Signaling-dependent immobilization of acylated proteins in the inner monolayer of the plasma membrane.
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.
Corbett-Nelson
Elaine F
EF
Division of Cell Biology, The Hospital for Sick Children, Department of Biochemistry, University of Toronto, M5G 1X8, Canada.
Mason
David
D
Marshall
John G
JG
Collette
Yves
Y
Grinstein
Sergio
S
D000199:Actins / Q000378:metabolism; D000215:Acylation; D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D002465:Cell Movement / Q000187:drug effects; D002784:Cholesterol / Q000172:deficiency; D003599:Cytoskeleton / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000378:metabolism; D050356:Lipid Metabolism; D008264:Macrophages / Q000166:cytology; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D010587:Phagocytosis; D010588:Phagosomes / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D015398:Signal Transduction
D000199:Actins; D008565:Membrane Proteins; D047428:Protein Kinase Inhibitors; D049452:Green Fluorescent Proteins; D019000:Phosphotyrosine; D002784:Cholesterol