Rab7 regulates phagosome maturation in Dictyostelium

Adam Rupper; Bryon Grove; James Cardelli

doi:10.1242/jcs.114.13.2449

Rab7 regulates phagosome maturation in Dictyostelium

Journal of Cell Science ◽

10.1242/jcs.114.13.2449 ◽

2001 ◽

Vol 114 (13) ◽

pp. 2449-2460

Author(s):

Adam Rupper ◽

Bryon Grove ◽

James Cardelli

Keyword(s):

Fluid Phase ◽

Integral Membrane Protein ◽

Dominant Negative ◽

Cysteine Proteinases ◽

Lysosomal Hydrolases ◽

Phagosomal Maturation ◽

Lysosomal System ◽

Phagocytic Cups ◽

In Cells

A Dictyostelium Rab7 homolog has been demonstrated to regulate fluid-phase influx, efflux, retention of lysosomal hydrolases and phagocytosis. Since Rab7 function appeared to be required for efficient phagocytosis, we sought to further characterize the role of Rab7 in phagosomal maturation. Expression of GFP-Rab7 resulted in labeling of both early and late phagosomes containing yeast, but not forming phagocytic cups. In order to determine if Rab7 played a role in regulating membrane traffic between the endo/lysosomal system and maturing phagosomes, latex bead containing (LBC) phagosomes were purified from wild-type cells at various times after internalization. Glycosidases, cysteine proteinases, Rab7 and lysosomally associated membrane proteins were delivered rapidly to nascent phagosomes in control cells. LBC phagosomes isolated from cells overexpressing dominant negative (DN) Rab7 contained very low levels of LmpA (lysosomal integral membrane protein) and α-mannosidase was not detectable. Interestingly, cysteine proteinases were delivered to phagosomes as apparent pro-forms in cells overexpressing DN Rab7. Despite these defects, phagosomes in cells overexpressing DN Rab7 matured to form multi-particle spacious phagosomes, except that these phagosomes remained significantly more acidic than control phagosomes. These results suggested that Rab7 regulates both an early and late steps of phagosomal maturation, similar to its role in the endo/lysosomal system.

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Involvement of the AP-1 Adaptor Complex in Early Steps of Phagocytosis and Macropinocytosis

Molecular Biology of the Cell ◽

10.1091/mbc.e03-06-0365 ◽

2004 ◽

Vol 15 (2) ◽

pp. 861-869 ◽

Cited By ~ 31

Author(s):

Yaya Lefkir ◽

Marilyne Malbouyres ◽

Daniel Gotthardt ◽

Adrian Ozinsky ◽

Sophie Cornillon ◽

...

Keyword(s):

Early Stage ◽

Fluid Phase ◽

Small Particles ◽

Medium Chain ◽

Murine Macrophages ◽

Fluid Phase Endocytosis ◽

Phagocytic Cups ◽

Golgi Network ◽

Mutant Cells

The best described function of the adaptor complex-1 (AP-1) is to participate in the budding of clathrin-coated vesicles from the trans-Golgi network and endosomes. Here, we show that AP-1 is also localized to phagocytic cups in murine macrophages as well as in Dictyostelium amoebae. AP-1 is recruited to phagosomal membranes at this early stage of phagosome formation and rapidly dissociates from maturing phagosomes. To establish the role of AP-1 in phagocytosis, we made used of Dictyostelium mutant cells (apm1-cells) disrupted for AP-1 medium chain. In this mutant, phagocytosis drops by 60%, indicating that AP-1 is necessary for efficient phagocytosis. Furthermore, phagocytosis in apm1-cells is more affected for large rather than small particles, and cells exhibiting incomplete engulfment are then often observed. This suggests that AP-1 could participate in the extension of the phagocytic cup. Interestingly, macropinocytosis, a process dedicated to fluid-phase endocytosis and related to phagocytosis, is also impaired in apm1-cells. In summary, our data suggest a new role of AP-1 at an early stage of phagosome and macropinosome formation.

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Evidence for a recycling role for Rab7 in regulating a late step in endocytosis and in retention of lysosomal enzymes in Dictyostelium discoideum.

Molecular Biology of the Cell ◽

10.1091/mbc.8.7.1343 ◽

1997 ◽

Vol 8 (7) ◽

pp. 1343-1360 ◽

Cited By ~ 57

Author(s):

G Buczynski ◽

J Bush ◽

L Zhang ◽

J Rodriguez-Paris ◽

J Cardelli

Keyword(s):

Dictyostelium Discoideum ◽

Cell Lines ◽

Lysosomal Enzymes ◽

Amino Acid Level ◽

Active Form ◽

Fluid Phase ◽

Retrograde Transport ◽

Dominant Negative ◽

Lysosomal Hydrolases ◽

Small Molecular Weight

The mammalian small molecular weight GTPase Rab7 (Ypt7 in yeast) has been implicated in regulating membrane traffic at postinternalization steps along the endosomal pathway. A cDNA encoding a protein 85% identical at the amino acid level to mammalian Rab7 has been cloned from Dictyostelium discoideum. Subcellular fractionation and immunofluorescence microscopy indicated that Rab7 was enriched in lysosomes, postlysosomes, and maturing phagosomes. Cell lines were generated that overexposed Rab7 wild-type (WT), Rab7 Q67L (constitutively active form), and Rab7 T22N (dominant negative form) proteins. The Rab7 T22N cell line internalized fluid phase markers and latex beads (phagocytosis) at one-third the rate of control cells, whereas Rab7 WT and Rab7 Q67L cell lines were normal in uptake rates but exocytosed fluid phase faster than control cells. In contrast, fluid phase markers resided in acidic compartments for longer periods of time and were more slowly exocytosed from Rab7 T22N cells as compared with control cells. Light microscopy indicated that Rab7-expressing cell lines contained morphologically altered endosomal compartments. Compared with control cells, Rab7 WT- and Rab7 Q67L-expressing cells contained a reduced number of vesicles, the size of postlysosomes (> 2.5 microns) and an increased number of smaller vesicles, many of which were nonacidic; in control cells, > 90% of the smaller vesicles were acidic. In contrast, Rab7 T22N cells contained an increased proportion of large acidic vesicles relative to nonacidic vesicles. Radiolabel pulse-chase experiments indicated that all of the cell lines processed and targeted lysosomal alpha-mannosidase normally, indicating the lack of a significant role for Rab7 in the targeting pathway; however, retention of mature lysosomal hydrolases was affected in Rab7 WT and Rab7 T22N cell lines. Contrary to the results observed for the fluid phase efflux experiments, Rab7 T22N cells oversecreted alpha-mannosidase, whereas Rab7 WT cells retained this hydrolase as compared with control cells. These data support a model that Rab7 may regulate retrograde transport of lysosomal enzymes and the V-type H(+)-ATPase from postlysosomes to lysosomes coupled with the efficient release of fluid phase from cells.

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PKCδ plays opposite roles in growth mediated by wild-type Kit and an oncogenic Kit mutant

Blood ◽

10.1182/blood-2004-04-1450 ◽

2005 ◽

Vol 105 (5) ◽

pp. 1923-1929 ◽

Cited By ~ 13

Author(s):

Tanya Jelacic ◽

Diana Linnekin

Keyword(s):

Receptor Tyrosine Kinase ◽

Catalytic Domain ◽

Dominant Negative ◽

Wild Type ◽

Aspartic Acid Residue ◽

Kit Receptor ◽

Murine Mast Cell ◽

Oncogenic Form ◽

In Cells

AbstractThe Kit receptor tyrosine kinase is critical for normal hematopoiesis. Mutation of the aspartic acid residue encoded by codon 816 of human c-kit or codon 814 of the murine gene results in an oncogenic form of Kit. Here we investigate the role of protein kinase Cδ (PKCδ) in responses mediated by wild-type murine Kit and the D814Y mutant in a murine mast cell-like line. PKCδ is activated after wild-type (WT) Kit binds stem cell factor (SCF), is constitutively active in cells expressing the Kit catalytic domain mutant, and coprecipitates with both forms of Kit. Inhibition of PKCδ had opposite effects on growth mediated by wild-type and mutant Kit. Both rottlerin and a dominant-negative PKCδ construct inhibited the growth of cells expressing mutant Kit, while SCF-induced growth of cells expressing wild-type Kit was not inhibited. Further, overexpression of PKCδ inhibited growth of cells expressing wild-type Kit and enhanced growth of cells expressing the Kit mutant. These data demonstrate that PKCδ contributes to factor-independent growth of cells expressing the D814Y mutant, but negatively regulates SCF-induced growth of cells expressing wild-type Kit. This is the first demonstration that PKCδ has different functions in cells expressing normal versus oncogenic forms of a receptor.

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An Assembly-incompetent Mutant Establishes a Requirement for Dynamin Self-assembly in Clathrin-mediated Endocytosis In Vivo

Molecular Biology of the Cell ◽

10.1091/mbc.e04-01-0015 ◽

2004 ◽

Vol 15 (5) ◽

pp. 2243-2252 ◽

Cited By ~ 57

Author(s):

Byeong Doo Song ◽

Defne Yarar ◽

Sandra L. Schmid

Keyword(s):

Self Assembly ◽

Point Mutations ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Gtpase Activity ◽

Insight Into ◽

Assay Conditions ◽

In Cells

Dynamin GTPase activity is required for its biological function in clathrin-mediated endocytosis; however, the role of self-assembly has not been unambiguously established. Indeed, overexpression of a dynamin mutant, Dyn1-K694A, with impaired ability to self-assemble has been shown to stimulate endocytosis in HeLa cells (Sever et al., Nature 1999, 398, 481). To identify new, assembly-incompetent mutants of dynamin 1, we made point mutations in the GTPase effector/assembly domain (GED) and tested for their effects on self-assembly and clathrin-mediated endocytosis. Mutation of three residues, I690, K694, and I697, suggests that interactions with an amphipathic helix in GED are required for self-assembly. In particular, Dyn1-I690K failed to exhibit detectable assembly-stimulated GTPase activity under all assay conditions. Overexpression of this assembly-incompetent mutant inhibited transferrin endocytosis as potently as the GTPase-defective dominant-negative mutant, Dyn1-K44A. However, worm-like endocytic intermediates accumulated in cells expressing Dyn1-I690K that were structurally distinct from long tubules that accumulated in cells expressing Dyn1-K44A. Together these results provide new structural insight into the role of GED in self-assembly and assembly-stimulated GTPase activity and establish that dynamin self-assembly is essential for clathrin-mediated endocytosis.

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Specific role of phosphoinositide 3-kinase p110α in the regulation of phagocytosis and pinocytosis in macrophages

Biochemical Journal ◽

10.1042/bj20090687 ◽

2009 ◽

Vol 423 (1) ◽

pp. 99-108 ◽

Cited By ~ 31

Author(s):

Namiko Tamura ◽

Kaoru Hazeki ◽

Natsumi Okazaki ◽

Yukiko Kametani ◽

Hiroki Murakami ◽

...

Keyword(s):

Molecular Mass ◽

Lucifer Yellow ◽

Density Lipoprotein ◽

Fluid Phase ◽

Specific Role ◽

Class I ◽

Raw 264.7 Cells ◽

Phosphoinositide 3 Kinase ◽

In Cells

PI3K (phosphoinositide 3-kinase) α has been implicated in phagocytosis and fluid-phase pinocytosis in macrophages. The subtype-specific role of PI3K in these processes is poorly understood. To elucidate this issue, we made Raw 264.7 cells (a mouse leukaemic monocyte–macrophage cell line) deficient in each of the class-I PI3K catalytic subunits: p110α, p110β, p110δ and p110γ. Among these cells, only the p110α-deficient cells exhibited lower phagocytosis of opsonized and non-opsonized zymosan. The p110α-deficient cells also showed the impaired phagocytosis of IgG-opsonized erythrocytes and the impaired fluid-phase pinocytosis of dextran (molecular mass of 40 kDa). Receptor-mediated pinocytosis of DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate)-labelled acetylated low-density lipoprotein and fluid-phase pinocytosis of Lucifer Yellow (molecular mass of 500 Da) were resistant to p110α depletion. None of these processes were impaired in cells lacking p110β, p110δ or p110γ, but were susceptible to a pan-PI3K inhibitor wortmannin. In cells deficient in the enzymes catalysing PtdIns(3,4,5)P3 breakdown [PTEN (phosphatase and tensin homologue deleted on chromosome 10) or SHIP-1 (Src-homology-2-domain-containing inositol phosphatase-1)], uptake of IgG-opsonized particles was enhanced. These results indicated that phagocytosis and fluid-phase pinocytosis of larger molecules are dependent on the lipid kinase activity of p110α, whereas pinocytosis via clathrin-coated and small non-coated vesicles may depend on subtypes of PI3Ks other than class I.

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Sequential Actions of Rab5 and Rab7 Regulate Endocytosis in the Xenopus Oocyte

The Journal of Cell Biology ◽

10.1083/jcb.136.6.1227 ◽

1997 ◽

Vol 136 (6) ◽

pp. 1227-1237 ◽

Cited By ~ 48

Author(s):

Amitabha Mukhopadhyay ◽

Alejandro M. Barbieri ◽

Kouichi Funato ◽

Richard Roberts ◽

Philip D. Stahl

Keyword(s):

Xenopus Oocytes ◽

Fluid Phase ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Aluminum Fluoride ◽

General Role ◽

Sensitive Factor ◽

Rate Limiting ◽

Stimulation Of

To explore the role of GTPases in endocytosis, we developed an assay using Xenopus oocytes injected with recombinant proteins to follow the uptake of the fluid phase marker HRP. HRP uptake was inhibited in cells injected with GTPγS or incubated with aluminum fluoride, suggesting a general role for GTPases in endocytosis. Injection of Rab5 into oocytes, as well as Rab5:Q79L, a mutant with decreased GTPase activity, increased HRP uptake. Injection of Rab5:S34N, the dominant-negative mutant, inhibited HRP uptake. Injection of N-ethylmaleimide–sensitive factor (NSF) stimulated HRP uptake, and ATPase-defective NSF mutants inhibited HRP uptake when coinjected with Rab5:Q79L, confirming a requirement for NSF in endocytosis. Surprisingly, injection of Rab7:WT stimulated both uptake and degradation/activation of HRP. The latter appears to be due to enhanced transport to a late endosomal/prelysosomal degradative compartment that is monensin sensitive. Enhancement of uptake by Rab7 appears to function via an Rab5-sensitive pathway in oocytes since the stimulatory effect of Rab7 was blocked by coinjection of Rab5:S34N. Stimulation of uptake by Rab5 was blocked by Rab5:S34N but not by Rab7:T22N. Our results suggest that Rab7, while functioning downstream of Rab5, may be rate limiting for endocytosis in oocytes.

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Role of clathrin in the regulated secretory pathway of pancreaticβ-cells

Journal of Cell Science ◽

10.1242/jcs.114.16.3059 ◽

2001 ◽

Vol 114 (16) ◽

pp. 3059-3066

Author(s):

Miguel Molinete ◽

Stéphane Dupuis ◽

Frances M. Brodsky ◽

Philippe A. Halban

Keyword(s):

Light Chain ◽

Fluorescent Protein ◽

Recombinant Adenovirus ◽

Secretory Granules ◽

Dominant Negative ◽

Detectable Effect ◽

Intense Staining ◽

C Peptide ◽

In Cells

The role of clathrin in the sorting of proinsulin to secretory granules,the formation of immature granules and their subsequent maturation is not known. To this end, primary rat pancreatic β-cells were infected with a recombinant adenovirus co-expressing the Hub fragment, a dominant-negative peptide of the clathrin heavy chain and enhanced green fluorescent protein(EGFP as a marker of infected cells). A population of cells expressing the highest levels of EGFP (and thus Hub) was obtained using a fluorescence-activated cell sorter (FACS). Control cells were infected with an adenovirus expressing EGFP alone. By immunofluorescence, control cells showed intense staining for both clathrin light chain and proinsulin in a perinuclear region. In cells expressing high levels of Hub, the clathrin light-chain signal was faint and diffuse in keeping with its displacement from membranes. There was, however, no detectable effect of Hub expression on proinsulin staining or disposition within the cell. Proinsulin sorting and conversion,and the fate (release and/or degradation) of insulin and C-peptide, was studied by pulse-chase and quantitative reverse phase HPLC. In both Hub-expressing and control cells, >99% of all newly synthesized proinsulin was sorted to the regulated pathway and there was no effect of Hub on proinsulin conversion to insulin. In presence of Hub there was, however, a significant increase in the percentage of C-peptide truncated to des-(27-31)-C-peptide at early times of chase as well as more extensive degradation of C-peptide thereafter. It is concluded that clathrin is not implicated in the sorting or processing of proinsulin or in regulated exocytosis of secretory granules. These results confirm a role for clathrin in the removal of proteases from maturing granules, thus explaining the increased truncation and degradation of C-peptide in cells expressing Hub.

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SNX17 regulates antigen internalization and phagosomal maturation by dendritic cells

10.21203/rs.3.rs-144634/v1 ◽

2021 ◽

Author(s):

Ignacio Cebrian ◽

Sofía Dinamarca ◽

Cristina Croce ◽

Anna Salvioni ◽

Facundo Garrido ◽

...

Keyword(s):

Dendritic Cells ◽

Immune Responses ◽

Mhc Class I ◽

Fluid Phase ◽

Antigenic Peptides ◽

Sorting Nexin ◽

Cross Presentation ◽

Phagosomal Maturation ◽

Fluid Phase Endocytosis

Abstract Cross-presentation is the process whereby antigenic peptides derived from exogenous antigens are associated to MHC class I molecules triggering the activation of CD8+ T lymphocytes. The endocytic route of dendritic cells (DCs) is strongly specialized to achieve antigen cross-presentation efficiently, which is crucial to initiate cytotoxic immune responses against many pathogens (i.e. Toxoplasma gondii) and tumors. Nevertheless, the endosomal molecular effectors involved in this process are not completely understood. In particular, the role of sorting nexin (SNX) proteins in cross-presentation has never been addressed. In this work, we identify the endosomal protein SNX17 as a key regulator of antigen internalization and cross-presentation by DCs. Our results demonstrate that SNX17 expression in DCs is essential to guarantee a normal cross-presentation of soluble, particulate and T. gondii-associated antigens. The silencing of SNX17 expression in DCs significantly affected the uptake of exogenous antigens by fluid-phase endocytosis and phagocytosis, but not by receptor-mediated endocytosis. Moreover, the knock-down of SNX17 impaired T. gondii invasion, CD11b integrin recycling and hampered the organization of the actin cytoskeleton. Finally, we show that SNX17 controls the proper maturation of DC phagosomes. Our findings provide compelling evidence that SNX17 plays a central role in the modulation of DC endocytic network, which is crucial for competent antigen internalization and cross-presentation.

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