Small GTPases and phosphoinositides in the regulatory mechanisms of macropinosome formation and maturation

Abstract

Macropinosome formation requires the sequential activation of numerous signaling pathways that coordinate the actin-driven formation of plasma membrane protrusions (ruffles) and circular ruffles (macropinocytic cups), followed by the closure of these macropinocytic cups into macropinosomes. In the process of macropinosome formation, localized productions of phosphoinositides such as PI(4,5)P2 and PI(3,4,5)P3 spatiotemporally orchestrate actin polymerization and rearrangement through recruiting and activating a variety of actin-associated proteins. In addition, the sequential activation of small GTPases, which are known to be master regulators of the actin cytoskeleton, plays a pivotal role in parallel with phosphoinositides. To complete macropinosome formation, phosphoinositide breakdown and Rho GTPase deactivation must occur in appropriate timings. After the nascent macropinosomes are formed, phosphoinositides and several Rab GTPases control macropinosome maturation by regulating vesicle trafficking and membrane fusion. In this review, we summarize recent advances in our understanding of the critical functions of phosphoinositide metabolism and small GTPases in association with their downstream effectors in macropinocytosis.

Keywords: Rac1; macropinocytosis; myotubularin-related proteins; optogenetics; phosphoinositides; small GTPases.

Figure 1

Proposed model for the sequential association of small GTPases and phosphoinositides with the processes of macropinocytosis. See text for explanations.

Figure 2

Macropinosome formation and maturation by optogenetic control of Rac1 activation and deactivation. (A) Local and reversible control of membrane ruffling and macropinosome formation through Rac1 photoactivation and deactivation. Diffraction interference contrast images of live RAW264 macrophages expressing PA-Rac1 were acquired by confocal laser microscopy with photoactivation. Time 0 indicates the initiation of blue laser irradiation in the area enclosed by the circle. At 12.4 min, ruffles were apparent within the irradiated region (blue circle). After 13 min, the irradiation was shifted to a different area of the same cell. After the irradiation was ceased, the ruffles immediately receded, and spherical macropinosomes were formed in the initial irradiation area (broken-lined circle) within 10 min. Marked ruffling was then induced in the newly irradiated area. The bar indicates 10 μm. (B,C) Time-lapse live-cell images of RAW264 macrophages expressing PA-Rac1 and GFP-Rab21 during photoactivation (B) and after photoactivation was ceased (C). The blue rectangular area in the cell was repeatedly irradiated using a blue laser for 12.2 min. The top left panel shows a merged image of the phase-contrast, mCherry, and GFP fluorescence signals obtained before the photoactivation of PA-Rac1, confirming the expression of mCherry-PA-Rac1 and GFP-Rab21. The other panels show selected time-lapse phase-contrast (upper) and GFP-Rab21 images (lower; green). Time 0 indicates the initiation of photoactivation (B) or deactivation. (C) The recruitment of Rab21, a maturation marker, to the macropinosomes was found after PA-Rac1 deactivation. The bar indicates 5 μm (adapted from Fujii et al., 2013).

Figure 3

Fluid-phase uptake in coelomocytes of Caenorhabditis elegans. (A) Caenorhabditis elegans generally possesses six coelomocytes (indicated by yellow) in its body cavity. This simple diagram of C. elegans is drawn from the side of the organism. Note that the other tissues of C. elegans are not shown for the sake of explanation. (B) Diagrams of the fluid-phase endocytosis assay. In wild-type worms containing myo-3p::ssGFP, GFP synthesized in the body wall muscle cells is secreted into the body cavity and taken up by coelomocytes. In wild-type worms, GFP signals are detected only in the coelomocytes. In CUP mutants containing myo-3p::ssGFP, GFP is not endocytosed by coelomocytes and is therefore accumulated in the body cavity. Note that the coelomocytes are magnified for the sake of explanation. (C) Images of wild-type worms and mtm-6(ok330)III mutants (one of the CUP mutants; see Supplementary Table 1). All worms contain myo-3p::ssGFP. Coelomocytes in the mtm-6 mutants do not have GFP-positive vesicles, and GFP accumulates in the body cavity (indicated by a white arrow). Coelomocytes are indicated by broken yellow lines. Scale bars: 10 μm (adapted from Maekawa et al., 2014).