Since their discovery in 1953, the precise role of caveolae has remained a matter of considerable debate. Freeze-fracture analysis of the surface membrane of smooth and striated muscle cells in the early 1970’s led to the first hypothesis that caveolae could flatten out under stretching conditions (Dulhunty and Franzini-Armstrong, 1975
; Prescott and Brightman, 1976
). Later studies have also associated caveolae with the mechanosensing response of the cell (Boyd et al., 2003
; Park et al., 2000
; Rizzo et al., 2003
; Sedding et al., 2005
; Kawamura et al., 2003
; Kozera et al., 2009
). Whether caveolae are directly involved in the cell response to mechanical stress, and by which mechanisms, still remain unknown. In this study, we establish that the primary cell response to an acute mechanical stress occurs through the rapid flattening of caveolae into the plasma membrane. Upon cell stretching or osmotic swelling, caveolae flattening provides the additional amount of membrane that inhibits any surge of membrane tension. This response occurs also in ATP depleted cells, and in membrane-derived vesicles devoid of actin, indicating that the ability to buffer membrane tension is intrinsic to the caveola structure. These results are in line with a theoretical model proposing that invaginated domains can serve as a membrane reservoir to regulate the membrane tension (Sens and Turner, 2006
). However, in contrast to this model, which predicted that flattening of existing caveolae or budding of new caveolae was driven by the deviation of the membrane tension with respect to the resting tension, our data indicate that the regulatory mechanism is asymmetric. While stress-induced flattening of caveolae is truly passive, the reassembly of new caveolae is assisted by ATP and actin dynamics. The energy landscape for flat and invaginated caveola is thus characterized by an activation energy. The transition between the two forms thus requires an energy barrier to be overcome or lowered. In this model, the flat configuration would be stabilized upon application of a mechanical force, whereas the formation of invaginated caveola is energetically favored through interactions with cortical actin in the presence of ATP. This is consistent with the known role of actin dynamics and various kinases in caveola function (Pelkmans et al., 2005
; Pelkmans and Zerial, 2005
). Our results further indicate that clathrin-coated pits, the other abundant invaginations present at the plasma membrane, are not involved in the fast response to membrane strain. Whereas CCPs exchange coat proteins within seconds, most caveolae are rather stable at the plasma membrane. The stability of caveolae combined with their low endocytic activity allows this membrane reservoir to be readily available to respond to sudden mechanical strains. The stability of the caveolae reservoir is a striking feature of cells that experience pulsating mechanical stresses in their lifetime (muscle cells, cardio-myocytes, endothelial cells), since they all express very high numbers of caveolae. Indeed, we could associate the lack of Cav3 expression at the surface of myotubes with impaired membrane tension buffering in patients bearing the Cav3 P28L mutation found in a form of human muscular dystrophy. Interestingly, P28L myotubes appear to be more fragile than wt myotubes when exposed to acute osmotic shock.
We have shown that the timescales of caveolae and actin cortex responses to osmotic shock are well separated (< 2 min and > 5 min, respectively). While the immediate response to mechanical stress relies primarily on caveola flattening, it is likely that other processes such as endocytosis, exocytosis and actin dynamics may prolong or complete the initial response at longer times. Whether, and how caveolae also contribute to long timescale regulation remains to be investigated. In endothelial cells, the application of chronic and repetitive shear stress tensions results in a several-fold increase of the number of caveolae at the plasma membrane, through the mobilization of the Cav1 pool associated with the Golgi complex (Boyd et al., 2003
; Park et al., 1998
). In agreement with the slow delivery rate of Cav1 from the Golgi complex (Tagawa et al., 2005
), we show that caveolae are reassembled independently from the Golgi complex when the mechanical stress is relaxed. It is thus important to distinguish between the short-term mechanical function of caveolae and the long-term adaptation of the cell to chronic stress. In this context, we also analyzed the contribution of caveolae to the setting of the membrane tension under resting conditions. At steady state, we measured a lower membrane tension in Cav1−/−
than in wt MLEC, however it was identical in wt and Cav1−/−
MEF, albeit to a lower level (Figure S8A
), raising the possibility of a peculiarity of MLEC. Furthermore, the resting tension was drastically affected by m-β-cyclodextrin, cytochalasin D or ATP depletion treatments in the different cell types, independently from the expression of caveolae (Figure S8B
). Although caveolae play a key role in cell tension homeostasis, their direct contribution to the resting tension remains intricate. As previously mentioned, the dynamic membrane-to-cytoskeleton adhesion, which is the main contribution to the apparent membrane tension, is likely to be regulated by cell line-dependent compensatory mechanisms.
The well-conserved scaffolding domain of caveolin (CSD) has been involved in both the assembly of caveolae and the interaction with several signaling effectors in vitro
(Parton et al., 2006
; Parton and Simons, 2007
). Since several of these effectors have been associated with mechanotransduction (Vogel and Sheetz, 2006
), stress-induced disassembly of caveolae may generate mechano-sensitive signals mediating the short and long-term cell response to mechanical challenges. It is tempting to speculate that the mechanical release of free Cav1 oligomers could favor the interaction between signaling effectors and CSD, which is otherwise hidden in the caveolar structure (Kirkham et al., 2008
; Parton et al., 2006
). This mechano-sensitive signaling would be terminated through the reassembly of free Cav1 oligomers into caveolae when the mechanical stress is relaxed. Endocytosis, which is favored for the retrieval of the excess of membrane during tension relaxation, may also contribute to signaling termination through the internalization of free Cav1 and its degradation in the endolysosomal pathway. The recently characterized Cavin1 protein, which was first described as a transcription factor (Jansa et al., 2001
) may also contribute to mechanosignaling regulation through the release from flattened caveola. Indeed, the redistribution of Cav1 to non-caveolar portions of the plasma membrane, the increased mobility of Cav1, and the decreased association of Cav1 and Cavin1 upon hypo-osmotic treatment are all consistent with the effect of Cavin1 knockdown on these parameters (Hill et al., 2008
), suggesting that dissociation of the Cav1-cavin module may be crucial in the caveolar response.
Our study establishes a new physiological mechanism by which cells can respond immediately to sudden variations in membrane tension induced by acute mechanical stress (). The different proposed roles of caveolae should therefore be reconsidered through this unique ability to respond to mechanical stress, especially in situations where cells experience physiological or pathological membrane strains such as osmotic swelling, shear stress or mechanical stretching.
Cells Respond to Acute Mechanical Stresses by Rapid Disassembly and Reassembly of Caveolae