Rit1-TBC1D10B signaling modulates FcγR-mediated phagosome formation in RAW264 macrophages.

Phagocytosis is an important immune response that protects the host from pathogen invasion. Rit1 GTPase is known to be involved in diverse cellular processes. However, its role in FcγR-mediated phagocytosis remains unclear. Our live-cell imaging analysis revealed that Rit1 was localized to the membranes of F-actin-rich phagocytic cups in RAW264 macrophages. Rit1 knockout and expression of the GDP-locked Rit1 mutant suppressed phagosome formation. We also found that TBC1D10B, a GAP for the Rab family GTPases, colocalizes with Rit1 in the membranes of phagocytic cups. Expression and knockout studies have shown that TBC1D10B decreases phagosome formation in both Rab-GAP activity-dependent and -independent manners. Notably, the expression of the GDP-locked Rit1 mutant or Rit1 knockout inhibited the dissociation of TBC1D10B from phagocytic cups. In addition, the expression of the GTP-locked Rit1 mutant promoted the dissociation of TBC1D10B in phagocytic cups and restored the rate of phagosome formation in TBC1D10B-expressing cells. These data suggest that Rit1-TBC1D10B signaling regulates FcγR-mediated phagosome formation in macrophages.

Rab10-Positive Tubular Structures Represent a Novel Endocytic Pathway That Diverges From Canonical Macropinocytosis in RAW264 Macrophages.

Using the optogenetic photo-manipulation of photoactivatable (PA)-Rac1, remarkable cell surface ruffling and the formation of a macropinocytic cup (premacropinosome) could be induced in the region of RAW264 macrophages irradiated with blue light due to the activation of PA-Rac1. However, the completion of macropinosome formation did not occur until Rac1 was deactivated by the removal of the light stimulus. Following PA-Rac1 deactivation, some premacropinosomes closed into intracellular macropinosomes, whereas many others transformed into long Rab10-positive tubules without forming typical macropinosomes. These Rab10-positive tubules moved centripetally towards the perinuclear Golgi region along microtubules. Surprisingly, these Rab10-positive tubules did not contain any endosome/lysosome compartment markers, such as Rab5, Rab7, or LAMP1, suggesting that the Rab10-positive tubules were not part of the degradation pathway for lysosomes. These Rab10-positive tubules were distinct from recycling endosomal compartments, which are labeled with Rab4, Rab11, or SNX1. These findings suggested that these Rab10-positive tubules may be a part of non-degradative endocytic pathway that has never been known. The formation of Rab10-positive tubules from premacropinosomes was also observed in control and phorbol myristate acetate (PMA)-stimulated macrophages, although their frequencies were low. Interestingly, the formation of Rab10-positive premacropinosomes and tubules was not inhibited by phosphoinositide 3-kinase (PI3K) inhibitors, while the classical macropinosome formation requires PI3K activity. Thus, this study provides evidence to support the existence of Rab10-positive tubules as a novel endocytic pathway that diverges from canonical macropinocytosis.

A novel DENND1B-localized structure found at the basal side of adherent cells.

Rab35 is a small G protein involved in various cellular events including clathrin-dependent endocytosis, phagocytosis, and autophagy. DENND1B, a DENN family member, acts as a guanine nucleotide exchange factor (GEF) for Rab35 to convert it to the GTP-bound active form from the GDP-bound inactive form. DENND1B contains the DENN domain which harbors GEF activity for Rab35 in the N-terminus, while the clathrin binding motif and adaptor protein-2-interaction motif are at the C-terminus. In this study, we investigated the intracellular localization of DENN1B in various cell types and found novel DENND1B-localized gathered line structures in BS-C-1 cells and in some other cell types. The localization of DENND1B to gathered line structures was dependent on a specific region located in the C-terminus of DENND1B protein. DENND1B-localized gathered lines were partially associated with microtubules but not with F-actin; instead, F-actin bundles surrounded the assembly of gathered lines. We also show that the gathered line structures appeared at the bottom of spreading lamellipodia and disappeared at the retracting site during cell motility in EGF-stimulated BS-C-1 cells. These results shed light on a new role for DENND1B in the regulation of cell migration.

Rab35 Targeting to the Plasma Membrane Is Dependent on the C-terminal Polybasic Cluster.

Rab35, a member of the Rab GTPase family, has been implicated in various cellular processes including cell motility and membrane trafficking. Although Rab35 is localized to the plasma membrane, Rab proteins that are identified to have high sequence homology with Rab35 exhibit distinct subcellular localization patterns. Comparing the amino acid sequences between Rab35 and its family members revealed a significant variation in an approximate 30-amino acid region of the C-terminus. This suggests that this region determines the subcellular localization of individual Rab proteins. To confirm this hypothesis, we constructed Rab35-Rab10 chimera proteins by exchanging their C-terminal domains with one another. Confocal microscopy of RAW264 cells expressing EGFP-fused Rab35-Rab10 chimeras has indicated that the C-terminal region of Rab35 is critical for its plasma membrane localization. Furthermore, we were able to determine that a basic amino acid cluster exists in the C-terminal region of Rab35 and that Rab35 localization shifts to the Golgi membrane when the number of basic amino acids in this region is reduced. Thus, it is likely that the approximate 30-amino acid C-terminal region containing basic clusters is responsible for Rab35 plasma membrane localization and that its preferential localization depends on the number of basic amino acids.

Transient recruitment of M-Ras GTPase to phagocytic cups in RAW264 macrophages during FcγR-mediated phagocytosis.

M-Ras, a member of the Ras superfamily, is known to be involved in diverse cellular processes. However, its involvement in FcγR-mediated phagocytosis remains unknown. We examined the spatiotemporal localization of M-Ras during the engulfment of IgG-opsonized erythrocytes (IgG-Es) in RAW264 macrophages. By the live-cell imaging of fluorescent protein-fused M-Ras, we found that M-Ras was localized to the membrane of phagocytic cups during the early stage of phagosome formation. Notably, ratiometric image analysis revealed that M-Ras was concentrated in the membrane of forming phagosomes. Moreover, our analysis of M-Ras mutant expression showed that phagosome formation was significantly inhibited in cells expressing GDP-locked mutant M-Ras-S27N. In contrast, the expression of wild-type M-Ras or GTP-locked mutant M-Ras-G22V facilitated the uptake of IgG-Es. These data suggest that M-Ras is a novel component of the FcγR-mediated phagocytic pathway and may regulate phagosome formation in macrophages.

RhoC regulates the actin remodeling required for phagosome formation during FcγR-mediated phagocytosis.

Phagosome formation is a complicated process that requires spatiotemporally regulated actin reorganization. We found that RhoC GTPase is a critical regulator of FcγR-mediated phagocytosis in macrophages. Our live-cell imaging revealed that RhoC, but not RhoA, is recruited to phagocytic cups engulfing IgG-opsonized erythrocytes (IgG-Es). RhoC silencing through RNAi, CRISPR/Cas-mediated RhoC knockout, and the expression of dominant-negative or constitutively active RhoC mutants suppressed the phagocytosis of IgG-Es. Moreover, RhoC-GTP pulldown experiments showed that endogenous RhoC is transiently activated during phagosome formation. Notably, actin-driven pseudopod extension, which is required for the formation of phagocytic cups, was severely impaired in cells expressing the constitutively active mutant RhoC-G14V, which induced abnormal F-actin accumulation underneath the plasma membrane. mDia1 (encoded by DIAPH1), a Rho-dependent actin nucleation factor, and RhoC were colocalized at the phagocytic cups. Similar to what was seen for RhoC, mDia1 silencing through RNAi inhibited phagosome formation. Additionally, the coexpression of mDia1 with constitutively active mutant RhoC-G14V or expression of active mutant mDia1-ΔN3 drastically inhibited the uptake of IgG-Es. These data suggest that RhoC modulates phagosome formation be modifying actin cytoskeletal remodeling via mDia1.

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

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.

Cofilin contributes to phagocytosis of IgG-opsonized particles but not non-opsonized particles in RAW264 macrophages.

Cofilin is an actin-binding protein that severs actin filaments. It plays a key role in regulating actin cytoskeletal remodeling, thereby contributing to diverse cellular functions. However, the involvement of cofilin in phagocytosis remains to be elucidated. We examined the spatiotemporal localization of cofilin during phagocytosis of IgG-opsonized erythrocytes, IgG-opsonized latex beads and non-opsonized latex beads. Live-cell imaging showed that GFP-cofilin accumulates in the sites of IgG-opsonized particle binding and in phagocytic cups. Moreover, immunofluorescence microscopy revealed that endogenous cofilin localizes to phagocytic cups engulfing IgG-opsonized particles, but not non-opsonized latex beads. Scanning electron microscopy demonstrated a notable difference in morphology between phagocytic structures in IgG-dependent and IgG-independent phagocytosis. In phagocytosis of IgG-opsonized particles, sheet-like pseudopodia extended along the surface of IgG-opsonized particles to form phagocytic cups. In contrast, in opsonin-independent phagocytosis, long finger-like filopodia captured non-opsonized latex beads. Importantly, non-opsonized beads sank into the cells without extending phagocytic cups. Our analysis of cofilin mutant expression demonstrates that phagocytosis of IgG-opsonized particles is enhanced in cells expressing wild-type cofilin or active mutant cofilin-S3A, whereas the uptake of non-opsonized latex beads is not. These data suggest that cofilin promotes actin cytoskeletal remodeling to form phagocytic cups by accelerating actin turnover and thereby facilitating phagosome formation. In contrast, cofilin is not involved in opsonin-independent phagocytosis of latex beads.

Molecular imaging analysis of Rab GTPases in the regulation of phagocytosis and macropinocytosis.

Phagocytosis and macropinocytosis, actin-dependent endocytic pathways that mediate the uptake of particles and fluid, respectively, are fundamental routes that enable cells to sample their environment, eliminate pathogens and endogenous cell debris, and contribute to immunoprotection and the maintenance of tissue homeostasis. These processes require a well-organized network of actin cytoskeletal remodeling and membrane transport, which are spatiotemporally regulated by small GTPases. The Rab family of small GTPases, which functions as molecular switches, plays central roles in intracellular membrane trafficking. Although multiple Rab proteins are localized to phagosomes and regulate phagosome maturation, the precise role of each Rab family member in Fcγ receptor (FcγR)-mediated phagocytosis is not fully characterized. Recently, we revealed that Rab35 and Rab20 are important regulators of phagosome formation and maturation, respectively. This review summarizes the functional implication of these Rab GTPases during FcγR-mediated phagocytosis in macrophages. Currently, compared with our knowledge of the regulatory mechanisms of receptor-mediated endocytosis including phagocytosis, the molecular components and signaling cascades of macropinocytosis remain poorly elucidated. Our time-lapse imaging showed that several Rab GTPases are sequentially recruited to the membrane of macropinosomes. Based on our observations, these findings regarding the spatiotemporal localization of Rab GTPases during macropinocytosis are introduced.

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.

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.

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.

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.

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.

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.

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.

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.

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)P(2) and PI(3,4,5)P(3) 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.

Characterization of Rab21-positive tubular endosomes induced by PI3K inhibitors.

We found that wortmannin, a potent phosphoinositide 3-kinase (PI3K) inhibitor, markedly induced the formation of Rab21-positive tubular compartments in A431 cells. By time-lapse fluorescence microscopy of live cells co-expressing fluorescent protein-fused Rab21 and other marker proteins, it was shown that the Rab21-positive tubules in wortmannin-treated cells were derived from Rab5-positive early endosomes, but not from late endosomes, recycling endosomes, lysosomes or the trans-Golgi network. The formation of Rab21-positive tubules was very dynamic and required microtubules. Rab21-positive tubules were also formed by the treatment of cells with 3-methyladenine (3-MA), which inhibits class III PI3K rather than class I PI3K. Furthermore, the loss of PI(3)P correlated with the tubulation of Rab21-positive endosomes in cells co-expressing fluorescent protein-fused Rab21 and a tandem FYVE domain. These results suggest that the lowering of PI(3)P as a result of class III PI3K inhibition may be an important cue for the morphological change of Rab21-positive early endosomes from vesicular to tubular form.

Phosphoinositide metabolism during membrane ruffling and macropinosome formation in EGF-stimulated A431 cells.

Inhibitors of phosphoinositide 3-kinase (PI3K) were found to perturb macropinosome formation without affecting the membrane ruffling and actin polymerization in epidermal growth factor-stimulated A431 cells. Live-cell imaging and quantitative image analysis of the fluorescence intensity ratio of the YFP-tagged phospholipase Cdelta1-pleckstrin homology domain (YFP-PLC-PH) relative to membrane-targeted CFP (CFP-Mem) demonstrated that the concentration of PI(4,5)P(2) in the membrane ruffles forming macropinocytic cups increased to more than double that in planar plasma membranes. The PI(4,5)P(2) level in the membrane reached its maximum just before macropinosome closure and rapidly fell as the macropinocytic cups closed. In contrast, the PI(3,4,5)P(3) concentrations visualized based on the YFP-Akt-PH or YFP-Bruton's tyrosine kinase (Btk)-PH/CFP-Mem ratio increased locally at the site of macropinosome formation and peaked at the time of macropinosome closure. The kinetics of PI(4,5)P(2) and PI(3,4,5)P(3) appeared to be mechanistically linked to actin remodeling during macropinocytosis. From the pharmacological data using inhibitors and synthetic phosphoinositides and other data, it could be concluded that both PI(4,5)P(2) elimination and PI(3,4,5)P(3) production by PI3K might be crucial for macropinosome formation from membrane ruffles. This study emphasizes that locally controlled levels of phosphoinositides are important for regulating the function of actin-binding proteins which effect changes in the membrane architecture.