Signaling-dependent immobilization of acylated proteins in the inner monolayer of the plasma membrane

Elaine F. Corbett-Nelson¹, David Mason^1,2, John G. Marshall¹, Yves Collette³, , and Sergio Grinstein^1,2

¹ Division of Cell Biology, The Hospital for Sick Children, Toronto, M5G 1X8, Canada
² Department of Biochemistry, University of Toronto, Toronto, M5S 1A8, Canada
³ Institut de Cancérologie de Marseille, UMR 599 Institut National de la Santé et de la Recherche Médicale, 13009 Marseille, France

Correspondence to Sergio Grinstein: sga{at}sickkids.ca

Abstract: Phospholipids play a critical role in the recruitment and activation of several adaptors and effectors during phagocytosis. Changes in lipid metabolism during phagocytosis are restricted to the phagocytic cup, the area of the plasmalemma lining the target particle. It is unclear how specific lipids and lipid-associated molecules are prevented from diffusing away from the cup during the course of phagocytosis, a process that often requires several minutes. We studied the mobility of lipid-associated proteins at the phagocytic cup by measuring fluorescence recovery after photobleaching. Lipid-anchored (diacylated) fluorescent proteins were freely mobile in the unstimulated membrane, but their mobility was severely restricted at sites of phagocytosis. Only probes anchored to the inner monolayer displayed reduced mobility, whereas those attached to the outer monolayer were unaffected. The immobilization persisted after depletion of plasmalemmal cholesterol, ruling out a role of conventional "rafts." Corralling of the probes by the actin cytoskeleton was similarly discounted. Instead, the change in mobility required activation of tyrosine kinases. We suggest that signaling-dependent recruitment of adaptors and effectors with lipid binding domains generates an annulus of lipids with restricted mobility.

E.F. Corbett-Nelson and D. Mason contributed equally to this paper.

Abbreviations used in this paper: BODIYP, boron dipyrromethene difluoride; DIC, differential interference contrast; GPI, glycosylphosphatidylinositol; LAT, linker for activation of T cells; LSM, laser-scanning microscopy; MßCD, methyl-ß-cyclodextrin; MF, mobile fraction; NBD, nitrobenzoxadiazole; PH, pleckstrin homology; PM, plasma membrane.

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