Some cells, such as red blood cells and muscular cells, must resist strong deformation. Disorders of these deformations are the basis of membrane fragility diseases such as spherocytosis (a disease of red blood cells) or Duchenne myopathy (which affects muscular cells). To understand cell deformation and associated disorders, it is essential to elucidate organization of membrane lipids, which are major membrane components.
Goals and cell models
Some cells impose their membranes large deformations during essential physiological processes; it is the case of red blood cells during filtration through very narrow pores of the spleen, of muscle cells during muscle contraction, of platelets during blood coagulation, of macrophages upon uptake of bacteria, of yeast during division, etc.. Lipids are the major membrane components. Elucidation of their organization is essential to understand membrane deformability, whose disorders are the basis of genetic membrane fragility diseases, such as hereditary spherocytosis that affects red blood cells and Duchenne muscular dystrophy that affects muscle cells.
Our current work focuses on red blood cells, as the simplest and best characterized living human cell model. The remarkable red blood cell deformability allows squeezing through narrow splenic pores, i.e. more than 10.000 times in the lifetime of a normal red blood cell. Strong membrane anchorage to cytoskeleton is crucial for red blood cell deformability. On the opposite, lack of deformation in spherocytosis, a genetic membrane fragility disease, causes hemolytic anemia, which eventually requires spleen removal. Muscular cell has also a highly structured cytoskeleton strongly anchored to the membrane, which is essential to avoid rupture during muscle contraction. Conversely, the lack of deformation in Duchenne muscular dystrophy causes degeneration of muscle fibers and comes quickly to muscle weakness and progressive paralysis. Our group addresses if organization of membrane lipids plays a key role in red blood cell and muscular cell deformations.
Organization of membrane lipids into domains
Until recently, membrane lipids were considered to form homogenous bilayers, except at small domains enriched in specific lipids, called short-lived nanometric lipid rafts. However, using high-resolution confocal imaging of red blood cells, our group discovered the existence of domains much larger and more stable than the nanometric lipid rafts, which were therefore called micrometric domains. These domains depend on temperature, membrane cholesterol content, membrane tension and anchoring of the red cell membrane to the underlying cytoskeleton. These domains are severely impaired in patients with spherocytosis. Our objectives are (i) to define the internal molecular organization of these micrometric domains, (ii) to determine their overall molecular lipid and protein composition, (iii) to assess how they contribute to the physiopathology of spherocytosis, and (iv) to extend our research to muscular cells.
Implication of membrane lipid domains in membrane fragility diseases
We already know that the organization of the red blood cell membrane in micrometric domains is impaired in patients with spherocytosis. We consider two possible impacts of these domains on membrane deformability. The first hypothesis considers these domains as sites of fragility, promoting the breakdown of abnormal red blood cells. The second hypothesis involves a membrane reservoir, which may include micrometric domains, promoting red blood cell deformability. Our research should clarify the physiopathology of membrane fragility disease of red blood cells and may provide preventive perspectives of hemolysis in patients with spherocytosis. We consider extending to Duchenne myopathy as an appealing perspective.
Membrane fluidity and stability are essential for mammalian cells. Until recently, membrane lipids were considered to form homogenous bilayers, except at small domains (short-lived lipid rafts and long-lived caveolae). However, using vital high-resolution confocal imaging, our group discovered that inserted exogenous fluorescent analogs of various membrane lipids or direct labeling of corresponding endogenous lipids using lipid-specific proteins derived from bacterial toxins label larger (micrometric) non-overlapping stable domains (Fig. 1). These domains depend on temperature, cholesterol, membrane tension as well as membrane:cytoskeleton anchorage, and are altered in spherocytosis, a genetic membrane fragility disease of red blood cells (Fig. 2). To understand the mechanisms underlying biogenesis, control and significance for physiopathology of micrometric lipid domains, our group addresses their (i) relation with lipid rafts; (ii) differential association with membrane proteins (proteomics of red blood cell membrane-derived microvesicles, imaging and screening in yeast); (iii) role for membrane deformability (during red blood cell deformation, yeast budding and migration of a muscular cell line); and (iv) contribution to physiopathology of membrane fragility diseases of red blood cells (spherocytosis) and muscle (Duchenne myopathy). Another part of the project aims at exploring drug activity and toxicity using red blood cells labeled with fluorescent lipids or toxins, as a new simple tool to study drug:membrane interaction. Investigations rely on cell culture, advanced bioimaging methods (live cell high-resolution confocal imaging, high-throughput confocal microscopy, transmission and scanning electron microscopy, dynamics by fluorescence recovery after photobleaching [FRAP], all available at PICT, see below), biochemistry (lipid content and metabolism, subcellular fractionation and lipid rafts extraction) and molecular biology (screening in yeast, proteomics).
Labelling of endogenous sphingomyelin, cholesterol and ganglioside GM1 by toxin fragments show submicrometric domains. Erythrocytes labelled by fluorescent lysenin (SM, a), theta toxin (cholesterol, b) or cholera toxin B subunit (ganglioside GM1, c).
Control of submicrometric domains labelled on erythrocytes upon insertion of fluorescent sphingomyelin (BODIPY-SM). (a) control erythrocyte; (b) cholesterol depletion (- 25%); (c) familial spherocytosis.
Our group works in tandem with the Platform for Imaging Cells and Tissues (PICT). PICT is not only a core facility providing access and training to high-throughput, high-resolution and multiphoton confocal imaging as well as scanning and transmission electron microscopy, but also an established tradition of expertise, advice and collaborations within the DDUV Institute, the health research campus of the UCL, as well as national and international partnerships, both academic and industrial. We are happy to share expertise in scientific projects addressing defined questions which can benefit from: (i) electron microscopy (scanning and transmission), including ultrastructural cytochemistry; (ii) high-resolution and high-throughput confocal microscopy, for live-cell imaging and immunolabelling; (iii) multiphoton microscopy to image tissues and organs for several hours without damage; and (iv) advanced applications by confocal microscopy, including dynamics of molecular movement by Fluorescence Recovery After Photobleaching (FRAP), protein dimerization by Bimolecular Fluorescence Complementation (BiFC), in situ detection of free radicals (ROS) at distinct subcellular compartments, and protein:protein interactions by FRET, etc. In addition, we propose advanced images analyses such as deconvolution, 3-D reconstruction, morphometry, objective co-localization, etc.
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INVOLVEMENT OF MEMBRANE LIPID ORGANIZATION IN CELL DEFORMATION AND MEMBRANE FRAGILITY DISEASES