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First published online 18 August 2009
doi: 10.1242/jcs.053207

Journal of Cell Science 122, 3250-3261 (2009)
Published by The Company of Biologists 2009
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Research Article

Sequential signaling in plasma-membrane domains during macropinosome formation in macrophages

Sei Yoshida1, Adam D. Hoppe1,2, Nobukazu Araki3 and Joel A. Swanson1,*

1 Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109, USA
2 Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007, USA
3 Department of Histology and Cell Biology, School of Medicine, Kagawa University, Miki, Kagawa 761-0793, Japan

* Author for correspondence (jswan{at}umich.edu)

Accepted 21 June 2009

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)P3] 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)P3 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)P3 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.

Key words: Macropinocytosis, Macrophage, PtdIns(3,4,5)P3, Rac1, FRET


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