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Joel A Swanson - One of the best experts on this subject based on the ideXlab platform.
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the structural dynamics of Macropinosome formation and pi3 kinase mediated sealing revealed by lattice light sheet microscopy
Nature Communications, 2021Co-Authors: Shayne E Quinn, Joel A Swanson, Lu Huang, Jason G Kerkvliet, Steve Smith, Adam D Hoppe, Robert B Anderson, Natalie W ThiexAbstract:Macropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure and dynamics of Macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce Macropinosomes and that the majority form through collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the Macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many Macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3′-phosphoinositides within ruffles plays a minor role in regulating their morphology. However, 3′-phosphoinositides are critical for the fusogenic activity that seals ruffles into Macropinosomes. Taken together, these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into Macropinosomes. Macropinocytosis is a cellular process for the uptake of extracellular fluid. Here, the authors use lattice light sheet microscopy to examine the spatial dynamics of the plasma membrane, PI3K activity, and structural differences of various macrophage cell types during macropinocytosis.
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the structural dynamics of Macropinosome formation and pi3 kinase mediated sealing revealed by lattice light sheet microscopy
bioRxiv, 2020Co-Authors: Shayne E Quinn, Joel A Swanson, Lu Huang, Jason G Kerkvliet, Steve Smith, Adam D Hoppe, Robert B Anderson, Natalie W ThiexAbstract:Abstract Macropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure and dynamics of Macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce Macropinosomes and that the majority form through non-specific collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the Macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many Macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3’-phosphoinositides within ruffles plays a minor in regulating their morphology. However, 3’-phosphoinositides are critical for the fusogenic activity that seals ruffles into Macropinosomes. Taken together these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into Macropinosomes.
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Video 2 from Macropinosomes as units of signal transduction
2018Co-Authors: Joel A Swanson, Sei YoshidaAbstract:Solute size-dependent delivery of dye from endolysosomes into Macropinosomes by piranhalysis. Time-lapse video of a macrophage stimulated with M-CSF after pre-loading endolysosomes with Lucifer yellow (LY) and Texas Red-labeled dextran (TRDx). One image set was collected every 20 sec. Top: Phase-contrast. Bottom: phase-contrast with blue overlay indicating endolysosomes containing both LY and TRDx fluorescence and red overlay indicating Macropinosomes containing increased concentrations of LY relative to TRDx. Preferential labeling of Macropinosomes with LY indicated molecular size-selective transfer of fluid-phase probes between the interacting organelles. Adapted from a supplementary movie described in Ref. (5)
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Differential signaling during macropinocytosis in response to M-CSF and PMA in macrophages.
Frontiers in physiology, 2015Co-Authors: Sei Yoshida, Isabella Gaeta, Regina Pacitto, Lydia Krienke, Olivia Alge, Brian Gregorka, Joel A SwansonAbstract:The cellular movements that construct a Macropinosome have a corresponding sequence of chemical transitions in the cup-shaped region of plasma membrane that becomes the Macropinosome. To determine the relative positions of type I phosphatidylinositol 3-kinase (PI3K) and phospholipase C (PLC) in this pathway, we analyzed macropinocytosis in macrophages stimulated by the growth factor macrophage-colony-stimulating factor (M-CSF) and by the diacylglycerol (DAG) analogue phorbol 12-myristate 13-acetate (PMA). In cells stimulated with M-CSF, microscopic imaging of fluorescent probes for intracellular lipids indicated that the PI3K product phosphatidylinositol (3,4,5)-trisphosphate (PIP3) appeared in cups just prior to DAG. We then tested the hypothesis that PMA and DAG function after PI3K and prior to Ras and protein kinase C (PKC) during Macropinosome formation in macrophages. Although the PI3K target Akt was activated by M-CSF, the Akt inhibitor MK-2206 did not inhibit macropinocytosis. The phospholipase C (PLC) inhibitor U73122 blocked macropinocytosis by M-CSF but not PMA. Macropinocytosis in response to M-CSF and PMA was inhibited by the Ras inhibitor farnesyl thiosalicylate (FTS), by the PKC inhibitor Calphostin C and by the broad specificity inhibitor rottlerin. These studies support a model in which M-CSF stimulates PI3K in macropinocytic cups, and the resulting increase in PIP3 activates PLC, which in turn generates DAG necessary for activation of PKC, Ras and the late stages of Macropinosome closure.
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ruffles limit diffusion in the plasma membrane during Macropinosome formation
Journal of Cell Science, 2011Co-Authors: Timothy P Welliver, Laura S Chang, Jennifer J Linderman, Joel A SwansonAbstract:In murine macrophages stimulated with macrophage-colony-stimulating factor (M-CSF), signals essential to Macropinosome formation are restricted to the domain of plasma membrane enclosed within cup-shaped, circular ruffles. Consistent with a role for these actin-rich structures in signal amplification, microscopic measures of Rac1 activity determined that disruption of actin polymerization by latrunculin B inhibited ruffling and the localized activation of Rac1 in response to M-CSF. To test the hypothesis that circular ruffles restrict the lateral diffusion of membrane proteins that are essential for signaling, we monitored diffusion of membrane-tethered, photoactivatable green fluorescent protein (PAGFP-MEM) in ruffling and non-ruffling regions of cells. Although diffusion within macropinocytic cups was not inhibited, circular ruffles retained photoactivated PAGFP-MEM inside cup domains. Confinement of membrane molecules by circular ruffles could explain how actin facilitates positive feedback amplification of Rac1 in these relatively large domains of the plasma membrane, thereby organizing the contractile activities that close Macropinosomes.
Natalie W Thiex - One of the best experts on this subject based on the ideXlab platform.
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the structural dynamics of Macropinosome formation and pi3 kinase mediated sealing revealed by lattice light sheet microscopy
Nature Communications, 2021Co-Authors: Shayne E Quinn, Joel A Swanson, Lu Huang, Jason G Kerkvliet, Steve Smith, Adam D Hoppe, Robert B Anderson, Natalie W ThiexAbstract:Macropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure and dynamics of Macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce Macropinosomes and that the majority form through collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the Macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many Macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3′-phosphoinositides within ruffles plays a minor role in regulating their morphology. However, 3′-phosphoinositides are critical for the fusogenic activity that seals ruffles into Macropinosomes. Taken together, these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into Macropinosomes. Macropinocytosis is a cellular process for the uptake of extracellular fluid. Here, the authors use lattice light sheet microscopy to examine the spatial dynamics of the plasma membrane, PI3K activity, and structural differences of various macrophage cell types during macropinocytosis.
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the structural dynamics of Macropinosome formation and pi3 kinase mediated sealing revealed by lattice light sheet microscopy
bioRxiv, 2020Co-Authors: Shayne E Quinn, Joel A Swanson, Lu Huang, Jason G Kerkvliet, Steve Smith, Adam D Hoppe, Robert B Anderson, Natalie W ThiexAbstract:Abstract Macropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure and dynamics of Macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce Macropinosomes and that the majority form through non-specific collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the Macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many Macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3’-phosphoinositides within ruffles plays a minor in regulating their morphology. However, 3’-phosphoinositides are critical for the fusogenic activity that seals ruffles into Macropinosomes. Taken together these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into Macropinosomes.
Sergio Grinstein - One of the best experts on this subject based on the ideXlab platform.
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rab5 regulates macropinocytosis by recruiting the inositol 5 phosphatases ocrl and inpp5b that hydrolyse ptdins 4 5 p2
Journal of Cell Science, 2021Co-Authors: Michelle E Maxson, Helen Sarantis, Allen Volchuk, John H Brumell, Sergio GrinsteinAbstract:Rab5 is required for Macropinosome formation, but its site and mode of action remain unknown. We report that Rab5 acts at the plasma membrane, downstream of ruffling, to promote Macropinosome sealing and scission. Dominant-negative Rab5, which obliterates macropinocytosis, had no effect on the development of membrane ruffles. However, Rab5-containing vesicles were recruited to circular membrane ruffles, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent endomembrane fusion was necessary for the completion of macropinocytosis. This fusion event coincided with the disappearance of PtdIns(4,5)P2 that accompanies Macropinosome closure. Counteracting the depletion of PtdIns(4,5)P2 by expression of phosphatidylinositol-4-phosphate 5-kinase impaired Macropinosome formation. Importantly, we found that the removal of PtdIns(4,5)P2 is dependent on Rab5, through the Rab5-mediated recruitment of the inositol 5-phosphatases OCRL and Inpp5b, via APPL1. Knockdown of OCRL and Inpp5b, or APPL1, prevented Macropinosome closure without affecting ruffling. We therefore propose that Rab5 is essential for the clearance of PtdIns(4,5)P2 needed to complete the scission of Macropinosomes or to prevent their back-fusion with the plasmalemma.
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rab5 regulates Macropinosome closure through recruitment of the inositol 5 phosphatases ocrl inpp5b and the hydrolysis of ptdins 4 5 p2
bioRxiv, 2020Co-Authors: Michelle E Maxson, Helen Sarantis, Allen Volchuk, John H Brumell, Sergio GrinsteinAbstract:Abstract Rab5 is required for Macropinosome formation, but its site and mode of action remain unknown. We report that Rab5 acts at the plasma membrane, downstream of ruffling, to promote Macropinosome sealing and scission. Dominant-negative Rab5, which obliterates macropinocytosis, had no effect on the development of membrane ruffles. However, Rab5-containing vesicles were recruited to circular membrane ruffles, and SNARE-dependent endomembrane fusion was necessary for completion of macropinocytosis. This fusion event coincided with the disappearance of PtdIns(4,5)P2 that accompanies Macropinosome closure. Counteracting the depletion of PtdIns(4,5)P2 by expression of phosphatidylinositol-4-phosphate 5-kinase impaired Macropinosome formation. Importantly, we found that removal of PtdIns(4,5)P2 is dependent on Rab5, through the Rab5-mediated recruitment of the inositol 5-phosphatases OCRL and Inpp5b, via APPL1. Knockdown of OCRL and Inpp5b, or APPL1 prevented Macropinosome closure, without affecting ruffling. We therefore propose that Rab5 is essential for the clearance of PtdIns(4,5)P2 needed to complete Macropinosome scission from the plasmalemma.
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rab5 regulates Macropinosome closure by recruitment of ocrl inpp5b and hydrolysis of ptdins 4 5 p2
bioRxiv, 2020Co-Authors: Sergio Grinstein, Allen Volchuk, Helen Sarantis, John H Brumell, Michelle E MaxsonAbstract:AO_SCPLOWBSTRACTC_SCPLOWRab5 is required for Macropinosome formation, but its site and mode of action remain unknown. We report that Rab5 acts at the plasma membrane, downstream of ruffling, to promote Macropinosome sealing and scission. Dominant-negative Rab5, which obliterates macropinocytosis, had no effect on the development of membrane ruffles. However, Rab5-containing vesicles were recruited to circular membrane ruffles, and SNARE-dependent endomembrane fusion was necessary for completion of macropinocytosis. This fusion event coincided with the disappearance of PtdIns(4,5)P2 that accompanies Macropinosome closure. Counteracting the depletion of PtdIns(4,5)P2 by expression of phosphatidylinositol-4-phosphate 5-kinase impaired Macropinosome formation. Importantly, we found that removal of PtdIns(4,5)P2 is dependent on Rab5, through the Rab5-mediated recruitment of the inositol 5-phosphatases OCRL and Inpp5b, via APPL1. Knockdown of OCRL and Inpp5b, or APPL1 prevented Macropinosome closure, without affecting ruffling. We therefore propose that Rab5 is essential for the clearance of PtdIns(4,5)P2 needed to complete Macropinosome scission from the plasmalemma.
Colin Watts - One of the best experts on this subject based on the ideXlab platform.
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rac is required for constitutive macropinocytosis by dendritic cells but does not control its downregulation
Current Biology, 2000Co-Authors: Michele A West, Alan R Prescott, Eevaliisa Eskelinen, Anne J Ridley, Colin WattsAbstract:Abstract 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.
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the coated pit and macropinocytic pathways serve distinct endosome populations
Journal of Cell Biology, 1994Co-Authors: Lindsay J Hewlett, Alan R Prescott, Colin WattsAbstract: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.
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The coated pit and macropinocytic pathways serve distinct endosome populations
1994Co-Authors: Lindsay J Hewlett, Alan R Prescott, Colin WattsAbstract:Abstract. 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 endosomeslysosomes as defined by labeling with transferrin or with LDL. Mixing of the contents of the macropino-somes and conventional endosomes was not induced by the addition of brefeldin A. Moreover, the morpholog
Nobukazu Araki - One of the best experts on this subject based on the ideXlab platform.
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Dynamic changes in the spatiotemporal localization of Rab21 in live RAW264 cells during macropinocytosis
2016Co-Authors: Youhei Egami, Nobukazu ArakiAbstract: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 wit
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small gtpases and phosphoinositides in the regulatory mechanisms of Macropinosome formation and maturation
Frontiers in Physiology, 2014Co-Authors: Youhei Egami, Tomohiko Taguchi, Masashi Maekawa, Hiroyuki Arai, Nobukazu ArakiAbstract: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.
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dissecting the roles of rac1 activation and deactivation in macropinocytosis using microscopic photo manipulation
Scientific Reports, 2013Co-Authors: Makoto Fujii, Youhei Egami, Katsuhisa Kawai, Nobukazu ArakiAbstract: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.
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AHCcta Histochemica et Cytochemica0044-599113 7 800Jap n Society of His ochemistry and CytochemistryTokyo, Japan12014.1267 / h.12014Regular Article Spatiotemporal Localization of Rab20 in Live RAW264 Macrophages
2012Co-Authors: During Macropinocytosis, Youhei Egami, Nobukazu ArakiAbstract:© 2012 The Japan Society of Histochemistry andRab20 is a memb r 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 macropino-somes 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. Key words: Rab20, macropinocytosis, macrophage, live-cell imaging, endocytosis I
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sequential signaling in plasma membrane domains during Macropinosome formation in macrophages
Journal of Cell Science, 2009Co-Authors: Sei Yoshida, Adam D Hoppe, Nobukazu Araki, Joel A SwansonAbstract:Macropinosomes are large endocytic vesicles that form in ruffling regions of plasma membrane. To analyze signal organization relative to ruffle closure into circular ruffles and cup closure into Macropinosomes, this study used quantitative microscopy to measure 3′ phosphoinositides and small-GTPase activities in a representative subset of forming Macropinosomes. Macropinocytosis was stimulated by the addition of macrophage colony-stimulating factor (M-CSF) to macrophages expressing fluorescent reporter proteins. Ratiometric and fluorescence resonance energy transfer (FRET) microscopy determined that Rac1 activity and phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] levels increased transiently, peaking 26-30 seconds after ruffle closure. Three-dimensional reconstruction of cells labeled with the fluorescent dye FM4-64 showed that PtdIns(3,4,5)P3 was restricted to open, circular cups in the plasma membrane. Quantitative fluorescence microscopic methods determined the timing of cup closure, which followed 40-100 seconds after Rac1 and PtdIns(3,4,5)P3 deactivation and coincided with accumulation of phosphatidylinositol 3-phosphate and Rab5a. Thus, ruffle closure creates a circular domain of plasma membrane that localizes the activation and deactivation of Rac1 and phosphoinositide 3-kinase (PI3K), followed by recruitment of Rab5a and the contractile activities of cup closure.
