Régulation de la macropinocytose constitutive dans les fibroblastes transformés par les oncogènes
(2001)
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- Authors
- Supervisors
- Courtoy, Pierre
- Abstract
- (en) Macropinocytosis refers to the formation of large primary endocytic vesicles of irregular size and shape, generated by actin-driven evaginations of the plasma membrane, whereby cells avidly incorporate extracellular solutes. Macropinosomes resemble "empty" phagosomes and show no difference with the "spacious phagosomes" triggered by the enteropathogenic bacteria Salmonella and Shigella. Macropinosomes are formed at the leading edge and appear tightly regulated. They may fuse with lysosomes or regurgitate their content back to the extracellular space. Transformation of fibroblasts by Src or Ras results into the constitutive formation of macropinosomes at "ruffling" zones. My thesis adresses the dynamic of the constitutive macropinocytosis and its potential regulators. The first part of the thesis deals with the fate of macropinosomes. We found that, in v-Src transformed fibroblasts, macropinosomes do not fuse with transferrin-containing endosomes and investigated the effects of cyclic AMP as a regulator of macropinocytosis in this cell system. The permeant analogs dibutyryl cyclic AMP and 8-bromo-cyclic AMP, as well as the pharmacological activator of adenylate cyclase, forskolin, similarly decreased by about 35% the net endocytic accumulation of the fluid-phase tracer, horseradish peroxidase, in v-Src-transformed cells but not in the non-transformed parental Rat-1 cell line. However, and in contrast to the phosphatidylinositol 3-kinase (PI3K) inhibitors and phosphatidylinositol-specific phospholipase C (PI-PLC), dibutyryl cyclic AMP neither returned the peroxidase accumulation rate of v-Src-transformed cells to that of parental Rat-1/control cells, nor prevented macropinosome formation, as shown by confocal microscopy. Detailed analysis of the kinetics of peroxidase entry and efflux in transformed cells revealed that dibutyryl cyclic AMP inhibited its accumulation only after intervals >5 minutes, due to accelerated regurgitation, but did not alter the rate of transferrin recycling. Taken together, these data indicate that, in v-Src-transformed fibroblasts, macropinocytosis and micropinocytosis serve different pathways and that cyclic AMP affects neither micropinocytosis nor the formation of macropinosomes, but selectively promotes regurgitation therefrom. The second part of my thesis deals with the regulation of macropinocytosis. Both transformation of Rat-1 fibroblasts by v-Src or K-Ras and stable transfection with an expression vector for wild-type PI3K regulatory subunit p85a, acting as dominant-positive PI3K, constitutively lead to stress fibers disruption, cortical actin recruitment, extensive ruffling and macropinosome formation, as measured by a selective acceleration of fluid-phase endocytosis. These alterations closely correlated with activation of PI3K and PI-PLC, as assayed by 3-phosphoinositides synthesis in situ and in vitro and IP3 steady state levels, respectively, and were abolished by stable transfection of Src-transformed cells with an expression vector for truncated p85a, acting as dominant-negative PI3K, as well as by pharmacological inhibitors of PI3K and PI-PLC, indicating requirement for both enzymes. These inhibitors strongly decreased the population of cells showing active membrane ruffling and did not detectably affect receptor-mediated endocytosis of transferrin. Interestingly, the effect of Src and dominant-positive p85a transfection on the actin cytoskeleton could be reversed within 30-60 min by the pharmacological inhibitor of PI3K, wortmannin. [...]
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Citations
Amyere, M. (2001). Régulation de la macropinocytose constitutive dans les fibroblastes transformés par les oncogènes. https://hdl.handle.net/2078.5/129645