of membrane tension induced by sudden and transient mechanical stress via a two-step process of rapid caveola disassembly and slower reassembly. RESULTS Mechanical Stress Leads to the Partial Disappearance of Caveolae from the Plasma Membrane We examined the response of caveolae when cells were exposed to acute mechanical stresses. Osmotic swelling causes an increase of the membrane tension of cells unless some additional membrane is delivered to the cell surface (Dai and Sheetz, 1995 Dai et al., 1998 Morris and Homann, 2001). Cav1-EGFP-transfected HeLa cells were exposed to hypo- osmotic medium (30 mOsm). We observed a 35% increase of the cell volume within the first 5 min and a slow decrease there- after (Figures 1A and 1B). Upon reversing back to iso-osmolarity (300 mOsm) after 30 min of hypotonic shock, the volume decreased below the initial cell volume. These observations support the existence of a compensatory mechanism known as regulatory volume decrease, which restores the osmotic balance by activating ion channels (D���Alessandro et al., 2002). Our data, however, suggest that this process is not dominant during the first 5 min following hypo-osmotic shock. To distin- guish caveolae at the plasma membrane from the internal Golgi pool of Cav1, we used total internal reflection fluorescence (TIRF) microscopy (Figure 1C and Figures S1A and S1B available online). Upon hypo-osmotic shock, we observed that the number of caveolae significantly decreased by 30% at the cell surface (Figures 1C and 1D) and that the loss correlated with the magnitude of the shock (Figure 1E). Importantly, the cell footprint and the adhesion between the cell and the glass surface were unaltered, as shown by reflection interference contrast microscopy (RICM) (Figure S1C). Because caveolae exhibit different types of dynamics at the plasma membrane (Pelkmans and Zerial, 2005), we also checked whether any particular pool was selectively affected. Within minutes of hypo-osmotic shock, slow-moving caveolae reduced their mobility (Figure S1D), and fast dynamics were abolished (Movie S1 and Movie S2), whereas caveolae displaying all kinds of mobility were reduced in number (Figures S1E and S1F). Similar results were obtained in mouse lung endothelial cells (MLEC) (Figure S1B). Although osmotic shocks have been extensively used to mimic the osmolarity changes that cells experience (Lang et al., 1998), we sought to rule out any indirect influence of cell swelling on caveolae. We developed a stretching device based on thin transparent silicone substrates to challenge the cell membrane with a different mechanical stress. It allowed imaging of caveolae by TIRF before and after stretch and was combined with micropatterning (Chen et al., 1997) to control the cell adhesion area and its orientation with the stretching axis. The number of caveolae present at the basal footprint of Cav1-EGFP HeLa cells decreased upon stretching (Figures 1F and 1G), and the loss correlated with the extent of stretch (Fig- ure 1H). Therefore, acute mechanical stress induced either by hypo-osmotic shock or membrane stretching leads to a rapid and significant loss of caveolae from the cell surface. We next performed electron microscopy (EM) on MLEC. These endothe- lial cells experience chronic cycles of shear stress from the blood flow in lungs��� vessels in vivo. MLEC immunostaining shows multiple subcellular Cav1 positive structures, which are localized predominantly at the plasma membrane and at the Golgi appa- ratus (Figure S3B Murata et al., 2007). In contrast, Cav1 / MLEC derived from cells knocked out for the CAV1 gene do not present any Cav1 staining. However, Cav1-EGFP expression can be induced by transfection in WT and Cav1 / MLEC (Fig- ure S3C). EM analysis showed a significant decrease of the number of caveolae (50%) upon a 5 min exposure of WT MLEC to a hypo-osmotic shock (Figures 2A and 2B). These data confirm the results obtained by TIRF imaging and extend our conclusions to endogenous caveolae present on the entire surface of the cell. Flattening and Disassembly of Caveolae upon Hypo-Osmotic Shock The contribution of caveolae to the general endocytic activity of the cell is believed to be minimal (Nabi and Le, 2003), and endo- cytosis is disfavored at high membrane tension (Dai et al., 1997). We still tested whether the loss of caveolae upon hypo-osmotic was due to increased caveola endocytosis. We used dynasore, an inhibitor of the dynamin GTPase that is involved in caveola internalization (Henley et al., 1998 Macia et al., 2006). Indeed, dynasore significantly increased the caveolae density at the plasma membrane, reflecting the efficient inhibition of caveola endocytosis (Figure 2C). However, upon hypo-osmotic shock, a similar loss of caveolae was measured (Figures 2C and 2D). We next examined Cavin1, which is part of the caveolar complex and is required to maintain caveola invagination (Hill et al., 2008 Hansen et al., 2009). Cavin1 does not bind to free Cav1 oligo- mers or to Cav1 present on the Golgi apparatus. We found a high level (64%) of Cav1-EGFP colocalization with Cavin1- mCherry (Figure 2E). Upon hypo-osmotic shock, there was a similar or even higher loss of Cavin1-labeled structures, con- firming the partial loss of caveolae (Figure 2F). We also observed a decreased colocalization with Cavin1 (35%) for the remaining caveolae, suggesting a loss of their invaginated structure. We quantified the interaction between Cav1 and Cavin1 using fluo- rescence lifetime imaging microscopy (FLIM). When Cavin1 is present in caveola, the close proximity of mRFP-Cav3 and Cavin1-EGFP results in FRET and a decrease in the EGFP Cell 144, 402���413, February 4, 2011 ��2011 Elsevier Inc. 403