Tuesday September 27th
11:30 am - 12:30 pm

Macropinosomes as Discrete Units of Signal Transduction

Amphithéâtre Antoine Lacassagne
Bâtiment Trouillet-Rossignol, 26 rue d'Ulm, Paris 5ème
Conférence en English

Macropinocytosis is an endocytic process which efficiently delivers extracellular solutes into lysosomes. It can be induced in cells by growth factors, phorbol esters, chemokines, and many microbial pathogens. Macropinosomes form in stimulated cells by actin-based ruffling and membrane closure into large plasma membrane-derived vesicles. We hypothesize that macropinosomes function as discrete units of signal transduction from cell surface receptors to the mechanistic target of rapamycin complex-1 (mTORC1), an essential regulator of cell growth and metabolism. In macrophages, acute stimulation of mTORC1 by the growth factor macrophage colony-stimulating factor (M-CSF) occurs by the rapid delivery of extracellular amino acids into endolysosomes via macropinocytosis (Yoshida, et al., 2015, J. Cell Biol. 211: 159-172). To examine the role of macropinocytosis in signaling by chemokines, we measured activation of mTORC1 and related signaling molecules in response to CXCL12. Like M-CSF, CXCL12 rapidly stimulated both macropinocytosis and the phosphorylation of S6K, an output of mTORC1 activity. Stimulation of mTORC1 by CXCL12 was inhibited by the macropinocytosis inhibitor ethylisopropyl amiloride (EIPA), by inhibition of actin dynamics, and by omission of extracellular amino acids. Various isoform-specific inhibitors of type 1 phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) showed similar patterns of inhibition of macropinocytosis and mTORC1 activity. However, unlike the response to M-CSF, Akt phosphorylation necessary for mTORC-1 activation in response to CXCL12 required the actin cytoskeleton and the formation of macropinocytic cups. Quantitative fluorescence microscopy showed that PIP3, a product of PI3K and an upstream activator of Akt, localized to membrane ruffles and macropinocytic cups, and that Akt phosphorylation occurred primarily in ruffles and cups. Together, these results indicate that: 1) the amino acid-laden macropinosome is an essential and discrete unit of CXCL12 signaling to mTORC1, and 2) the cup-shaped domain of plasma membrane localizes PI3K signal amplification and Akt phosphorylation. Furthermore, these studies suggest that, during continuous exposure to growth factors, receptor-dependent signaling occurs primarily through the stochastic and transient construction of macropinosomes.

Joel A.
Professor of Microbiology and Immunology, University of Michigan Medical School (Ann Arbor, Michigan, USA)