The coated pit and macropinocytic pathways serve distinct endosome populations

Clathrin-coated vesicle endocytosis and macropinocytosis are distinct endocytic pathways demonstrable in several cell types including human epidermoid A431 cells (West, M.A., M.S. Bretscher, and C. Watts. 1989. J. Cell Biol. 109:2731-2739). Here we analyze the extent of mixing of macropinocytic endosome (macropinosome) content with that of conventional endosomes served by coated vesicle endocytosis. Using laser scanning confocal fluorescence microscopy we detected very little delivery of macropinosome content to either early or late endosomes- lysosomes as defined by labeling with transferrin or with LDL. Mixing of the contents of the macropinosomes and conventional endosomes was not induced by the addition of brefeldin A. Moreover, the morphology of macropinosomes was not grossly altered in the presence of brefeldin A, whilst in the same cells there were dramatic tubulation effects on conventional endosomes as reported by others. Although refractory to fusion with conventional endosomes, macropinosomes were nonetheless dynamic structures which sometimes exhibited vesiculo-tubular morphology in living cells and were capable of fusing with each other. We suggest that different endocytic mechanisms can give rise to distinct endosome populations.     

A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages

Phosphoinositide 3-kinase (PI 3-kinase) has been implicated in growth factor signal transduction and vesicular membrane traffic. It is thought to mediate the earliest steps leading from ligation of cell surface receptors to increased cell surface ruffling. We show here that inhibitors of PI 3-kinase inhibit endocytosis in macrophages, not by interfering with the initiation of the process but rather by preventing its completion. Consistent with earlier studies, the inhibitors wortmannin and LY294002 inhibited fluid-phase pinocytosis and Fc receptor-mediated phagocytosis, but they had little effect on the receptor-mediated endocytosis of diI-labeled, acetylated, low density lipoprotein. Large solute probes of endocytosis reported greater inhibition by wortmannin than smaller probes did, indicating that macropinocytosis was affected more than micropinocytosis. Since macropinocytosis and phagocytosis are actin-mediated processes, we expected that their inhibition by wortmannin resulted from deficient signaling from macrophage colony-stimulating factor (M-CSF) receptors or Fc receptors to the actin cytoskeleton. However, video microscopy showed cell surface ruffling in wortmannin-treated cells, and increased ruffling after addition of M-CSF or phorbol myristate acetate. Quantitative measurements of video data reported slightly diminished ruffling in wortmannin-treated cells. Remarkably, the ruffles that formed in wortmannin-treated macrophages all receded into the cytoplasm without closing into macropinosomes. Similarly, wortmannin and LY294002 did not inhibit the extension of actin-rich pseudopodia along IgG- opsonized sheep erythrocytes, but instead prevented them from closing into phagosomes. These findings indicate that PI 3-kinase is not necessary for receptor-mediated stimulation of pseudopod extension, but rather functions in the closure of macropinosomes and phagosomes into intracellular organelles.     

Roles of phosphatidylinositol 3-kinase in root hair growth

The root hair is a model system for understanding plant cell tip growth. As phosphatidylinositol 3-phosphate [PtdIns(3)P] has been shown in other plant cell types to regulate factors that affect root hair growth, including reactive oxygen species (ROS) levels, cytoskeleton, and endosomal movement, we hypothesized that PtdIns(3)P is also important for root hair elongation. The enzyme that generates PtdIns(3)P, phosphatidylinositol 3-kinase (PI3K), was expressed in root hair cells of transgenic plants containing the PI3K promoter:beta-glucuronidase reporter construct. To obtain genetic evidence for the role of PtdIns(3)P in root hair elongation, we attempted to isolate Arabidopsis (Arabidopsis thaliana) mutant plants that did not express the gene VPS34 encoding the PI3K enzyme. However, the homozygous mutant was lethal due to gametophytic defects, and heterozygous plants were not discernibly different from wild-type plants. Alternatively, we made transgenic plants expressing the PtdIns(3)P-binding FYVE domain in the root hair cell to block signal transduction downstream of PtdIns(3)P. These transgenic plants had shorter root hairs and a reduced hair growth rate compared with wild-type plants. In addition, LY294002, a PI3K-specific inhibitor, inhibited root hair elongation but not initiation. In LY294002-treated root hair cells, endocytosis at the stage of final fusion of the late endosomes to the tonoplast was inhibited and ROS level decreased in a dose-dependent manner. Surprisingly, the LY294002 effects on ROS and root hair elongation were similar in rhd2 mutant plants, suggesting that RHD2 was not the major ROS generator in the PtdIns(3)P-mediated root hair elongation process. Collectively, these results suggest that PtdIns(3)P is required for maintenance of the processes essential for root hair cell elongation.     

Regulation of intracellular trafficking of the EGF receptor by Rab5 in the absence of phosphatidylinositol 3-kinase activity

Rab5 and phosphatidylinositol 3-kinase (PI3K) have been proposed to co-regulate receptor endocytosis by controlling early endosome fusion. However, in this report we demonstrate that inhibition of epidermal growth factor (EGF)-stimulated PI3K activity by expression of the kinase-deficient PI3K p110 subunit (p110Δkin) does not block the lysosomal targeting and degradation of the EGF receptor (EGFR). Moreover, inhibition of total PI3K activity by wortmannin or LY294002 significantly enlarges EGFR-containing endosomes and dissociates the early-endosomal autoantigen EEA1 from membrane fractions. However, this does not block the lysosomal targeting and degradation of EGFR. In contrast, transfection of cells with mutant Rab5 S34N or microinjection of anti-Rabaptin5 antibodies inhibits EGFR endocytosis. Our results, therefore, demonstrate that PI3K is not universally required for the regulation of receptor intracellular trafficking. The present work suggests that the intracellular trafficking of EGFR is controlled by a novel endosome fusion pathway that is regulated by Rab5 in the absence of PI3K, rather than by the previously defined endosome fusion pathway that is co-regulated by Rab5 and PI3K.     

Macropinocytosis is the endocytic pathway that mediates macrophage foam cell formation with native low density lipoprotein

Previously, we reported that fluid-phase endocytosis of native LDL by PMA-activated human monocytederived macrophages converted these macrophages into cholesterol-enriched foam cells (Kruth, H. S., Huang, W., Ishii, I., and Zhang, W. Y. (2002) J. Biol. Chem. 277, 34573-34580). Uptake of fluid by cells can occur either by micropinocytosis within vesicles (<0.1 microm diameter) or by macropinocytosis within vacuoles ( approximately 0.5-5.0 microm) named macropinosomes. The current investigation has identified macropinocytosis as the pathway for fluid-phase LDL endocytosis and determined signaling and cytoskeletal components involved in this LDL endocytosis. The phosphatidylinositol 3-kinase inhibitor, LY294002, which inhibits macropinocytosis but does not inhibit micropinocytosis, completely blocked PMA-activated macrophage uptake of fluid and LDL. Also, nystatin and filipin, inhibitors of micropinocytosis from lipid-raft plasma membrane domains, both failed to inhibit PMA-stimulated macrophage cholesterol accumulation. Time-lapse video phase-contrast microscopy and time-lapse digital confocal-fluorescence microscopy with fluorescent DiI-LDL showed that PMA-activated macrophages took up LDL in the fluid phase by macropinocytosis. Macropinocytosis of LDL depended on Rho GTPase signaling, actin, and microtubules. Bafilomycin A1, the vacuolar H+-ATPase inhibitor, inhibited degradation of LDL and caused accumulation of undegraded LDL within macropinosomes and multivesicular body endosomes. LDL in multivesicular body endosomes was concentrated >40-fold over its concentration in the culture medium consistent with macropinosome shrinkage by maturation into multivesicular body endosomes. Macropinocytosis of LDL taken up in the fluid phase without receptor-mediated binding of LDL is a novel endocytic pathway that generates macrophage foam cells. Macropinocytosis in macrophages and possibly other vascular cells is a new pathway to target for modulating foam cell formation in atherosclerosis.     

Regulation of endosome fusion.

Homotypic fusion between early endosomes can be reconstituted in vitro. By using wortmannin and LY294002, inhibitors of phosphatidylinositol (Pl) 3-kinase, a requirement for this activity has been established in order for fusion to proceed efficiently. It has been shown that Pl 3-kinase activity is required downstream of rab5 activation, although a large excess of activated rab5 can overcome wortmannin inhibition. A series of experiments have also been performed which indicate a role for early endosomal autoantigen 1 (EEA1) in determining fusion efficiency. EEA1 dissociates from membranes following wortmannin treatment. It is proposed that the requirement of endosome fusion for Pl 3-kinase activity is to promote the association of EEA1 with endosomes.     

3-Methyladenine specifically inhibits retrograde transport of cation-independent mannose 6-phosphate/insulin-like growth factor II receptor from the early endosome to the TGN

3-Methyladenine (3-MA), a well-known inhibitor of autophagic sequestration, can also prevent class III phosphatidylinositide (PI) 3-kinase activity, which is required for many processes in endosomal membrane trafficking. Although much is known about the effects of other PI 3-kinase inhibitors, such as wortmannin and LY294002, on endosomal membrane trafficking, little is known about those of 3-MA. Here we show that the treatment of cells with 3-MA results in a specific redistribution of the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (MPR300) from the trans-Golgi network (TGN) to early/recycling endosomal compartments containing internalized transferrin. Importantly, in contrast to wortmannin and LY294002, 3-MA did not cause the enlargement of late endosomal/lysosomal compartments. The results suggest that the effect of 3-MA is restricted to the retrieval of MPR300 from early/recycling endosomes.     

Regulation of epidermal growth factor receptor endocytosis by wortmannin through activation of Rab5 rather than inhibition of phosphatidylinositol 3-kinase

The involvement of phosphatidylinositol 3-kinase (PI3K) in membrane trafficking in mammalian cells has largely come from experiments with wortmannin. This compound inhibits endosome fusion in vitro, possibly by inhibiting the production of phosphatidylinositol (PtdIns)-3-P, which co-regulates EEA1 with Rab5. However, the results from wortmannin inhibition experiments performed in vivo differ significantly. We have recently shown that wortmannin enlarges endosomes containing the epidermal growth factor receptor (EGFR) and enhances the lysosomal degradation of EGFR. In this report, we demonstrate that addition of the PI3K reaction products does not suppress wortmannin-induced enlargement of EGFR-containing endosomes and enhancement of EGFR degradation. Moreover, the effects of wortmannin on the intracellular trafficking of EGFR mimic those of the permanently activated Rab5 mutant, Rab5 Q79L, which stimulates endosome fusion. We also found that an inactive Rab5 mutant, Rab5 S34N, blocks wortmannin-induced endosome enlargement and that wortmannin stimulates the activation of Rab5. We further showed that wortmannin reduced the membrane association of p120 Ras GTPase-activating protein (GAP) and inhibited the interaction between Rab5 and p120 Ras GAP. We conclude that wortmannin alters intracellular trafficking of EGFR by activating Rab5 rather than by inhibiting PI3K.     

Phosphatidylinositol 3- and 4-phosphate are required for normal stomatal movements

Phosphatidylinositol (PI) metabolism plays a central role in signaling pathways in both animals and higher plants. Stomatal guard cells have been reported to contain PI 3-phosphate (PI3P) and PI 4-phosphate (PI4P), the products of PI 3-kinase (PI3K) and PI 4-kinase (PI4K) activities. In this study, we tested the roles of PI3P and PI4P in stomatal movements. Both wortmannin (WM) and LY294002 inhibited PI3K and PI4K activities in guard cells and promoted stomatal opening induced by white light or the circadian clock. WM and LY294002 also inhibited stomatal closing induced by abscisic acid (ABA). Furthermore, overexpression in guard cells of GFP:EBD (green fluorescent protein:endosome binding domain of human EEA1) or GFP:FAPP1PH (PI-four-P adaptor protein-1 pleckstrin homology domain), which bind to PI3P and PI4P, respectively, increased stomatal apertures under darkness and white light and partially inhibited stomatal closing induced by ABA. The reduction in ABA-induced stomatal closing with reduced levels of PI monophosphate seemed to be attributable, at least in part, to impaired Ca(2+) signaling, because WM and LY294002 inhibited ABA-induced cytosolic Ca(2+) increases in guard cells. These results suggest that PI3P and PI4P play an important role in the modulation of stomatal closing and that reductions in the levels of functional PI3P and PI4P enhance stomatal opening.     

The phosphoinositol-3-kinase-protein kinase B/Akt pathway is critical for Pseudomonas aeruginosa strain PAK internalization

Several Pseudomonas aeruginosa strains are internalized by epithelial cells in vitro and in vivo, but the host pathways usurped by the bacteria to enter nonphagocytic cells are not clearly understood. Here, we report that internalization of strain PAK into epithelial cells triggers and requires activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B/Akt (Akt). Incubation of Madin-Darby canine kidney (MDCK) or HeLa cells with the PI3K inhibitors LY294002 (LY) or wortmannin abrogated PAK uptake. Addition of the PI3K product phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] to polarized MDCK cells was sufficient to increase PAK internalization. PtdIns(3,4,5)P3 accumulated at the site of bacterial binding in an LY-dependent manner. Akt phosphorylation correlated with PAK invasion. The specific Akt phosphorylation inhibitor SH-5 inhibited PAK uptake; internalization also was inhibited by small interfering RNA-mediated depletion of Akt phosphorylation. Expression of constitutively active Akt was sufficient to restore invasion when PI3K signaling was inhibited. Together, these results demonstrate that the PI3K signaling pathway is necessary and sufficient for the P. aeruginosa entry and provide the first example of a bacterium that requires Akt for uptake into epithelial cells.     

A role for phosphatidylinositol 3-phosphate in abscisic acid-induced reactive oxygen species generation in guard cells

Guard cells generate reactive oxygen species (ROS) in response to abscisic acid (ABA), which leads to stomatal closing. The upstream steps of the ABA-induced ROS generation pathway remain largely unknown. In animal cells, ROS generation in neutrophils is activated by phosphatidylinositol 3-phosphate (PI3P). Stomatal guard cells contain PI3P and PI 3-kinase activity. In this study, we tested whether PI3P has a role in ROS generation in guard cells exposed to ABA. We found that PI 3-kinase inhibitors wortmannin or LY294002 inhibited ABA-induced ROS generation and stomatal closing. Endosome-binding domain (of human EEA1), which specifically binds to PI3P, also inhibited ABA-induced ROS generation and stomatal closing when overexpressed in guard cells. Hydrogen peroxide partially reversed the effects of wortmannin or LY294002 on ABA-induced stomatal closing. These results support a role for PI3P in ABA-induced ROS generation and stomatal closing movement.     

Fluid-Phase Pinocytosis of Native Low Density Lipoprotein Promotes Murine M-CSF Differentiated Macrophage Foam Cell Formation

During atherosclerosis, low-density lipoprotein (LDL)-derived cholesterol accumulates in macrophages to form foam cells. Macrophage uptake of LDL promotes foam cell formation but the mechanism mediating this process is not clear. The present study investigates the mechanism of LDL uptake for macrophage colony-stimulating factor (M-CSF)-differentiated murine bone marrow-derived macrophages. LDL receptor-null (LDLR−/−) macrophages incubated with LDL showed non-saturable accumulation of cholesterol that did not down-regulate for the 24 h examined. Incubation of LDLR−/− macrophages with increasing concentrations of ¹²⁵I-LDL showed non-saturable macrophage LDL uptake. A 20-fold excess of unlabeled LDL had no effect on ¹²⁵I-LDL uptake by wild-type macrophages and genetic deletion of the macrophage scavenger receptors CD36 and SRA did not affect ¹²⁵I-LDL uptake, showing that LDL uptake occurred by fluid-phase pinocytosis independently of receptors. Cholesterol accumulation was inhibited approximately 50% in wild-type and LDLR−/− mice treated with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or wortmannin, inhibitors of all classes of phosphoinositide 3-kinases (PI3K). Time-lapse, phase-contrast microscopy showed that macropinocytosis, an important fluid-phase uptake pathway in macrophages, was blocked almost completely by PI3K inhibition with wortmannin. Pharmacological inhibition of the class I PI3K isoforms alpha, beta, gamma or delta did not affect macrophage LDL-derived cholesterol accumulation or macropinocytosis. Furthermore, macrophages from mice expressing kinase-dead class I PI3K beta, gamma or delta isoforms showed no decrease in cholesterol accumulation or macropinocytosis when compared with wild-type macrophages. Thus, non-class I PI3K isoforms mediated macropinocytosis in these macrophages. Further characterization of the components necessary for LDL uptake, cholesterol accumulation, and macropinocytosis identified dynamin, microtubules, actin, and vacuolar type H(+)-ATPase as contributing to uptake. However, Pak1, Rac1, and Src-family kinases, which mediate fluid-phase pinocytosis in certain other cell types, were unnecessary. In conclusion, our findings provide evidence that targeting those components mediating macrophage macropinocytosis with inhibitors may be an effective strategy to limit macrophage accumulation of LDL-derived cholesterol in arteries.     

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.     

Class III Phosphoinositide 3‐Kinase/VPS34 and Dynamin are Critical for Apical Endocytic Recycling

Recycling is a limiting step for receptor‐mediated endocytosis. We first report three in vitro or in vivo evidences that class III PI3K/VPS34 is the key PI3K isoform regulating apical recycling. A substractive approach, comparing in Opossum Kidney (OK) cells a pan‐class I/II/III PI3K inhibitor (LY294002) with a class I/II PI3K inhibitor (ZSTK474), suggested that class III PI3K/VPS34 inhibition induced selective apical endosome swelling and sequestration of the endocytic receptor, megalin/LRP‐2, causing surface down‐regulation. GFP‐(FYVE)x2 overexpression to sequester PI(3)P caused undistinguishable apical endosome swelling. In mouse kidney proximal tubular cells, conditional Vps34 inactivation also led to vacuolation and intracellular megalin redistribution. We next report that removal of LY294002 from LY294002‐treated OK cells induced a spectacular burst of recycling tubules and restoration of megalin surface pool. Acute triggering of recycling tubules revealed recruitment of dynamin‐GFP and dependence of dynamin‐GTPase, guidance directionality by microtubules, and suggested that a microfilamentous net constrained endosomal swelling. We conclude that (i) besides its role in endosome fusion, PI3K‐III is essential for endosome fission/recycling; and (ii) besides its role in endocytic entry, dynamin also supports tubulation of recycling endosomes. The unleashing of recycling upon acute reversal of PI3K inhibition may help study its dynamics and associated machineries.     

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.     

Cholesterol is required for endocytosis and endosomal escape of adenovirus type 2

The species C adenovirus type 2 (Ad2) and Ad5 bind the coxsackievirus B Ad receptor and alphav integrin coreceptors and enter epithelial cells by clathrin-mediated endocytosis. This pathway is rapid and efficient. It leads to cell activation and the cholesterol-dependent formation of macropinosomes. Macropinosomes are triggered to release their contents when incoming Ad2 escapes from endosomes. Here, we show that cholesterol extraction of epithelial cells by methyl-beta-cyclodextrin (mbetaCD) treatment reduced Ad5-mediated luciferase expression approximately 4-fold. The addition of cholesterol to normal cells increased gene expression in a dose-dependent manner up to threefold, but it did not restore gene expression in mbetaCD-treated cells. mbetaCD had no effect in the presence of excess cholesterol, indicating that the inhibition of gene expression was due specifically to cholesterol depletion. Cholesterol depletion inhibited rapid Ad2 endocytosis, endosomal escape, and nuclear targeting, consistent with the notion that clathrin-dependent endocytosis of Ad2 is cholesterol dependent. In cholesterol-reduced cells, Ad2 internalized at a low rate, suggestive of an alternative, clathrin-independent, low-capacity entry pathway. While exogenous cholesterol completely restored rapid Ad2 endocytosis, macropinocytosis, and macropinosome disruption, it did not, surprisingly, restore viral escape from endosomes. Our results indicate that macropinosome disruption and endosomal escape of Ad2 are independent events in cells depleted of and then refilled with cholesterol, suggesting that viral escape from endosomes requires lipid-controlled membrane homeostasis, trafficking, or signaling.     

The SNX-PX-BAR family in macropinocytosis: the regulation of macropinosome formation by SNX-PX-BAR proteins

Background

Macropinocytosis is an actin-driven endocytic process, whereby membrane ruffles fold back onto the plasma membrane to form large (>0.2 µm in diameter) endocytic organelles called macropinosomes. Relative to other endocytic pathways, little is known about the molecular mechanisms involved in macropinocytosis. Recently, members of the Sorting Nexin (SNX) family have been localized to the cell surface and early macropinosomes, and implicated in macropinosome formation. SNX-PX-BAR proteins form a subset of the SNX family and their lipid-binding (PX) and membrane-curvature sensing (BAR) domain architecture further implicates their functional involvement in macropinosome formation.

Methodology/Principal Findings

We exploited the tractability of macropinosomes through image-based screening and systematic overexpression of SNX-PX-BAR proteins to quantitate their effect on macropinosome formation. SNX1 (40.9+/−3.19 macropinosomes), SNX5 (36.99+/−4.48 macropinosomes), SNX9 (37.55+/−2.4 macropinosomes), SNX18 (88.2+/−8 macropinosomes), SNX33 (65.25+/−6.95 macropinosomes) all exhibited statistically significant (p<0.05) increases in average macropinosome numbers per 100 transfected cells as compared to control cells (24.44+/−1.81 macropinosomes). SNX1, SNX5, SNX9, and SNX18 were also found to associate with early-stage macropinosomes within 5 minutes following organelle formation. The modulation of intracellular PI(3,4,5)P₃ levels through overexpression of PTEN or a lipid phosphatase-deficient mutant PTEN(G129E) was also observed to significantly reduce or elevate macropinosome formation respectively; coexpression of PTEN(G129E) with SNX9 or SNX18 synergistically elevated macropinosome formation to 119.4+/−7.13 and 91.4+/−6.37 macropinosomes respectively (p<0.05).

Conclusions/Significance

SNX1, SNX5, SNX9, SNX18, and SNX33 were all found to elevate macropinosome formation and (with the exception of SNX33) associate with early-stage macropinosomes. Moreover the effects of SNX9 and SNX18 overexpression in elevating macropinocytosis is likely to be synergistic with the increase in PI(3,4,5)P₃ levels, which is known to accumulate on the cell surface and early-stage macropinocytic cups. Together these findings represent the first systematic functional study into the impact of the SNX-PX-BAR family on macropinocytosis.     

Characterization of a G protein-activated phosphoinositide 3-kinase in vascular smooth muscle cell nuclei

Recent studies highlight the existence of an autonomous nuclear polyphosphoinositide metabolism related to cellular proliferation and differentiation. However, only few data document the nuclear production of the putative second messengers, the 3-phosphorylated phosphoinositides, by the phosphoinositide 3-kinase (PI3K). In the present paper, we examine whether GTP-binding proteins can directly modulate 3-phosphorylated phosphoinositide metabolism in membrane-free nuclei isolated from pig aorta smooth muscle cells (VSMCs). In vitro PI3K assays performed without the addition of any exogenous substrates revealed that guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) specifically stimulated the nuclear synthesis of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), whereas guanosine 5'-(beta-thio)diphosphate was ineffective. PI3K inhibitors wortmannin and LY294002 prevented GTPgammaS-induced PtdIns(3,4,5)P(3) synthesis. Moreover, pertussis toxin inhibited partially PtdIns(3,4,5)P(3) accumulation, suggesting that nuclear G(i)/G(0) proteins are involved in the activation of PI3K. Immunoblot experiments showed the presence of Galpha(0) proteins in VSMC nuclei. In contrast with previous reports, immunoblots and indirect immunofluorescence failed to detect the p85alpha subunit of the heterodimeric PI3K within VSMC nuclei. By contrast, we have detected the presence of a 117-kDa protein immunologically related to the PI3Kgamma. These results indicate the existence of a G protein-activated PI3K inside VSMC nucleus that might be involved in the control of VSMC proliferation and in the pathogenesis of vascular proliferative disorders.     

Vps34p differentially regulates endocytosis from the apical and basolateral domains in polarized hepatic cells

Using a microinjection approach to study apical plasma membrane protein trafficking in hepatic cells, we found that specific inhibition of Vps34p, a class III phosphoinositide 3 (PI-3) kinase, nearly perfectly recapitulated the defects we reported for wortmannin-treated cells (Tuma, P.L., C.M. Finnegan, J.-H Yi, and A.L. Hubbard. 1999. J. Cell Biol. 145:1089-1102). Both wortmannin and injection of inhibitory Vps34p antibodies led to the accumulation of resident apical proteins in enlarged prelysosomes, whereas transcytosing apical proteins and recycling basolateral receptors transiently accumulated in basolateral early endosomes. To understand how the Vps34p catalytic product, PI3P, was differentially regulating endocytosis from the two domains, we examined the PI3P binding protein early endosomal antigen 1 (EEA1). We determined that EEA1 distributed to two biochemically distinct endosomal populations: basolateral early endosomes and subapical endosomes. Both contained rab5, although the latter also contained late endosomal markers but was distinct from the transcytotic intermediate, the subapical compartment. When PI3P was depleted, EEA1 dissociated from basolateral endosomes, whereas it remained on subapical endosomes. From these results, we conclude that PI3P, via EEA1, regulates early steps in endocytosis from the basolateral surface in polarized WIF-B cells. However, PI3P must use different machinery in its regulation of the apical endocytic pathway, since later steps are affected by Vps34p inhibition.     

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.