title: Sequential signaling in plasma-membrane domains during macropinosome formation in macrophages.
abstract: Macropinosomes are large endocytic vesicles that form in ruffling regions of plasma membrane. To analyze signal organization relative to ruffle closure into circular ruffles and cup closure into macropinosomes, this study used quantitative microscopy to measure 3' phosphoinositides and small-GTPase activities in a representative subset of forming macropinosomes. Macropinocytosis was stimulated by the addition of macrophage colony-stimulating factor (M-CSF) to macrophages expressing fluorescent reporter proteins. Ratiometric and fluorescence resonance energy transfer (FRET) microscopy determined that Rac1 activity and phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] levels increased transiently, peaking 26-30 seconds after ruffle closure. Three-dimensional reconstruction of cells labeled with the fluorescent dye FM4-64 showed that PtdIns(3,4,5)P(3) was restricted to open, circular cups in the plasma membrane. Quantitative fluorescence microscopic methods determined the timing of cup closure, which followed 40-100 seconds after Rac1 and PtdIns(3,4,5)P(3) deactivation and coincided with accumulation of phosphatidylinositol 3-phosphate and Rab5a. Thus, ruffle closure creates a circular domain of plasma membrane that localizes the activation and deactivation of Rac1 and phosphoinositide 3-kinase (PI3K), followed by recruitment of Rab5a and the contractile activities of cup closure.
authors.affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
authors.forename: Sei
authors.initials: S
authors.lastname: Yoshida
authors.forename: Adam D
authors.initials: AD
authors.lastname: Hoppe
authors.forename: Nobukazu
authors.initials: N
authors.lastname: Araki
authors.forename: Joel A
authors.initials: JA
authors.lastname: Swanson
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism*; D005260:Female; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010873:Pinocytosis; D015398:Signal Transduction; D013997:Time Factors; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000378:metabolism
chemical_list: D018129:Phosphatidylinositol Phosphates; C060974:phosphatidylinositol 3,4,5-triphosphate; D019869:Phosphatidylinositol 3-Kinases; D020764:cdc42 GTP-Binding Protein; D020696:rab5 GTP-Binding Proteins; D020744:rac GTP-Binding Proteins
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