Youhei Egami

Rab35 regulates phagosome formation through recruitment of ACAP2 in macrophages during FcγR-mediated phagocytosis

Phagosome formation and subsequent maturation are complex sequences of events that involve actin cytoskeleton remodeling and membrane trafficking. Here, we demonstrate that the Ras-related protein Rab35 is involved in the early stage of FcγR-mediated phagocytosis in macrophages. Live-cell image analysis revealed that Rab35 was markedly concentrated at the membrane where IgG-opsonized erythrocytes (IgG-Es) are bound. Rab35 silencing by RNA interference (RNAi) or the expression of GDP- or GTP-locked Rab35 mutant drastically reduced the rate of phagocytosis of IgG-Es. Actin-mediated pseudopod extension to form phagocytic cups was disturbed by the Rab35 silencing or the expression of GDP-Rab35, although initial actin assembly at the IgG-E binding sites was not inhibited. Furthermore, GTP-Rab35-dependent recruitment of ACAP2, an ARF6 GTPase-activating protein, was shown in the phagocytic cup formation. Concomitantly, overexpression of ACAP2 along with GTP-locked Rab35 showed a synergistic inhibitory effect on phagocytosis. It is likely that Rab35 regulates actin-dependent phagosome formation by recruiting ACAP2, which might control actin remodeling and membrane traffic through ARF6.

Dissecting the roles of Rac1 activation and deactivation in macropinocytosis using microscopic photo-manipulation

Macropinocytosis, a fluid-phase endocytosis, is a crucial pathway for antigen uptake and presentation in macrophages. We attempted to characterise the activation and deactivation of a small GTPase molecular switch, Rac1, in macropinocytosis using microscopic photo-manipulation. Expression of genetically encoded photoactivatable-Rac1 (PA-Rac1) in RAW264 macrophages enabled the local, reversible control of macropinocytosis using blue laser irradiation. Marked membrane ruffling and unclosed pre-macropinosomes were observed in the irradiated region of macrophages under the persistent activation of PA-Rac1. Although phosphatidylinositol 4,5-bisphosphate and actin were also localised to this region, the recruitment of maturating endosome markers, such as phosphatidylinositol 3-phosphate and Rab21, was restricted until PA-Rac1 deactivation. After deactivating PA-Rac1 by ceasing irradiation, membrane ruffling immediately receded, and the macropinosomes acquired maturation markers. These data suggest that activation of Rac1 is sufficient to induce membrane ruffling and macropinocytic cup formation, but subsequent deactivation of Rac1 is required for macropinosome closure and further maturation.

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

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.

Dynamic changes in the spatiotemporal localization of Rab21 in live RAW264 cells during macropinocytosis.

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.

Rab20 Regulates Phagosome Maturation in RAW264 Macrophages during Fc Gamma Receptor-Mediated Phagocytosis

Rab20, a member of the Rab GTPase family, is known to be involved in membrane trafficking, however its implication in FcγR-mediated phagocytosis is unclear. We examined the spatiotemporal localization of Rab20 during phagocytosis of IgG-opsonized erythrocytes (IgG-Es) in RAW264 macrophages. By the live-cell imaging of fluorescent protein-fused Rab20, it was shown that Rab20 was transiently associated with the phagosomal membranes. During the early stage of phagosome formation, Rab20 was not localized on the membranes of phagocytic cups, but was gradually recruited to the newly formed phagosomes. Although Rab20 was colocalized with Rab5 to some extent, the association of Rab20 with the phagosomes persisted even after the loss of Rab5 from the phagosomal membranes. Then, Rab20 was colocalized with Rab7 and Lamp1, late endosomal/lysosomal markers, on the internalized phagosomes. Moreover, our analysis of Rab20 mutant expression revealed that the maturation of phagosomes was significantly delayed in cells expressing the GDP-bound mutant Rab20-T19N. These data suggest that Rab20 is an important component of phagosome and regulates the phagosome maturation during FcγR-mediated phagocytosis.

Activation-Inactivation Cycling of Rab35 and ARF6 Is Required for Phagocytosis of Zymosan in RAW264 Macrophages

Phagocytosis of zymosan by phagocytes is a widely used model of microbial recognition by the innate immune system. Live-cell imaging showed that fluorescent protein-fused Rab35 accumulated in the membranes of phagocytic cups and then dissociated from the membranes of newly formed phagosomes. By our novel pull-down assay for Rab35 activity, we found that Rab35 is deactivated immediately after zymosan internalization into the cells. Phagosome formation was inhibited in cells expressing the GDP- or GTP-locked Rab35 mutant. Moreover, the simultaneous expression of ACAP2—a Rab35 effector protein—with GTP-locked Rab35 or the expression of plasma membrane-targeted ACAP2 showed a marked inhibitory effect on phagocytosis through ARF6 inactivation by the GAP activity of ACAP2. ARF6, a substrate for ACAP2, was also localized on the phagocytic cups and dissociated from the membranes of internalized phagosomes. In support of the microscopic observations, ARF6-GTP pull-down experiments showed that ARF6 is transiently activated during phagosome formation. Furthermore, the expression of GDP- or GTP-locked ARF6 mutants also suppresses the uptake of zymosan. These data suggest that the activation-inactivation cycles of Rab35 and ARF6 are required for the uptake of zymosan and that ACAP2 is an important component that links Rab35/ARF6 signaling during phagocytosis of zymosan.

Rac1-Dependent Lamellipodial Motility in Prostate Cancer PC-3 Cells Revealed by Optogenetic Control of Rac1 Activity

The lamellipodium, an essential structure for cell migration, plays an important role in the invasion and metastasis of cancer cells. Although Rac1 recognized as a key player in the formation of lamellipodia, the molecular mechanisms underlying lamellipodial motility are not fully understood. Optogenetic technology enabled us to spatiotemporally control the activity of photoactivatable Rac1 (PA-Rac1) in living cells. Using this system, we revealed the role of phosphatidylinositol 3-kinase (PI3K) in Rac1-dependent lamellipodial motility in PC-3 prostate cancer cells. Through local blue laser irradiation of PA-Rac1-expressing cells, lamellipodial motility was reversibly induced. First, outward extension of a lamellipodium parallel to the substratum was observed. The extended lamellipodium then showed ruffling activity at the periphery. Notably, PI(3,4,5)P3 and WAVE2 were localized in the extending lamellipodium in a PI3K-dependent manner. We confirmed that the inhibition of PI3K activity greatly suppressed lamellipodial extension, while the ruffling activity was less affected. These results suggest that Rac1-induced lamellipodial motility consists of two distinct activities, PI3K-dependent outward extension and PI3K-independent ruffling.

Spatiotemporal Localization of Rab20 in Live RAW264 Macrophages during Macropinocytosis.

Rab20 is a member of the Rab GTPase family, but its implication in macropinocytosis is unclear. We examined the spatiotemporal localization of Rab20 in RAW264 macrophages by the live-cell imaging of fluorescent protein-fused Rab20. It was shown that Rab20 was transiently associated with macropinosomal membranes. During the early stage of macropinosome formation, Rab20 was slightly localized on the circular ruffles (macropinocytic cups), the precursor forms of macropinosomes, and was increasingly recruited to the newly formed macropinosomes. Although Rab20 was colocalized with Rab5 and Rab21 on macropinosomal membranes, the association of Rab20 with macropinosomes persisted even after the dissociations of Rab5 and Rab21 from macropinosomal membranes. Rab20 was then colocalized with Rab7 and Lamp1, late endosomal/lysosomal markers, on macropinosomes for a while. Our data indicate that Rab20 is a novel component of macropinocytic pathway and functions at long-standing stages from early to late macropinosome maturation.

Rac1 switching at the right time and location is essential for Fcγ receptor-mediated phagosome formation

ABSTRACT Lamellipodia are sheet-like cell protrusions driven by actin polymerization mainly through Rac1, a GTPase molecular switch. In Fcγ receptor-mediated phagocytosis of IgG-opsonized erythrocytes (IgG-Es), Rac1 activation is required for lamellipodial extension along the surface of IgG-Es. However, the significance of Rac1 deactivation in phagosome formation is poorly understood. Our live-cell imaging and electron microscopy revealed that RAW264 macrophages expressing a constitutively active Rac1 mutant showed defects in phagocytic cup formation, while lamellipodia were formed around IgG-Es. Because activated Rac1 reduced the phosphorylation levels of myosin light chains, failure of the cup formation is probably due to inhibition of actin/myosin II contractility. Reversible photo-manipulation of the Rac1 switch in macrophages fed with IgG-Es could phenocopy two lamellipodial motilities: outward-extension and cup-constriction by Rac1 ON and OFF, respectively. In conjunction with fluorescence resonance energy transfer imaging of Rac1 activity, we provide a novel mechanistic model of phagosome formation spatiotemporally controlled by Rac1 switching within a phagocytic cup. Highlighted Article: A novel mechanistic model for phagosome formation that is spatiotemporally controlled by Rac1 switching within a phagocytic cup.

Development of an automated fluorescence microscopy system for photomanipulation of genetically encoded photoactivatable proteins (optogenetics) in live cells

Photomanipulation of genetically encoded light-sensitive protein activity, also known as optogenetics, is one of the most innovative recent microscopy techniques in the fields of cell biology and neurobiology. Although photomanipulation is usually performed by diverting the photobleaching mode of a confocal laser microscope, photobleaching by the laser scanning unit is not always suitable for photoactivation. We have developed a simple automated wide-field fluorescence microscopy system for the photomanipulation of genetically encoded photoactivatable proteins in live cells. An electrically automated fluorescence microscope can be controlled through MetaMorph imaging software, making it possible to acquire time-lapse, multiwavelength images of live cells. Using the journal (macro recording) function of MetaMorph, we wrote a macro program to change the excitation filter for photoactivation and illumination area during the intervals of image acquisition. When this program was run on the wide-field fluorescence microscope, cells expressing genetically encoded photoactivatable Rac1, which is activated under blue light, showed morphological changes such as lamellipodial extension and cell surface ruffling in the illuminated region. Using software-based development, we successfully constructed a fully automated photoactivation microscopy system for a mercury lamp-based fluorescence microscope.