Burkholderia cenocepacia Induces Macropinocytosis to Enter Macrophages.
ABSTRACT: Burkholderia cenocepacia is an opportunistic pathogen that infects individuals with cystic fibrosis, chronic granulomatous disease, and other immunocompromised states. B. cenocepacia survives in macrophages in membrane-bound vacuoles; however, the mechanism by which B. cenocepacia gains entry into macrophages remains unknown. After macrophage internalization, survival of B. cenocepacia within a bacteria-containing membrane vacuole (BcCV) is associated with its ability to arrest the maturation of the BcCV. In this study, we show that B. cenocepacia induces localized membrane ruffling, macropinocytosis, and macropinosomes-like compartments upon contact with the macrophage. The Type 3 Secretion System (T3SS) of B. cenocepacia contributes to macrophage entry and macropinosome-like compartment formation. These data demonstrate the ability of Burkholderia to enter macrophages through the induction of macropinocytosis.
Project description:Macropinocytosis mediates the bulk endocytosis of solute molecules, nutrients and antigens. As this endocytic pathway is considered important in functions associated with immune responses, the molecular mechanisms regulating this pathway in immune cells is of particular significance. However, the regulators of macropinocytosis in primary cells remain poorly defined. Members of the sorting nexin (SNX) family have been implicated in macropinosome biogenesis in cultured cells and here we have analyzed the role of two SNX family members, SNX1 and its binding partner SNX5, in macropinocytosis of mouse primary macrophages. We show that endogenous SNX1 and SNX5 are localised to newly-formed macropinosomes in primary mouse macrophages and, moreover, demonstrate that SNX5 plays an essential role in macropinosome biogenesis. Depletion of SNX5 in bone marrow-derived macrophages dramatically decreased both the number and size of macropinosomes. Depletion of SNX5 also resulted in dramatic reduction in uptake and processing of soluble ovalbumin in macrophages, indicating that the majority of antigen uptake and delivery to late endosomes is via macropinocytosis. By contrast, the absence of SNX1 had no effect on endogenous SNX5 localisation and macropinosome biogenesis using macrophages from SNX1 knockout mice. Therefore, SNX5 can function independently of SNX1 and is a modulator of macropinocytosis that influences the uptake and processing of soluble antigen in primary mouse macrophages.
Project description:Macropinocytosis is a prevalent and essential pathway in macrophages where it contributes to anti-microbial responses and innate immune cell functions. Cell surface ruffles give rise to phagosomes and to macropinosomes as multi-functional compartments that contribute to environmental sampling, pathogen entry, plasma membrane turnover and receptor signalling. Rapid, high resolution, lattice light sheet imaging demonstrates the dynamic nature of macrophage ruffling. Pathogen-mediated activation of surface and endosomal Toll-like receptors (TLRs) in macrophages upregulates macropinocytosis. Here, using multiple forms of imaging and microscopy, we track membrane-associated, fluorescently-tagged Rab8a expressed in live macrophages, using a variety of cell markers to demonstrate Rab8a localization and its enrichment on early macropinosomes. Production of a novel biosensor and its use for quantitative FRET analysis in live cells, pinpoints macropinosomes as the site for TLR-induced activation of Rab8a. We have previously shown that TLR signalling, cytokine outputs and macrophage programming are regulated by the GTPase Rab8a with PI3 K? as its effector. Finally, we highlight another effector, the phosphatase OCRL, which is located on macropinosomes and interacts with Rab8a, suggesting that Rab8a may operate on multiple levels to modulate phosphoinositides in macropinosomes. These findings extend our understanding of macropinosomes as regulatory compartments for innate immune function in macrophages. This article is part of the Theo Murphy meeting issue 'Macropinocytosis'.
Project description: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.
Project description: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.
Project description:Pathogen-mediated activation of macrophages arms innate immune responses that include enhanced surface ruffling and macropinocytosis for environmental sampling and receptor internalization and signaling. Activation of macrophages with bacterial lipopolysaccharide (LPS) generates prominent dorsal ruffles, which are precursors for macropinosomes. Very rapid, high-resolution imaging of live macrophages with lattice light sheet microscopy (LLSM) reveals new features and actions of dorsal ruffles, which redefine the process of macropinosome formation and closure. We offer a new model in which ruffles are erected and supported by F-actin tent poles that cross over and twist to constrict the forming macropinosomes. This process allows for formation of large macropinosomes induced by LPS. We further describe the enrichment of active Rab13 on tent pole ruffles and show that CRISPR deletion of Rab13 results in aberrant tent pole ruffles and blocks the formation of large LPS-induced macropinosomes. Based on the exquisite temporal and spatial resolution of LLSM, we can redefine the ruffling and macropinosome processes that underpin innate immune responses.
Project description:Internalization of extracellular fluid and its solute by macropinocytosis requires dynamic reorganization of actin cytoskeleton, membrane ruffling, and formation of large endocytic vacuolar compartments, called macropinosomes, inside the cell. Although instigators of macropinocytosis, such as growth factors and phorbol esters, stimulate NADPH oxidase (Nox) activation and signal transduction mediators upstream of Nox assembly, including Rac1 and protein kinase C (PKC), are involved in macropinocytosis, the role of Nox enzymes in macropinocytosis has never been investigated. This study was designed to examine the role of Nox2 and the potential downstream redox signaling involved in macropinocytosis.Phorbol myristate acetate activation of human and murine macrophages stimulated membrane ruffling, macropinosome formation, and subsequent uptake of macromolecules by macropinocytosis. Mechanistically, we found that pharmacological blockade of PKC, transcriptional knockdown of Nox2, and scavenging of intracellular superoxide anion abolished phorbol ester-induced macropinocytosis. We observed that Nox2-derived reactive oxygen species via inhibition of phosphatase and tensin homolog and activation of the phosphoinositide-3-kinase (PI3K)/Akt pathway lead to activation of actin-binding protein cofilin, membrane ruffling, and macropinocytosis. Similarly, activation of macropinocytosis by macrophage colony-stimulating factor involves Nox2-mediated cofilin activation. Furthermore, peritoneal chimera experiments indicate that macropinocytotic uptake of lipids in hypercholesterolemic ApoE-/- mice was attenuated in Nox2y/- macrophages compared with wild-type controls. Innovation and Conclusion: In summary, these findings demonstrate a novel Nox2-mediated mechanism of solute uptake via macropinocytosis, with broad implications for both general cellular physiology and pathological processes. The redox mechanism described here may also identify new targets in atherosclerosis and other disease conditions involving macropinocytosis. Antioxid. Redox Signal. 26, 902-916.
Project description:Macropinocytosis is an ancient mechanism that allows cells to harvest nutrients from extracellular media, which also allows immune cells to sample antigens from their surroundings. During macropinosome formation, bulk plasma membrane is internalized with all its integral proteins. It is vital for cells to salvage these proteins before degradation, but the mechanisms for sorting them are not known. Here we describe the evolutionarily conserved recruitment of the WASH (WASP and SCAR homolog) complex to both macropinosomes and phagosomes within a minute of internalization. Using Dictyostelium, we demonstrate that WASH drives protein sorting and recycling from macropinosomes and is thus essential to maintain surface receptor levels and sustain phagocytosis. WASH functionally interacts with the retromer complex at both early and late phases of macropinosome maturation, but mediates recycling via retromer-dependent and -independent pathways. WASH mutants consequently have decreased membrane levels of integrins and other surface proteins. This study reveals an important pathway enabling cells to sustain macropinocytosis without bulk degradation of plasma membrane components.
Project description:Activation of the macrophage colony stimulating factor-1 receptor (CSF-1R) by CSF-1 stimulates pronounced macropinocytosis and drives proliferation of macrophages. Although the role of macropinocytosis in CSF-1R signaling remains unknown, we show here that, despite internalizing large quantities of plasma membrane, macropinosomes contribute little to the internalization of the CSF-1-CSF-1R complex. Rather, internalization of the CSF-1R in small endocytic vesicles that are sensitive to clathrin disruption, outcompetes macropinosomes for CSF-1R endocytosis. Following internalization, small vesicles carrying the CSF-1R underwent homotypic fusion and then trafficked to newly formed macropinosomes bearing Rab5. As these macropinosomes matured, acquiring Rab7, the CSF-1R was transported into their lumen and degraded. Inhibition of macropinocytosis delayed receptor degradation despite no disruption to CSF-1R endocytosis. These data indicate that CSF-1-stimulated macropinosomes are sites of multivesicular body formation and accelerate CSF-1R degradation. Furthermore, we demonstrate that macropinocytosis and cell growth have a matching dose dependence on CSF-1, suggesting that macropinosomes might be a central mechanism coupling CSF-1R signaling and macrophage growth.
Project description: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.
Project description: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 ? (DGK?), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show DGK? is required for optimal macropinocytosis induced by growth factor stimulation of mouse embryonic fibroblasts. Time-lapse imaging of live cells and quantitative analysis revealed DGK? was associated with membrane ruffles and nascent macropinosomes. Macropinocytosis was attenuated in DGK?-null cells, as determined by live imaging and vaccinia virus uptake experiments. Moreover, macropinosomes that did form in DGK?-null cells were smaller than those found in wild type cells. Rescue of this defect required DGK? catalytic activity, consistent with it also being required for Rac1 activation. A constitutively membrane bound DGK? mutant substantially increased the size of macropinosomes and potentiated the effect of a constitutively active Rac1 mutant on macropinocytosis. Collectively, our results suggest DGK? functions in concert with Rac1 to regulate macropinocytosis.