Dynamic Changes in the Spatiotemporal Localization of Rab21 in Live RAW264 Cells during Macropinocytosis

Youhei Egami,Nobukazu Araki,Joshua Z Rappoport

doi:10.1371/journal.pone.0006689

Abstract

Rab21, a member of the Rab GTPase family, is known to be involved in membrane trafficking, but its implication in macropinocytosis is unclear. We analyzed the spatiotemporal localization of Rab21 in M-CSF-stimulated RAW264 macrophages by the live-cell imaging of fluorescent protein-fused Rab21. It was demonstrated that wild-type Rab21 was transiently associated with macropinosomes. Rab21 was recruited to the macropinosomes after a decrease in PI(4,5)P2 and PI(3,4,5)P3 levels. Although Rab21 was largely colocalized with Rab5, the recruitment of Rab21 to the macropinosomes lagged a minute behind that of Rab5, and preceded that of Rab7. Then, Rab21 was dissociated from the macropinosomes prior to the accumulation of Lamp1, a late endosomal/lysosomal marker. Our analysis of Rab21 mutants revealed that the GTP-bound mutant, Rab21-Q78L, was recruited to the macropinosomes, similarly to wild-type Rab21. However, the GDP-bound mutant, Rab21-T33N, did not localize on the formed macropinosomes, suggesting that the binding of GTP to Rab21 is required for the proper recruitment of Rab21 onto the macropinosomes. However, neither mutation of Rab21 significantly affected the rate of macropinosome formation. These data indicate that Rab21 is a transient component of early and intermediate stages of macropinocytosis, and probably functions in macropinosome maturation before fusing with lysosomal compartments.

Highlights

Macropinocytosis is a clathrin-independent endocytosis which accounts for the bulk fluid-phase uptake from the extracellular environment, and is an essential aspect of normal cell function
We found that Rab21 is associated with macropinosomes in RAW264 macrophages expressing Rab21 fused with green fluorescent protein (GFP) variants
We examined the association of Rab21 with macropinosomes in live RAW264 macrophages expressing GFP-Rab21 by phase-contrast and fluorescence microscopy

Summary

Introduction

Macropinocytosis is a clathrin-independent endocytosis which accounts for the bulk fluid-phase uptake from the extracellular environment, and is an essential aspect of normal cell function. Some pathogenic bacteria and viruses exploit macropinocytosis to invade their host [7,8,9,10]. Owing to this high important physiopathological relevance of macropinocytosis, the regulatory mechanisms of macropinosome formation and maturation have recently been receiving increasing attention. Newly formed macropinosomes gradually mature and merge with lysosomes [11] Both phosphoinositides and small GTPases, such as Cdc, Rac, ARF6 and Rab, are known to regulate actin polymerization and remodeling in membrane ruffling and macropinocytosis [3,12,13,14,15,16,17]. Knowledge about macropinocytosis has increased; the complex regulatory molecular components and signaling pathways of macropinocytosis still require detailed investigation

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Conclusion