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Caveolae-mediated mechanotransduction: Role in intracellular signaling and cell cholesterol homeostasis – MECHANOCAV

Role of caveolae, small structures of the cell surface during mechanical stress

To understand the dynamics of caveolae flattening in response to different types of mechanical stress <br />To understand the role of caveolae flattening in the activation of intracellular signaling pathways and the control of cholesterol metabolism

Links between flattening of caveolae and signaling and cell metabolism

Through a unique combination of biophysics and cell biology, we have recently shown that caveolae, these characteristic small invaginations (60 nm) of the plasma membrane of the cells, are mechanosensors that immediately respond to mechanical stress by flattening. This flattening leads to the release of the protein components of caveolae i.e. caveolin (Cav1) and its Cavin-1 partner. <br />We propose that the mechanically dependent cycle of caveolae disassembly / reassembly and its molecular consequences is a key molecular event, which should have a major impact on intracellular signaling pathways and metabolism. <br />We have three main objectives: <br />1) develop new micromechanical devices for applying different types of mechanical stress on the cells and study the mechanical cellular response through caveolae (P1: Pierre Nassoy, Institute of Optics, Bordeaux) <br />2) study the role of caveolae flattening in the intracellular fate of Cav1 and intracellular signaling, focusing on the JAK / STAT pathway (P2. Christophe Lamaze U1143 INSERM / CNRS UMR 3666 / I Curie) <br />3) study the potentially existing reciprocal relationship between cholesterol metabolism and caveolar mechanical function (P3: Cardiovascular Biochemistry Unit, Prof JL Paul; HEGP, Faculty of Pharmacy, Paris 11)

We use the expertise of our colleague physicist to develop new micromechanical devices allowing on the one hand to reproduce mechanical stresses as close as possible to the physiological or pathophysiological cell environment and on the other hand to simultaneously observe caveolae dynamics on a single living cell using sophisticated microscopy techniques at high resolution. Our colleague has already developed a new device for uniaxial stretching, miniaturized and easier to use, and that can reproduce the mechanical stretching observed in muscle cells. In parallel, he has validated the design of a microfluidic hydrodynamic shear stress device that allows to compare cells subjected to different shear stress in the same area of observation and in a range of physiological shear values such as those exerted on the vascular endothelial cells by blood flow. With these unique systems, we can observe the dynamics of caveolae at the surface of the cells and their role in signaling and cholesterol metabolism. The signaling pathways will be identified through high throughput screening to detect protein activation changes involved in cell activation. Cholesterol metabolism will be analyzed using the techniques developed in the cardiovascular biochemistry department of the Georges Pompidou European Hospital.

Thanks to the sophisticated devices developed by our physicist colleague, we were able to study the fate of the caveolae components under different types of stress namely hypo-osmotic shock and uniaxial stretching at short and longer times. We were able to visualize the intracellular fate of the caveolae components using single cell microscopy techniques such as confocal spinning disc and evanescent waves microscopy. We then identified the critical role of a particular enzyme present in caveolae in the mechanical response of the cells and the stability of the caveolae reservoir at the plasma membrane. The role of this enzyme in the regulation of the membrane tension of the cell has been investigated using the nanotube pulling technique in collaboration with physicists at the Institut Curie. These results led the team to develop new «edited genome« cell lines that express the enzyme at endogenous levels. Moreover, we began exploring the caveolae response in intracellular signaling. All of these experiences will be repeated and compared under different types of mechanical stress.
Our hospital biochemists colleagues have also studied the role of the caveolae response in the regulation of cholesterol metabolism (CT). They analyzed the gene regulation of so-called sterol-dependent, following an excess or lack of CT in human endothelial cells. For the sake of physiological relevance, the team has adapted this experience to observe the effect of shear forces corresponding to blood flow in the arteries.

The results obtained in this research program should allow the understanding of a new function of caveolae, those structures present in many cells of the body that are implicated in several diseases without knowing the precise mechanisms. This research program will have a potentially significant economic and societal impact. The role of mechanical forces in the regulation of cholesterol metabolism in endothelial cells of blood vessels is certainly linked to atherosclerosis, a major public health problem in Western societies. Similarly, more and more studies relate the progression of cancerous tumors to mechanical forces. Finally myopathies are connected to abnormal mechanical response of the muscle cells.

We published with two partners of the program a research article in a very good specialized American journal (Traffic), some of our results that highlight a new regulation of intracellular trafficking of cholesterol. We plan to patent the new micromechanical devices that have been developed during this program because they will be useful to our colleagues interested in similar studies. Preliminary results obtained in this program have already been presented at prestigious international conferences and scientific meetings reflecting the importance and interest of our research.

Cells perceive their microenvironment not only through soluble signals, receptors, but also through physical and mechanical cues, such as extracellular matrix (ECM) stiffness, confined adhesiveness and shear pressure of blood in endothelial cells. Cells translate these stimuli by mechanotransduction into biochemical signals controlling multiple cellular aspects, such as proliferation, growth and differentiation. Hence, mechanotransduction couples the mechanics and dynamics of mechanosensation-mechanotransmission with specific mechanoresponses that depend on the nature, strength, and duration of the mechanical force. The key machineries and players in mechanosensing and the link between mechanotransduction and the associated signaling pathways are poorly understood.
It has been suggested that cells can accommodate moderate mechanical stretching through a “membrane reservoir” that buffers changes in membrane tension. Recently, through a unique combination of Physics and Cell Biology, we have established that caveolae, those specialized invaginations at the plasma membrane, play such a role. We could thus demonstrate for the first time that caveolae act as mechanosensors that respond immediately to mechanical stress, by flattening out into the plasma membrane (Sinha et al., Cell 2011). This results in the disassembly of the caveolar structure, which provides the additional membrane stored in the caveolar invagination, maintaining thereby membrane tension homeostasis during mechanical stress . Caveola disassembly leads also to the release of caveolin and its partner, Cavin-1, which are freed from the caveola structure at the plasma membrane.
We propose that the mechano-dependent cycle of caveolae disassembly/reassembly, and its molecular consequences, is a key molecular event, which will have a major impact on cell signaling and cell metabolism.
To elucidate the molecular mechanisms controlling this new aspect of caveolae function, we propose a transdisciplinary project that relies on three major conceptual and technical advances present in our laboratories: 1) the recent demonstration that caveolae act as mechanosensors and adapt the cell response to mechanical constraints 2) the development by the physicists of sophisticated optico-mechanical assays to measure cell mechanical properties and to apply various mechanical forces 3) the preliminary results showing that caveola flattening selectively controls the JAK/STAT signaling pathway.

We will investigate:

1) The impact of new types of mechanical stress in the mechanical cell response through caveolae (team 1: Pierre Nassoy, Membrane Physics Dept, UMR 168 CNRS/Curie)

2) The role of caveola flattening in caveolin trafficking and cell signaling with a particular focus on the JAK/STAT signaling pathway (team 2: Christophe Lamaze, Cell Biology Dept, UMR 144 CNRS/Curie)

3) The reciprocal relationship that is likely to exist between cholesterol homeostasis and caveolae function (team 3: Cardiovascular Biochemistry Unit, Pr JL Paul and coll. HEGP Hospital / School of Pharmacy, Paris 11 University)

In summary, the basic line of this proposal is to employ ‘the state of the art’ biophysical and cell biology technologies developed at the Curie Institute to address the molecular consequences of caveola flattening in signaling and cholesterol homeostasis in endothelial cells.

This project associates cell biologists, physicists and clinical biochemists, to address these unexplored issues while controlling parameters that condition membrane dynamics and mechanics. This interdisciplinary collaboration should provide very important new information on the cell response to mechanical stress and the role of membrane mechanics, which are emerging as key factors in several pathologies, including cancer, atherosclerosis and muscle diseases, but remain largely unexplored at the molecular level.

Project coordinator

Monsieur Christophe Lamaze (Institut Curie) – christophe.lamaze@curie.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

LP2N Laboratoire Photonique Numérique et Nanosciences (LP2N)
HEGP Service de Biochimie HEGP Hôpitaux Universitaires Paris Ouest
IC Institut Curie

Help of the ANR 579,453 euros
Beginning and duration of the scientific project: December 2012 - 36 Months

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