FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2007630314> ?p ?o ?g. }

• W2007630314 endingPage "11396" @default.
• W2007630314 startingPage "11384" @default.
• W2007630314 abstract "Cellular remodeling during differentiation is essential for lifecycle progression of many unicellular eukaryotic pathogens such as Leishmania, but the mechanisms involved are largely uncharacterized. The role of endosomal sorting in differentiation was analyzed in Leishmania major by overexpression of a dominant-negative ATPase, VPS4. VPS4E235Q accumulated in vesicles from the endocytic pathway, and the mutant L. major was deficient in endosome sorting. Mutant parasites failed to differentiate to the obligate infective metacyclic promastigote form. Furthermore, the autophagy pathway, monitored via the expression of autophagosome marker GFP-ATG8, and shown to normally peak during initiation of metacyclogenesis, was disrupted in the mutants. The defect in late endosome-autophagosome function in the VPS4E235Q parasites made them less able to withstand starvation than wild-type L. major. In addition, a L. major ATG4-deficient mutant was found also to be defective in the ability to differentiate. This finding, that transformation to the infective metacyclic form is dependent on late endosome function and, more directly, autophagy, makes L. major a good model for studying the roles of these processes in differentiation. Cellular remodeling during differentiation is essential for lifecycle progression of many unicellular eukaryotic pathogens such as Leishmania, but the mechanisms involved are largely uncharacterized. The role of endosomal sorting in differentiation was analyzed in Leishmania major by overexpression of a dominant-negative ATPase, VPS4. VPS4E235Q accumulated in vesicles from the endocytic pathway, and the mutant L. major was deficient in endosome sorting. Mutant parasites failed to differentiate to the obligate infective metacyclic promastigote form. Furthermore, the autophagy pathway, monitored via the expression of autophagosome marker GFP-ATG8, and shown to normally peak during initiation of metacyclogenesis, was disrupted in the mutants. The defect in late endosome-autophagosome function in the VPS4E235Q parasites made them less able to withstand starvation than wild-type L. major. In addition, a L. major ATG4-deficient mutant was found also to be defective in the ability to differentiate. This finding, that transformation to the infective metacyclic form is dependent on late endosome function and, more directly, autophagy, makes L. major a good model for studying the roles of these processes in differentiation. Cargo destined for the lysosome/vacuole compartment in eukaryotic cells requires sorting from the biosynthetic, secretory, and endocytic pathways. Transport of newly synthesized lysosomal proteins from the trans-Golgi network to the lysosomal compartment involves the delivery of cargo into the endosomal system, which serves as a sorting compartment as well as a collection site for endocytosed materials. The endocytic process, which is important for internalization of portions of the plasma membrane as well as extracellular fluids, involves a number of mechanisms (many of them receptor-mediated), including phagocytosis, macropinocytosis, caveolae, and clathrin-dependent or clathrin-independent endocytosis (1Conner S.D. Schmid S.L. Nature. 2003; 422: 37-44Crossref PubMed Scopus (3070) Google Scholar). Once internalized, receptor-bound ligands, solutes, and lipids are subject to complex intracellular trafficking pathways and can be recycled back to the plasma membrane or degraded in the lysosomal compartment. The endosomal system itself is complex, and several types of vacuoles have been identified, including late endosomes having a multivesicular aspect and called multivesicular bodies (MVB 2The abbreviations used are: MVB, multivesicular bodies; VPS, vacuolar protein sorting; ESCRT, endosomal sorting complexes required for transport; MVT, multivesicular tubule; GFP, green fluorescent protein; PE, phosphatidylethanolamine; EF1α, elongation factor 1α; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PBS, phosphate-buffered saline. ; see Refs. 2Katzmann D.J. Odorizzi G. Emr S.D. Nat. Rev. Mol. Cell. Biol. 2002; 3: 893-905Crossref PubMed Scopus (1021) Google Scholar and references therein for a review). A screen for vacuolar protein sorting (Vps) mutants in yeast led to the discovery of the molecular machinery responsible for MVB formation and, in particular, the class E group of Vps mutants, which contained a large multilamellar cisternal compartment thought to represent an endosome unable to form intraluminal vesicles (3Raymond C.K. Howald-Stevenson I. Vater C.A. Stevens T.H. Mol. Biol. Cell. 1992; 3: 1389-1402Crossref PubMed Scopus (680) Google Scholar, 4Rieder S.E. Banta L.M. Kohrer J.M. McCaffery J.M. Emr S.D. Mol. Biol. Cell. 1996; 7: 985-999Crossref PubMed Scopus (239) Google Scholar). Vps class E proteins are organized in several sub-complexes named “endosomal sorting complexes required for transport” (ESCRT) I, II, and III (5Babst M. Katzmann D.J. Snyder W.B. Wendland B. Emr S.D. Dev. Cell. 2002; 3: 283-289Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar, 6Babst M. Katzmann D.J. Estepa-Sabal E.J. Meerloo T. Emr S.D. Dev. Cell. 2002; 3: 271-282Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar, 7Conibear E. Mol. Cell. 2002; 10: 215-216Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 8Katzmann D.J. Babst M. Emr S.D. Cell. 2001; 106: 145-155Abstract Full Text Full Text PDF PubMed Scopus (1129) Google Scholar). The ESCRT proteins are recruited from the cytoplasm to the endosomal membrane where they function sequentially in the formation of MVB vesicles and the sorting of proteins into the MVB pathway. It has been proposed that a multimeric AAA-type ATPase, Vps4, binds to ESCRT III and catalyzes the disassembly of this complex in an ATP-dependent manner (9Babst M. Wendland B. Estepa E.J. Emr S.D. EMBO J. 1998; 17: 2982-2993Crossref PubMed Scopus (625) Google Scholar). This Vps4-dependent dissociation of the ESCRT machinery is the final step of protein sorting into the MVB and is a prerequisite for vesicle formation (6Babst M. Katzmann D.J. Estepa-Sabal E.J. Meerloo T. Emr S.D. Dev. Cell. 2002; 3: 271-282Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar). The Vps4 homologue in mammals, SKD1, is involved in membrane transport through endosomes and overexpression of a dominant negative mutant, SKD1E235Q, resulted in the production of aberrant endosomes defective in membrane transport between late endosomes and lysosomes (10Yoshimori T. Yamagata F. Yamamoto A. Mizushima N. Kabeya Y. Nara A. Miwako I. Ohashi M. Ohsumi M. Ohsumi Y. Mol. Biol. Cell. 2000; 11: 747-763Crossref PubMed Scopus (176) Google Scholar). In the protozoan parasite Leishmania, as in related kinetoplastid flagellates such as Trypanosoma, there is a complex membrane network highly polarized around the flagellar pocket, an invagination of the plasma membrane where the flagellum emerges from the cell body (11Landfear S.M. Ignatushchenko M. Mol. Biochem. Parasitol. 2001; 115: 1-17Crossref PubMed Scopus (107) Google Scholar). This is an important zone of interaction between the parasite and its environment, because it is the only site in the cell for endocytosis and exocytosis and so is responsible for crucial exchanges such as uptake of nutrients via receptor-mediated endocytosis (12Krishnamurthy G. Vikram R. Singh S.B. Patel N. Agarwal S. Mukhopadhyay G. Basu S.K. Mukhopadhyay A. J. Biol. Chem. 2005; 280: 5884-5891Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) and secretion of virulence factors that can interact with the host. Another characteristic of Leishmania parasites is that their promastigote (insect stage) form possesses a rather unusual lysosomal compartment, named the multivesicular tubule (MVT)-lysosome (13Mullin K.A. Foth B.J. Ilgoutz S.C. Callaghan J.M. Zawadzki J.L. McFadden G.I. McConville M.J. Mol. Biol. Cell. 2001; 12: 2364-2377Crossref PubMed Scopus (92) Google Scholar, 14Weise F. Stierhof Y.D. Kühn C. Wiese M. Overath P. J. Cell Sci. 2000; 113: 4587-4603Crossref PubMed Google Scholar, 15Ghedin E. Debrabant A. Engel J.C. Dwyer D.M. Traffic. 2001; 2: 175-188Crossref PubMed Scopus (59) Google Scholar). This compartment has a low lytic capacity and a relatively high luminal pH in multiplicative procyclic promastigotes but acquires the properties of mature lysosomes as the parasite differentiates into its infective, but non-replicative, metacyclic form (13Mullin K.A. Foth B.J. Ilgoutz S.C. Callaghan J.M. Zawadzki J.L. McFadden G.I. McConville M.J. Mol. Biol. Cell. 2001; 12: 2364-2377Crossref PubMed Scopus (92) Google Scholar). So far, only a few components of the Leishmania endosomal machinery have been characterized at the molecular level such as earlyendosomal Rab5 (16Singh S.B. Tandon R. Krishnamurthy G. Vikram R. Sharma N. Basu S.K. Mukhopadhyay A. EMBO J. 2003; 22: 5712-5722Crossref PubMed Scopus (53) Google Scholar) and late-endosomal Rab7 (17Denny P.W. Lewis S. Tempero J.E. Goulding D. Ivens A.C. Field M.C. Smith D.F. Mol. Biochem. Parasitol. 2002; 123: 105-113Crossref PubMed Scopus (27) Google Scholar). Indeed, although it has been shown that the MVT-lysosome is downstream of a MVB-like network of vesicular endosomes that surrounds the flagellar pocket (13Mullin K.A. Foth B.J. Ilgoutz S.C. Callaghan J.M. Zawadzki J.L. McFadden G.I. McConville M.J. Mol. Biol. Cell. 2001; 12: 2364-2377Crossref PubMed Scopus (92) Google Scholar), very little is known about them in Leishmania. Among the pathways involving vesicular traffic in eukaryotic cells is autophagy, a process that is important for protein and organelle degradation during cellular differentiation and also as a defense against starvation conditions (18Levine B. Klionsky D.J. Dev. Cell. 2004; 6: 463-477Abstract Full Text Full Text PDF PubMed Scopus (3214) Google Scholar). The autophagic pathways of yeast and mammals have been characterized, and, although they have many common features, they appear to differ in some ways (19Klionsky D.J. Autophagy. Landes Bioscience, Georgetown, TX2004Google Scholar, 20Reggiori F. Klionsky D.J. Eukaryot. Cell. 2002; 1: 11-21Crossref PubMed Scopus (474) Google Scholar). Central to the process in both is a vesicular compartment called the autophagosome, which is formed by a membranous structure that engulfs the cytoplasm/organelles that are to be degraded. The genesis of autophagosomal structures requires the activity of two protein conjugation systems, one involving ubiquitin-like protein Atg8 proteolytically processed by Atg4, and a second involving ubiquitin-like protein Atg12, covalently attached to Atg5 (18Levine B. Klionsky D.J. Dev. Cell. 2004; 6: 463-477Abstract Full Text Full Text PDF PubMed Scopus (3214) Google Scholar, 20Reggiori F. Klionsky D.J. Eukaryot. Cell. 2002; 1: 11-21Crossref PubMed Scopus (474) Google Scholar, 21Stromhaug P.E. Klionsky D.J. Traffic. 2001; 2: 524-531Crossref PubMed Scopus (138) Google Scholar). The autophagosome delivers the internalized material to the lysosomal compartment for degradation. It seems that, at least in mammals, but perhaps not in yeast, autophagosomes first fuse with endosomal vesicles (22Mizushima N. Ohsumi Y. Yoshimori T. Cell Struct. Funct. 2002; 27: 421-429Crossref PubMed Scopus (757) Google Scholar, 23Nara A. Mizushima N. Yamamoto A. Kabeya Y. Ohsumi Y. Yoshimori T. Cell Struct. Funct. 2002; 27: 29-37Crossref PubMed Scopus (123) Google Scholar, 24Shirahama K. Noda T. Ohsumi Y. Cell Struct. Funct. 1997; 22: 501-509Crossref PubMed Scopus (56) Google Scholar). Thus in this case, the autophagosome requires strong interactions with the endosomal compartments to reach its full degradative potential. Among the cellular functions proposed for autophagy is a role in the architectural changes occurring during development and differentiation, although experimental evidence supporting this hypothesis is not extensive (18Levine B. Klionsky D.J. Dev. Cell. 2004; 6: 463-477Abstract Full Text Full Text PDF PubMed Scopus (3214) Google Scholar). Recent reports, however, have shown that stress-induced differentiation is impaired in autophagy mutants of Dictyostelium discoideum (25Otto G.P. Wu M.Y. Kazgan N. Anderson O.R. Kessin R.H. J. Biol. Chem. 2003; 278: 17636-17645Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), Caenorhabditis elegans (26Melendez A. Talloczy Z. Seaman M. Eskelinen E.L. Hall D.H. Levine B. Science. 2003; 301: 1387-1391Crossref PubMed Scopus (1033) Google Scholar), and even yeast, where autophagy genes seem to be important for sporulation (27Deutschbauer A.M. Williams R.M. Chu A.M. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15530-15535Crossref PubMed Scopus (135) Google Scholar). In this study, we investigated the function of MVBs in Leishmania major by characterizing the leishmanial Vps4 homologue. A dominantnegative VPS4 mutant (VPS4E235Q) accumulated the mutated protein around vesicular structures of the endocytic system and showed a defect in transport to the MVT-lysosome. This is similar to what has been observed in yeast and mammalian Vps4 mutants, suggesting a conservation of role for this protein in MVB architecture from the early branching kinetoplastid flagellate lineage to mammals. Moreover, L. major-overexpressing VPS4E235Q were impaired in their differentiation in culture and their resistance to starvation, suggesting a crucial role for VPS4 and the MVB compartment in these processes. In addition, by demonstrating the presence of autophagosomes in Leishmania using ATG8 as a marker and showing that these structures accumulated and were not functional in the VPS4E235Q mutant, we have revealed that the autophagic pathway is intimately involved with the endosomal system. Production of an L. major ATG4.2 null mutant, which could not undergo metacyclogenesis, further confirms that the lack of a functional autophagic pathway correlates with, and probably mediates, the differentiation defect of the VPS4 mutant parasite that impairs its virulence. Parasites—L. major (MHOM/JL/80/Friedlin) promastigotes were grown in modified Eagle's medium with 10% (v/v) heat-inactivated fetal calf serum (designated complete HOMEM medium) at 25 °C. Metacyclic promastigotes were isolated by the peanut agglutinin method (28Sacks D.L. Hieny S. Sher A. J. Immunol. 1985; 135: 564-569PubMed Google Scholar) from cultures that had been in stationary growth phase for 2-5 days. When referring to the stage of growth of the different cell lines throughout this report, early log phase corresponds to ∼5 × 105 parasites/ml and mid-log phase to ∼5 × 106 parasites/ml. Early stationary phase corresponds to ∼9 × 106 parasites/ml for pXG-VPS4E235Q and to ∼2 × 107 parasites/ml for pXG-VPS4 or wild type L. major. Unless otherwise stated, the pXG-VPS4 and pXG-VPS4E235Q cell lines were maintained in culture with 12.5 μg/ml G418. Generation of VPS4-expressing L. major Cell Lines—LmjVPS4 was obtained by PCR from L. major genomic DNA with primers OL1478 and OL1479 (Table 1) containing PvuII and BamHI sites, respectively. The 1340-bp fragment was then digested by PvuII/BamHI and cloned into SmaI/BamHI-digested pXG vector (29Ha D.S. Schwarz J.K. Turco S.J. Beverley S.M. Mol. Biochem. Parasitol. 1996; 77: 57-64Crossref PubMed Scopus (221) Google Scholar), yielding plasmid pXG-VPS4. Plasmid expressing LmjVPS4E235Q was produced by site-directed mutagenesis of pXG-VPS4, using the QuikChange mutagenesis kit (Stratagene) with primers OL1480 and OL1481 to yield plasmid pXG-VPS4E235Q.TABLE 1Primers used in this studyOL14785′-CAATTCAGCTGATGTCCGTCGACTTCAC-′OL14795′-CTCATGGATCCTTAGCCCTCCTGACCA-3′OL14805′-CCATCATCTTTGTGGATCAGATCGACTCTCTCGTC-3′OL14815′-GACGAGAGAGTCGATCTGATCCACAAAGATGATGG-3′OL15435′-CTCATAAGATCTATGTCCGTCGACTTCA-3′OL15445′-CACAAGGGTACCTTAGCCCTCCTGACCA-3′OL15065′-ACCTCGAGATCTATGTCTTCCAGAGTA-3′OL15075′-GCCATTCTCGAGCTAGTGCAGCCCCTGC-3′NT1585′-CATATGCTCCGCTACGTGCAAGATT-3′NT1595′-CTCGAGATCCAGATACTCCCACGAA-3′NT1685′-AAGCTTGGTGTGGTGTGCGTGCGTCACGAGCCTCTGT-3′NT1695′-GTCGACCGATGTATGCTGGGGCAGGGAGGGAGGGCA-3′NT1705′-CCCGGGTGATGCTGCTGCTGCTGCTCCACGCGCCCA-3′NT1715′-AGATCTAAAAGAGGCATGTGCGCCAAGAGAGAGGTG-3′ Open table in a new tab The GFP fusion constructs were obtained as follows. LmjVPS4 was obtained by PCR from plasmid pXG-VPS4 with primers OL1543 and OL1544 and cloned into BglII/KpnI-digested GFP-containing pNUS-GFPnH vector (30Tetaud E. Lecuix I. Sheldrake T. Baltz T. Fairlamb A.H. Mol. Biochem. Parasitol. 2002; 120: 195-204Crossref PubMed Scopus (56) Google Scholar). The resulting plasmid was named GFP-VPS4 and contained LmjVPS4 in-frame with the 3′-end of GFP. A plasmid (GFP-VPS4E235Q) containing LmjVPS4E235Q was obtained by site-directed mutagenesis on plasmid GFP-VPS4, as described above. L. major ATG8 was amplified by PCR from genomic DNA with primers OL1506 and OL1507 and cloned into BglII/XhoI-digested pNUS-GFPnH vector to give GFP-ATG8. Generation of a L. major ATG4.2 Null Mutant and Re-expressing Cell Lines—The 1005-bp 5′-flank fragment of LmATG4.2 (LmjF30.0270) was generated by PCR from L. major genomic DNA with primers NT168 and NT169 (Table 1), digested with HindIII and SalI, and inserted into HindIII/SalI-digested pGL345-HYG (31Mottram J.C. Souza A.E. Hutchison J.E. Carter R. Frame M.J. Coombs G.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6008-6013Crossref PubMed Scopus (206) Google Scholar) to give pGL345ATG4.2-HYG5′. The 3′-fragment was generated by PCR using primers NT170 and NT171. The resulting 1104-bp fragment was digested by SmaI and BglII and cloned into pGL345ATG4.2-HYG5′ to give pGLATG4.2-HYG5′3′. The cassette used for transfection was released by HindIII/BglII digestion. pGLATG4.2-BSD5′3′ plasmid, used for the replacement of the second ATG4.2 allele, was generated from plasmid pGLATG4.2-HYG5′3′ by replacing the SpeI/BamHI cassette containing the hygromycin resistance gene by a SpeI/BamHI cassette containing the blasticidin S deaminase gene. A population of parasites resistant to hygromycin was generated after transfection with pGLATG4.2-HYG5′3′. This population was used for a second round of transfection with the pGLATG4.2-BSD5′3′ construct. Two blasticidin-resistant clones from independent transfection events, designated Δatg4.2A1 and Δatg4.2B4, were selected for analysis. For the re-expression experiments, the ATG4.2 gene, modified with a poly-histidine tag at its C-terminal end, was inserted into the pNUS episomal vector. PCR using the primers NT158 and NT159 produced the poly-histidine-tagged version of ATG4.2. The resulting 1185-bp fragment was digested by NdeI/XhoI and ligated into pNUS-HnN plasmid (30Tetaud E. Lecuix I. Sheldrake T. Baltz T. Fairlamb A.H. Mol. Biochem. Parasitol. 2002; 120: 195-204Crossref PubMed Scopus (56) Google Scholar), previously digested by the same enzymes, to give the pN-ATG4.2 plasmid. L. major wild-type promastigotes were electroporated with 15 μg of the episome, and transfectants were selected with the appropriate antibiotic (Geneticin, G148, Invitrogen). Complement-mediated Lysis and Macrophage Infections—The sensitivity of procyclic and metacyclic promastigotes to complement lysis was assessed with a protocol modified from (32Franke E.D. McGreevy P.B. Katz S.P. Sacks D.L. J. Immunol. 1985; 134: 2713-2718PubMed Google Scholar). Briefly, 107 parasites were incubated at 37 °C in PBS mixed with an appropriate volume of fresh normal human serum. After 20 min, the samples were diluted 2-fold in PBS. The percentage of lysis, assessed by loss of promastigote motility and morphological changes, was calculated relative to control samples incubated in heat-inactivated serum, in which all cells remained viable. Peritoneal macrophages from CD1 mice were adhered overnight in RPMI medium (Sigma) at 37 °C in 5% CO2/95% air onto eight-chamber tissue culture plastic slides (Labtech) and then infected using late stationary phase promastigotes or peanut lectin-purified metacyclics at a ratio of 10 promastigotes per macrophage. After incubation for 3 h at 35 °C in 5% CO2/95% air, non-phagocytosed promastigotes were removed by washing gently with RPMI four times, and the cultures were then incubated at 35 °C in 5% CO2/95% air. The initial uptake of promastigotes by macrophages and their subsequent intracellular survival and growth as amastigotes was determined by counting the number of infected macrophages and the number of intracellular parasites in stained slides 3, 24, and 120 h after infection. Slides were fixed in methanol and stained with Giemsa to identify the parasites. The number of L. major per 100 macrophages was determined by examination of ∼200 macrophages per assay. Monitoring Autophagy—Live promastigotes of wild-type or VPS4-expressing L. major expressing GFP-ATG8 were observed daily by fluorescence microscopy, and the proportion of autophagosome-bearing cells as well as the number of these structures per cell were assessed. At least four series of 200 cells were counted per experiment for each point. To inhibit the formation of autophagosomes, promastigotes were grown with either 10 μm wortmannin (Sigma, 1 mm stock solution in Me2SO) or 10 mm 3-methyladenine (Sigma, 30 mg/ml stock solution in water). To investigate responses to starvation conditions, promastigotes from early log phase cultures were sedimented by centrifugation and washed twice in PBS before being resuspended in pre-warmed PBS and incubated at 25 °C for up to 2 h. Parasite Viability Assay—Viability of L. major during starvation was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma) assay as adapted for Leishmania species (33Kiderlen A.F. Kaye P.M. J. Immunol. Methods. 1990; 127: 11-18Crossref PubMed Scopus (79) Google Scholar). Briefly, 5 × 106 promastigotes were starved in PBS as described above and then incubated for 45 min at 37 °C with MTT to a final concentration of 1 mg/ml, and the absorbance was measured at 620 nm using a microtiter plate reader. Control cells were grown in complete medium during the same period of time and similarly assessed with MTT. The results were expressed as a fraction of the values obtained for the starved cells relative to the values of the control cells (in percentages). Antibodies and Immunoblotting—Anti-LmjVPS4 antibodies were raised in a rabbit using a peptide comprising the 15-amino acid C-terminal sequence (residues 361-375: CHFKRVVGPDPHDPTR). 10 mg of the peptide was linked to an Aminolink column (Pierce) on which the antibodies were affinity-purified according to the manufacturer's protocol. Western blot analysis was performed as described previously (34Besteiro S. Coombs G.H. Mottram J.C. Mol. Microbiol. 2004; 54: 1224-1236Crossref PubMed Scopus (53) Google Scholar). The primary antibodies used and their respective dilutions were rabbit anti-LmjVPS4 (1/1,000), rabbit anti-HASPB (1/5,000) (35Flinn H.M. Rangarajan D. Smith D.F. Mol. Biochem. Parasitol. 1994; 65: 259-270Crossref PubMed Scopus (64) Google Scholar), rabbit anti-SHERP (1/5,000) (36Knuepfer E. Stierhof Y.D. McKean P.G. Smith D.F. Biochem. J. 2001; 356: 335-344Crossref PubMed Scopus (43) Google Scholar), and mouse monoclonal anti-TbEF1α (Upstate, 1/10,000). The West-Pico chemiluminescence detection system (Pierce) was used to visualize antigens. Fluorescent Staining of Cells—107 L. major promastigotes were harvested by centrifugation and washed once in serum-free HOMEM. For FM4-64 labeling, the cells were incubated with 40 μm FM4-64 (from a 12 mm stock solution in Me2SO; Invitrogen) for 15 min at 4 °C and then washed in fresh medium and incubated for various times at 25 °C. Cells were then washed in cold PBS and processed for microscopy. For dextran endocytosis, cells were incubated with Alexa Fluor 594-conjugated dextran at 500 μg/ml (Mr 10,000, Invitrogen) in complete HOMEM medium for various times at 25 °C. Cells were then washed five times in cold PBS and processed for microscopy. For tomato lectin labeling, cells were resuspended in 100 μl of cold serum-free HOMEM, and 1 μl of fluorescein isothiocyanate-conjugated tomato lectin (1.3 mg/ml solution, Sigma) was added for 10 min at 4 °C. Cells were then washed and fixed for fluorescence after a 30-min further incubation in the absence of tomato lectin at 25 °C. Cells were fixed with 2% (v/v) formaldehyde in PBS for 30 min at 4 °C, resuspended in PBS, and allowed to dry onto glass slides. For concanavalin A lectin labeling, cells were resuspended in 100 μl of cold serum-free HOMEM, 1 μl of Texas red-conjugated concanavalin A (5 mg/ml solution, Invitrogen) was added, and cells were incubated for 2 h at 25 °C. They were then washed in cold PBS and processed as described above for microscopic observation. Statistical Analyses—Values were expressed as mean ± S.D. Data from macrophage infections, MTT assay, GFP-ATG8 puncta, and motile cell analyses during starvation were pooled (n = 15) for comparison using unpaired t-tests. A p value of <0.05 was used as the level of significance. Expression Profile of L. major VPS4 and Generation of a Dominantnegative Mutant—The L. major VPS4 (LmjF29.2500 gene) was identified by searching the L. major-predicted proteins data base (www.genedb.org/) with the yeast Vps4 (P52917) as a query. Sequence comparisons of LmjVPS4 with yeast (Vps4) and mouse (SKD1) orthologues revealed 53.7% and 52.9% amino acid identity, respectively. SKD1/VPS4 proteins belong to the AAA ATPase family (supplementary Figs. S1 and S2). An antiserum raised to a peptide located in the C-terminal region of LmjVPS4 detected the 50-kDa VPS4 protein in lysates of L. major promastigotes from various growth stages (Fig. 1A, insets, and Fig. 2A, arrowed), as well as a cross-reacting protein of ∼25 kDa. The only difference in VPS4 levels during in vitro growth was an apparent downregulation of the protein in the non-dividing metacyclic form of the parasite. To investigate the function of the VPS4 protein in L. major,a line overexpressing a LmjVPS4E235Q mutant was generated. This Leishmania mutant is equivalent to the Vps4E233Q yeast mutant (37Finken-Eigen M. Röhricht R.A. Köhrer K. Curr. Genet. 1997; 31: 469-480Crossref PubMed Scopus (41) Google Scholar) and the SKD1E235Q mouse mutant (10Yoshimori T. Yamagata F. Yamamoto A. Mizushima N. Kabeya Y. Nara A. Miwako I. Ohashi M. Ohsumi M. Ohsumi Y. Mol. Biol. Cell. 2000; 11: 747-763Crossref PubMed Scopus (176) Google Scholar), and the amino acid change introduced was expected to cause a defect in ATP hydrolysis (9Babst M. Wendland B. Estepa E.J. Emr S.D. EMBO J. 1998; 17: 2982-2993Crossref PubMed Scopus (625) Google Scholar). L. major promastigotes were transfected with pXG plasmid expressing VPS4 and VPS4E235Q. The wild-type parasite and cell line containing pXG-VPS4 grew normally as promastigotes in vitro, but the pXG-VPS4E235Q mutant showed a premature exit from exponential growth phase. Indeed, the promastigotes overexpressing pXG-VPS4E235Q grew similarly to wild-type parasites throughout most of the logarithmic phase of growth in vitro but went into stationary phase at a lower density than wild-type or pXG-VPS4 promastigotes (Fig. 1B). During this growth phase in culture, an increasing number of promastigotes expressing VPS4E235Q showed morphological peculiarities and became broader and shorter (for example see GFP-fused VPS4E235Q in Fig. 2B, right). The use of an episomal vector for the expression of the VPS4 gene allowed us to increase expression levels with higher amounts of selective drug. This way we have shown that the growth defect was even more pronounced when the pXG-VPS4E235Q mutant was grown in higher concentrations of G418 (Fig. 1A, lower panel), with growth being hindered even in early logarithmic phase, and this correlated with increased amounts of VPS4E235Q protein at the higher G418 concentrations (Fig. 1A, lower panel inset). In contrast, cells transfected with the empty vector (pXG cell line) could withstand concentrations of G418 up to 50 μg/ml with little or no effect on growth (Fig. 1A, upper panel), confirming that neither the vector nor the resistance marker per se were toxic to the cells. Moreover, overexpression of VPS4 was shown not to be toxic to the cells as the pXG-VPS4 cell line grew normally in the presence of increased amounts of G418 and only showed a slight growth defect at 50 μg/ml (Fig. 1A, middle panel).FIGURE 2VPS4 expression and localization. A, Western blot analyses performed with the anti-VPS4 antibodies on lysates from 2 × 107 promastigotes from early-log (EL), mid-log (ML), early-stationary (ES), and late-stationary (LS) phase cultures of wild-type L. major and late-log phase GFP fusion-expressing cell lines. The arrow and arrowhead indicate VPS4 protein and GFP-fused VPS4 proteins, respectively. Molecular mass is shown on the right (in kDa). B, localization of GFP-fused VPS4 (left) and GFP-fused VPS4E235Q (right) in log phase promastigotes. The GFP fluorescence image (green) has been merged with the equivalent DAPI image (blue; n, nucleus; k, kinetoplast). Differential interference contrast image is shown in the inset panel. Scale bar = 10 μm. C, colocalization of GFP-fused VPS4 or GFP-fused VPS4E235Q with endocytosed tomato lectin (left) and concanavalin A (right). GFP signal is shown in green, and the fluorescent-lectin signal is red; the merged images are magnified. Scale bar = 10 μm. Fp, flagellar pocket; L, late endosomes.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Localization of VPS4 and VPS4E235Q—L. major promastigotes were transfected with N-terminal GFP fusions of VPS4 and VPS4E235Q and expression of the proteins confirmed with anti-" @default.
• W2007630314 created "2016-06-24" @default.
• W2007630314 creator A5000896896 @default.
• W2007630314 creator A5005274850 @default.
• W2007630314 creator A5024173243 @default.
• W2007630314 creator A5025425229 @default.
• W2007630314 creator A5037988773 @default.
• W2007630314 date "2006-04-01" @default.
• W2007630314 modified "2023-10-12" @default.
• W2007630314 title "Endosome Sorting and Autophagy Are Essential for Differentiation and Virulence of Leishmania major" @default.
• W2007630314 cites W1495275933 @default.
• W2007630314 cites W1497136418 @default.
• W2007630314 cites W1548019309 @default.
• W2007630314 cites W1555054913 @default.
• W2007630314 cites W1797888712 @default.
• W2007630314 cites W1838334543 @default.
• W2007630314 cites W1855297813 @default.
• W2007630314 cites W1958092593 @default.
• W2007630314 cites W1969305980 @default.
• W2007630314 cites W1973214920 @default.
• W2007630314 cites W1973626807 @default.
• W2007630314 cites W1978791549 @default.
• W2007630314 cites W1985747385 @default.
• W2007630314 cites W1989821468 @default.
• W2007630314 cites W1990247684 @default.
• W2007630314 cites W1993548033 @default.
• W2007630314 cites W1999256145 @default.
• W2007630314 cites W1999357416 @default.
• W2007630314 cites W1999836892 @default.
• W2007630314 cites W2001819758 @default.
• W2007630314 cites W2003979167 @default.
• W2007630314 cites W2007809227 @default.
• W2007630314 cites W2019215631 @default.
• W2007630314 cites W2019987740 @default.
• W2007630314 cites W2024373248 @default.
• W2007630314 cites W2027109238 @default.
• W2007630314 cites W2039999903 @default.
• W2007630314 cites W2043964141 @default.
• W2007630314 cites W2060864992 @default.
• W2007630314 cites W2061700186 @default.
• W2007630314 cites W2066727453 @default.
• W2007630314 cites W2067692987 @default.
• W2007630314 cites W2068587263 @default.
• W2007630314 cites W2069938223 @default.
• W2007630314 cites W2075027086 @default.
• W2007630314 cites W2075234136 @default.
• W2007630314 cites W2076874733 @default.
• W2007630314 cites W2079998088 @default.
• W2007630314 cites W2093530916 @default.
• W2007630314 cites W2096794529 @default.
• W2007630314 cites W2100312575 @default.
• W2007630314 cites W2110522042 @default.
• W2007630314 cites W2129247085 @default.
• W2007630314 cites W2145625720 @default.
• W2007630314 cites W2145823322 @default.
• W2007630314 cites W2146183220 @default.
• W2007630314 cites W2151413638 @default.
• W2007630314 cites W2156414842 @default.
• W2007630314 cites W2160575327 @default.
• W2007630314 cites W2164785771 @default.
• W2007630314 cites W2167806590 @default.
• W2007630314 cites W4253116674 @default.
• W2007630314 doi "https://doi.org/10.1074/jbc.m512307200" @default.
• W2007630314 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16497676" @default.
• W2007630314 hasPublicationYear "2006" @default.
• W2007630314 type Work @default.
• W2007630314 sameAs 2007630314 @default.
• W2007630314 citedByCount "199" @default.
• W2007630314 countsByYear W20076303142012 @default.
• W2007630314 countsByYear W20076303142013 @default.
• W2007630314 countsByYear W20076303142014 @default.
• W2007630314 countsByYear W20076303142015 @default.
• W2007630314 countsByYear W20076303142016 @default.
• W2007630314 countsByYear W20076303142017 @default.
• W2007630314 countsByYear W20076303142018 @default.
• W2007630314 countsByYear W20076303142019 @default.
• W2007630314 countsByYear W20076303142020 @default.
• W2007630314 countsByYear W20076303142021 @default.
• W2007630314 countsByYear W20076303142022 @default.
• W2007630314 countsByYear W20076303142023 @default.
• W2007630314 crossrefType "journal-article" @default.
• W2007630314 hasAuthorship W2007630314A5000896896 @default.
• W2007630314 hasAuthorship W2007630314A5005274850 @default.
• W2007630314 hasAuthorship W2007630314A5024173243 @default.
• W2007630314 hasAuthorship W2007630314A5025425229 @default.
• W2007630314 hasAuthorship W2007630314A5037988773 @default.
• W2007630314 hasBestOaLocation W20076303141 @default.
• W2007630314 hasConcept C102747710 @default.
• W2007630314 hasConcept C104317684 @default.
• W2007630314 hasConcept C111696304 @default.
• W2007630314 hasConcept C136764020 @default.
• W2007630314 hasConcept C190283241 @default.
• W2007630314 hasConcept C199360897 @default.
• W2007630314 hasConcept C203522944 @default.
• W2007630314 hasConcept C2780599195 @default.
• W2007630314 hasConcept C2781092759 @default.
• W2007630314 hasConcept C41008148 @default.
• W2007630314 hasConcept C54355233 @default.

• next