Rac is required for constitutive macropinocytosis by dendritic cells but does not control its downregulation

BACKGROUND: Dendritic cells use constitutive macropinocytosis to capture exogenous antigens for presentation on MHC molecules. Upon exposure to inflammatory stimuli or bacterial products such as lipopolysaccharide (LPS), macropinocytosis is dramatically downregulated as part of a developmental programme leading to dendritic cell maturation, migration and activation of T cells. It is not known, however, how macropinocytosis is sustained in dendritic cells in the absence of exogenous stimuli, nor how it is downregulated upon maturation. We have tested the possibility that one or more members of the Rho family of GTPases are involved in and control pinocytosis in dendritic cells. RESULTS: We established dendritic cell populations that show constitutive macropinocytosis that was downregulated by LPS treatment. Microinjection of immature cells with dominant-negative Rac (N17Rac1) or treatment with Clostridium difficile toxin B, the phosphoinositide 3-kinase (PI3-K) inhibitor wortmannin, or LPS all inhibited the formation of macropinosomes but, surprisingly, did not eliminate membrane ruffling. Microinjection of N17Cdc42 or the Rho inhibitor C3 transferase eliminated actin plaques/podosomes and actin cables, respectively, but had little effect on the formation of macropinosomes. Surprisingly, dendritic cells matured with LPS had equivalent or even somewhat higher levels of active Rac than immature cells. Moreover, microinjection of a constitutively active form of Rac (V12Rac1) into mature dendritic cells did not reactivate macropinocytosis. CONCLUSIONS: Rac has an important role in the constitutive formation of macropinosomes in dendritic cells but may be required downstream of membrane ruffling. Furthermore, regulation of Rac activity does not appear to be the control point in the physiological downregulation of dendritic cell pinocytosis. Instead, one or more downstream effectors may be modulated to allow Rac to continue to regulate other cellular functions.     

SWAP-70 associates transiently with macropinosomes

Cells accomplish the non-selective uptake of extracellular fluids, antigens and pathogens by the endocytic process of macropinocytosis. The protein SWAP-70 is a widely expressed, pleckstrin-homology (PH) domain-containing protein that marks a transitional subset of actin filaments in motile cells. Here we report that the protein SWAP-70 associates transiently with macropinosomes in dendritic cells and NIH/3T3 fibroblasts. The association of SWAP-70 with macropinosomes is preceded by the accumulation of Rac-GTP and followed by that of Rab5. Three regions of SWAP-70, the N-terminal region, the PH domain and the C-terminal region, contribute in a combinatorial manner to the transient association with newly formed macropinosomes in the cell periphery and occasionally with aged macropinosomes on their passage to the cell center. These data identify SWAP-70 as a transient component of early macropinosomes.     

Inactivation of lmpA, encoding a LIMPII-related endosomal protein, suppresses the internalization and endosomal trafficking defects in profilin-null mutants

Profilin is a key phosphoinositide and actin-binding protein connecting and coordinating changes in signal transduction pathways with alterations in the actin cytoskeleton. Using biochemical assays and microscopic approaches, we demonstrate that profilin-null cells are defective in macropinocytosis, fluid phase efflux, and secretion of lysosomal enzymes but are unexpectedly more efficient in phagocytosis than wild-type cells. Disruption of the lmpA gene encoding a protein (DdLIMP) belonging to the CD36/LIMPII family suppressed, to different degrees, most of the profilin-minus defects, including the increase in F-actin, but did not rescue the secretion defect. Immunofluorescence microscopy indicated that DdLIMP, which is also capable of binding phosphoinositides, was associated with macropinosomes but was not detected in the plasma membrane. Also, inactivation of the lmpA gene in wild-type strains resulted in defects in macropinocytosis and fluid phase efflux but not in phagocytosis. These results suggest an important role for profilin in regulating the internalization of fluid and particles and the movement of material along the endosomal pathway; they also demonstrate a functional interaction between profilin and DdLIMP that may connect phosphoinositide-based signaling through the actin cytoskeleton with endolysosomal membrane trafficking events.     

Akt/PKB localisation and 3' phosphoinositide generation at sites of epithelial cell-matrix and cell-cell interaction

Protein kinase B (PKB or Akt) is a mitogen-regulated protein kinase involved in the protection of cells from apoptosis, the promotion of cell proliferation and diverse metabolic responses [1]. Its activation is initiated by the binding of 3' phosphorylated phosphoinositide lipids to its pleckstrin homology (PH) domain, resulting in the induction of activating phosphorylation at residues Thr308 and Ser473 by upstream kinases such as phosphoinositide-dependent protein kinase-1 (PDK1) [2]. Adhesion of epithelial cells to extracellular matrix leads to protection from apoptosis via the activation of phosphoinositide (PI) 3-kinase and Akt/PKB through an unknown mechanism [3] [4]. Here, we use the localisation of Akt/PKB within the cell to probe the sites of induction of PI 3-kinase activity. In fibroblasts, immunofluorescence microscopy showed that endogenous Akt/PKB localised to membrane ruffles at the outer edge of the cell following mitogen treatment as did green fluorescent protein (GFP) fusions with full-length Akt/PKB or its PH domain alone. In epithelial cells, the PH domain of Akt/PKB localised to sites of cell-cell and cell-matrix contact, distinct from focal contacts, even in the absence of serum. As this localisation was disrupted by PI 3-kinase inhibitory drugs and by mutations that inhibit interaction with phosphoinositides, it is likely to represent the sites of constitutive 3' phosphoinositide generation that provide a cellular survival signal. We propose that the attachment-induced, PI-3-kinase-mediated survival signal in epithelial cells is generated not only by cell-matrix interaction but also by cell-cell interaction.     

A novel phosphatidylinositol 4,5-bisphosphate-binding domain targeting the Phg2 kinase to the membrane in Dictyostelium cells

Phg2 is a ser/thr kinase involved in adhesion, motility, actin cytoskeleton dynamics, and phagocytosis in Dictyostelium cells. In a search for Phg2 domains required for its localization to the plasma membrane, we identified a new domain interacting with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and phosphatidylinositol 4-phosphate (PI(4)P) membrane phosphoinositides. Deletion of this domain prevented membrane recruitment of Phg2 and proper function of the protein in the phagocytic process. Moreover, the overexpression of this PI(4,5)P₂-binding domain specifically had a dominant-negative effect by inhibiting phagocytosis. Therefore, plasma membrane recruitment of Phg2 is essential for its function. The PI(4,5)P₂-binding domain fused to GFP (green fluorescent protein) (GFP-Nt-Phg2) was also used to monitor the dynamics of PI(4,5)P₂ during macropinocytosis and phagocytosis. GFP-Nt-Phg2 disappeared from macropinosomes immediately after their closure. During phagocytosis, PI(4,5)P₂ disappeared even before the sealing of phagosomes as it was already observed in mammalian cells. Together these results demonstrate that PI(4,5)P₂ metabolism regulates the dynamics and the function of Phg2.     

N‐Cadherin Regulates Cell Migration Through a Rab5‐Dependent Temporal Control of Macropinocytosis

Macropinocytosis is a clathrin‐independent endocytic pathway implicated in fluid uptake, pathogen invasion and cell migration. During collective cell migration, macropinocytosis occurs primarily at membrane ruffles arising from the leading edges of migrating cells. We report here that N‐cadherin (Ncad) regulates the tempo of macropinocytosis and thereby influences wound‐induced collective cell migration. Using live‐cell and super‐resolution imaging techniques, we observed that Ncad formed clusters at the membrane ruffles and macropinosomes. De‐clustering of Ncad by an interfering antibody impaired the recruitment of Rab5‐an early endosomal marker‐to the macropinosomes. Moreover, we demonstrated that Ncad interacts with Rab5, and laser ablation of Ncad caused Rab5 to dissociate from the macropinosomes. Although Rab5 detached from macropinosomes upon the de‐clustering of Ncad, the recruitment of late endosomal marker Rab7 occurred earlier. Consequently, both centripetal trafficking of macropinosomes and collective migration were accelerated due to de‐clustering of Ncad. Thus, our results suggest that Ncad is involved in the maturation of macropinocytosis through Rab5 recruitment, linking macropinocytosis and cell migration through a novel function of Ncad.      

Effect of 3-methyladenine on the fusion process of macropinosomes in EGF-stimulated A431 cells

In the process of receptor-mediated endocytosis, the fusion of endosomes in vitro is known to be inhibited by wortmannin or LY294002; inhibitors of phosphoinositide 3-kinase (PI3K), suggesting that the activity of PI3K is required for the fusion of early endosomes. In macropinocytosis, a process of bulk fluid-phase endocytosis, however, it remains unclear whether PI3K is required for the fusion of macropinosomes, since the macropinosome formation is inhibited by the PI3K inhibitors. In this study, we examined the effect of 3-methlyadenine (3-MA), which shows a distinct specificity to the PI3K classes from wortmannin and LY294002, on the macropinosome formation and fusion in EGF-stimulated A431 cells. Unlike wortmannin or LY294002, 3-MA did not inhibit the uptake of fluorescent dextran by macropinocytosis. However, the fusion of macropinosomes was inhibited by 3-MA. By imaging of live-cells expressing fluorescent protein-fused tandem FYVE domains, we found that PtdIns(3)P appeared on the macropinosomal membrane shortly after the closure of macropinocytic cups and remained on macropinosomes even at 60-min age. The production of PtdIns(3)P and the recruitment of EEA1 to macropinosomes were abolished by the 3-MA treatment. Therefore, it is likely that 3-MA impairs recruitment of EEA1 by inhibiting PtdIns(3)P production and resultantly blocks the fusion of macropinosomes. These results suggest that the local production of PtdIns(3)P implicates the fusion of macropinosomes via EEA1 as well as conventional early endosomes. However, the long association of PtdIns(3)P with macropinosomes may well be a cell-type specific feature of A431 cells.     

A unique platform for H-Ras signaling involving clathrin-independent endocytosis

Trafficking of H-Ras was examined to determine whether it can enter cells through clathrin-independent endocytosis (CIE). H-Ras colocalized with the CIE cargo protein, class I major histocompatibility complex, and it was sequestered in vacuoles that formed upon expression of an active mutant of Arf6, Q67L. Activation of Ras, either through epidermal growth factor stimulation or the expression of an active mutant of Ras, G12V, induced plasma membrane ruffling and macropinocytosis, a stimulated form of CIE. Live imaging of cells expressing H-RasG12V and fluorescent protein chimeras with pleckstrin homology domains that recognize specific phosphoinositides showed that incoming macropinosomes contained phosphatidylinositol 4,5-bisphosphate (PIP(2)) and phosphatiylinositol 3,4,5-trisphosphate (PIP(3)). PIP(2) loss from the macropinosome was followed by the recruitment of Rab5, a downstream target of Ras, and then PIP(3) loss. Our studies support a model whereby Ras can signal on macropinosomes that pass through three distinct stages: PIP(2)/PIP(3), PIP(3)/Rab5, and Rab5. Vacuoles that form in cells expressing Arf6Q67L trap Ras signaling in the first stage, recruiting the active form of the Ras effectors extracellular signal-regulated kinase and protein kinase B (Akt) but not Rab5. Arf6 stimulation of macropinocytosis also involves passage through the distinct lipid phases, but recruitment of Akt is not observed.     

Cortactin is essential for F-actin assembly in enteropathogenic Escherichia coli (EPEC)- and enterohaemorrhagic E. coli (EHEC)-induced pedestals and the alpha-helical region is involved in the localization of cortactin to bacterial attachment sites

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are important human pathogens. Upon attachment to host cells, EPEC and EHEC are able to induce actin polymerization, which accumulates, forming a pedestal-like structure beneath the attached bacteria. Using siRNA, we show here that EPEC- and EHEC-induced pedestals are dependent on cortactin, an F-actin-binding protein found in the mammalian cell cortex. Knock-down of cortactin by siRNA resulted in a dramatic reduction of the pedestal formation induced by both pathogens. We also show that disruption of the Src homology 3 (SH3) domain of cortactin, or its downregulation by specific point mutations, negatively affects pedestal formation, suggesting that this domain is important for regulation of F-actin assembly by EPEC and EHEC. Green fluorescent protein (GFP) fused with the SH3 domain (GFP-SH3), proline-rich region (GFP-PRR) or alpha-helical region of cortactin markedly reduced the amount of F-actin at the bacterial attachment sites. Interestingly, neither GFP-SH3 nor GFP-PRR was recruited to the vicinity of the bacterial adherence sites; however, GFP fused to the alpha-helical region was efficiently recruited and colocalized with the attached bacteria. These results demonstrate that cortactin is a requirement for pedestal formation and suggest a novel function for the predicted alpha-helical region of cortactin in actin assembly induced by EPEC and EHEC.     

Macropinocytosis: an endocytic pathway for internalising large gulps

Macropinocytosis is a regulated form of endocytosis that mediates the non-selective uptake of solute molecules, nutrients and antigens. It is an actin-dependent process initiated from surface membrane ruffles that give rise to large endocytic vacuoles called macropinosomes. Macropinocytosis is important in a range of physiological processes; it is highly active in macrophages and dendritic cells where it is a major pathway for the capture of antigens, it is relevant to cell migration and tumour metastasis and it represents a portal of cell entry exploited by a range of pathogens. The molecular basis for the formation and maturation of macropinosomes has only recently begun to be defined. Here, we review the general characteristics of macropinocytosis, describe some of the regulators of this pathway, which have been identified to date and highlight strategies to explore the relevance of this endocytosis pathway in vivo.     

Phagocytosis and macropinocytosis in Dictyostelium: phosphoinositide-based processes, biochemically distinct

Phagocytosis and macropinocytosis are actin-dependent clathrin-independent processes primarily performed by cells like neutrophils and macrophages that result in the internalization of particles or the formation of fluid-filled macropinosomes, respectively. Phagocytosis consists of a number of stages, including attachment of particles to cell surface receptors, engulfment of the particle dependent on actin polymerization and membrane exocytosis, and formation of phago-lysosomes. In contrast, the molecular steps regulating macropinocytosis are only just now being deciphered. Much remains to be learned concerning the signaling pathways that regulate these processes. Dictyostelium is a genetically and biochemically tractable professional phagocyte that has proven to be a powerful system with which to determine the nature of the molecular steps involved in regulating these internalization processes. This review summarizes what is currently understood concerning the molecular mechanisms governing phagocytosis and macropinocytosis in Dictyostelium and describes recent data concerning the common and distinct pathways that regulate these processes.     

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.     

Dynamics of phosphatidylinositol 4,5-bisphosphate in actin-rich structures

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is known to regulate a wide range of molecular targets and cellular processes, from ion channels to actin polymerization [1] [2] [3] [4] [5] [6]. Recent studies have used the phospholipase C-delta1 (PLC-delta1) pleckstrin-homology (PH) domain fused to green fluorescent protein (GFP) as a detector for PI(4,5)P(2) in vivo [7] [8] [9] [10]. Although these studies demonstrated that PI(4,5)P(2) is concentrated in the plasma membrane, its association with actin-containing structures was not reported. In the present study, fluorescence imaging of living NIH-3T3 fibroblasts expressing the PLC-delta1 PH domain linked to enhanced green fluorescent protein (PH-EGFP) reveals intense, non-uniform fluorescence in distinct structures at the cell periphery. Corresponding fluorescence and phase-contrast imaging over time shows that these fluorescent structures correlate with dynamic, phase-dense features identified as ruffles and with microvillus-like protrusions from the cell's dorsal surface. Imaging of fixed and permeabilized cells shows co-localization of PH-EGFP with F-actin in ruffles, but not with vinculin in focal adhesions. The selective concentration of the PH-EGFP fusion protein in highly dynamic regions of the plasma membrane that are rich in F-actin supports the hypothesis that localized synthesis and lateral segregation of PI(4,5)P(2) spatially restricts actin polymerization and thereby affects cell spreading and retraction.     

Coxsackievirus entry across epithelial tight junctions requires occludin and the small GTPases Rab34 and Rab5

The major group B coxsackievirus (CVB) receptor is a component of the epithelial tight junction (TJ), a protein complex that regulates the selective passage of ions and molecules across the epithelium. CVB enters polarized epithelial cells from the TJ, causing a transient disruption of TJ integrity. Here we show that CVB does not induce major reorganization of the TJ, but stimulates the specific internalization of occludin-a TJ integral membrane component-within macropinosomes. Although occludin does not interact directly with virus, depletion of occludin prevents CVB entry into the cytoplasm and inhibits infection. Both occludin internalization and CVB entry require caveolin but not dynamin; both are blocked by inhibitors of macropinocytosis and require the activity of Rab34, Ras, and Rab5, GTPases known to regulate macropinocytosis. Thus, CVB entry depends on occludin and occurs by a process that combines aspects of caveolar endocytosis with features characteristic of macropinocytosis.     

Regulation of Macropinocytosis by Diacylglycerol Kinase ζ

Macropinosomes arise from the closure of plasma membrane ruffles to bring about the non-selective uptake of nutrients and solutes into cells. The morphological changes underlying ruffle formation and macropinosome biogenesis are driven by actin cytoskeleton rearrangements under the control of the Rho GTPase Rac1. We showed previously that Rac1 is activated by diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show DGKζ is required for optimal macropinocytosis induced by growth factor stimulation of mouse embryonic fibroblasts. Time-lapse imaging of live cells and quantitative analysis revealed DGKζ was associated with membrane ruffles and nascent macropinosomes. Macropinocytosis was attenuated in DGKζ-null cells, as determined by live imaging and vaccinia virus uptake experiments. Moreover, macropinosomes that did form in DGKζ-null cells were smaller than those found in wild type cells. Rescue of this defect required DGKζ catalytic activity, consistent with it also being required for Rac1 activation. A constitutively membrane bound DGKζ mutant substantially increased the size of macropinosomes and potentiated the effect of a constitutively active Rac1 mutant on macropinocytosis. Collectively, our results suggest DGKζ functions in concert with Rac1 to regulate macropinocytosis.     

The tumor suppressor PTEN positively regulates macroautophagy by inhibiting the phosphatidylinositol 3-kinase/protein kinase B pathway

The tumor suppressor PTEN is a dual protein and phosphoinositide phosphatase that negatively controls the phosphatidylinositol (PI) 3-kinase/protein kinase B (Akt/PKB) signaling pathway. Interleukin-13 via the activation of the class I PI 3-kinase has been shown to inhibit the macroautophagic pathway in the human colon cancer HT-29 cells. Here we demonstrate that the wild-type PTEN is expressed in this cell line. Its overexpression directed by an inducible promoter counteracts the interleukin-13 down-regulation of macroautophagy. This effect was dependent upon the phosphoinositide phosphatase activity of PTEN as determined by using the mutant G129E, which has only protein phosphatase activity. The role of Akt/PKB in the signaling control of interleukin-13-dependent macroautophagy was investigated by expressing a constitutively active form of the kinase ((Myr)PKB). Under these conditions a dramatic inhibition of macroautophagy was observed. By contrast a high rate of autophagy was observed in cells expressing a dominant negative form of PKB. These data demonstrate that the signaling control of macroautophagy overlaps with the well known PI 3-kinase/PKB survival pathway and that the loss of PTEN function in cancer cells inhibits a major catabolic pathway.     

Synaptopodin, a molecule involved in the formation of the dendritic spine apparatus, is a dual actin/alpha-actinin binding protein

Synaptopodin (SYNPO) is a cytoskeletal protein that is preferentially located in mature dendritic spines, where it accumulates in the spine neck and closely associates with the spine apparatus. Formation of the spine apparatus critically depends on SYNPO. To further determine its molecular action, we screened for cellular binding partners. Using the yeast two-hybrid system and biochemical assays, SYNPO was found to associate with both F-actin and alpha-actinin. Ectopic expression of SYNPO in neuronal and non-neuronal cells induced actin aggregates, thus confirming a cytoplasmic interaction with the actin cytoskeleton. Whereas F-actin association is mediated by a central SYNPO motif, binding to alpha-actinin requires the C-terminal domain. Notably, the alpha-actinin binding domain is also essential for dendritic targeting and postsynaptic accumulation of SYNPO in primary neurons. Taken together, our data suggest that dendritic spine accumulation of SYNPO critically depends on its interaction with postsynaptic alpha-actinin and that SYNPO may regulate spine morphology, motility and function via its distinct modes of association with the actin cytoskeleton.