Late Endosomal Cholesterol Accumulation Leads to Impaired Intra-Endosomal Trafficking
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{"title"=>"Late endosomal cholesterol accumulation leads to impaired intra-endosomal trafficking", "type"=>"journal", "authors"=>[{"first_name"=>"Komla", "last_name"=>"Sobo", "scopus_author_id"=>"6504092527"}, {"first_name"=>"Isabelle", "last_name"=>"Le Blanc", "scopus_author_id"=>"36914418100"}, {"first_name"=>"Pierre Philippe", "last_name"=>"Luyet", "scopus_author_id"=>"8518847600"}, {"first_name"=>"Marc", "last_name"=>"Fivaz", "scopus_author_id"=>"6701510254"}, {"first_name"=>"Charles", "last_name"=>"Ferguson", "scopus_author_id"=>"7201459577"}, {"first_name"=>"Robert G.", "last_name"=>"Parton", "scopus_author_id"=>"7101912368"}, {"first_name"=>"Jean", "last_name"=>"Gruenberg", "scopus_author_id"=>"7005949878"}, {"first_name"=>"F. Gisou", "last_name"=>"Van der Goot", "scopus_author_id"=>"35560623700"}], "year"=>2007, "source"=>"PLoS ONE", "identifiers"=>{"sgr"=>"38049011141", "doi"=>"10.1371/journal.pone.0000851", "issn"=>"19326203", "pui"=>"351420358", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "pmid"=>"17786222", "scopus"=>"2-s2.0-38049011141"}, "id"=>"c723f7f1-d6de-38fd-bd0b-53e6b0965ca1", "abstract"=>"BACKGROUND: Pathological accumulation of cholesterol in late endosomes is observed in lysosomal storage diseases such as Niemann-Pick type C. We here analyzed the effects of cholesterol accumulation in NPC cells, or as phenocopied by the drug U18666A, on late endosomes membrane organization and dynamics. METHODOLOGY/PRINCIPAL FINDINGS: Cholesterol accumulation did not lead to an increase in the raft to non-raft membrane ratio as anticipated. Strikingly, we observed a 2-3 fold increase in the size of the compartment. Most importantly, properties and dynamics of late endosomal intralumenal vesicles were altered as revealed by reduced late endosomal vacuolation induced by the mutant pore-forming toxin ASSP, reduced intoxication by the anthrax lethal toxin and inhibition of infection by the Vesicular Stomatitis Virus. CONCLUSIONS/SIGNIFICANCE: These results suggest that back fusion of intralumenal vesicles with the limiting membrane of late endosomes is dramatically perturbed upon cholesterol accumulation.", "link"=>"http://www.mendeley.com/research/late-endosomal-cholesterol-accumulation-leads-impaired-intraendosomal-trafficking", "reader_count"=>67, "reader_count_by_academic_status"=>{"Unspecified"=>2, "Professor > Associate Professor"=>7, "Researcher"=>17, "Student > Doctoral Student"=>5, "Student > Ph. D. Student"=>18, "Student > Master"=>7, "Other"=>1, "Student > Bachelor"=>5, "Lecturer"=>2, "Lecturer > Senior Lecturer"=>1, "Professor"=>2}, "reader_count_by_user_role"=>{"Unspecified"=>2, "Professor > Associate Professor"=>7, "Researcher"=>17, "Student > Doctoral Student"=>5, "Student > Ph. D. Student"=>18, "Student > Master"=>7, "Other"=>1, "Student > Bachelor"=>5, "Lecturer"=>2, "Lecturer > Senior Lecturer"=>1, "Professor"=>2}, "reader_count_by_subject_area"=>{"Unspecified"=>3, "Biochemistry, Genetics and Molecular Biology"=>12, "Agricultural and Biological Sciences"=>36, "Medicine and Dentistry"=>7, "Neuroscience"=>3, "Chemistry"=>2, "Computer Science"=>1, "Immunology and Microbiology"=>3}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>7}, "Neuroscience"=>{"Neuroscience"=>3}, "Chemistry"=>{"Chemistry"=>2}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>3}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>36}, "Computer Science"=>{"Computer Science"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>12}, "Unspecified"=>{"Unspecified"=>3}}, "reader_count_by_country"=>{"Argentina"=>1, "United States"=>1, "Finland"=>1, "Egypt"=>1, "Australia"=>1, "Indonesia"=>1}, "group_count"=>3}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/945986"], "description"=>"<p>A: Late endosomes were isolated from control and U18666A treated BHK cells. Fractions were analyzed by SDS-PAGE either in terms of equal volume (Yield, same fraction of total late endosomes) or equal amount of protein (enrichment) and immunoblotted for the presence of Lamp-1, Rab7, flotillin-1 (Flot-1) or GPI-anchored proteins, which were revealed using an aerolysin overlay. BC: For each of these proteins, the change in abundance upon U18666A treatment vs. control was determined through quantitation by densitometry. Quantifications were performed both for analysis by Yield and by Enrichment. Error bars represent the standard deviation (n = 3). * represent statistically significant differences (p<0.05). D: DRMs were prepared from late endosomes (BHK cells +/− U18666A) by flotation on an OptiPrep gradient. Six 400 µl fractions were collected and their protein content determined. Each fraction protein content is expressed as percentage of the total amount of proteins along the gradient. Error bars represent the standard deviation (n = 3).</p>", "links"=>[], "tags"=>["u18666a", "endosomal"], "article_id"=>616350, "categories"=>["Microbiology", "Cell Biology"], "users"=>["Komla Sobo", "Isabelle Le Blanc", "Pierre-Philippe Luyet", "Marc Fivaz", "Charles Ferguson", "Robert G. Parton", "Jean Gruenberg", "F. Gisou van der Goot"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0000851.g006", "stats"=>{"downloads"=>0, "page_views"=>16, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effect_of_U18666A_on_late_endosomal_proteins_/616350", "title"=>"Effect of U18666A on late endosomal proteins.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 12:01:50"}
  • {"files"=>["https://ndownloader.figshare.com/files/945787"], "description"=>"<p>A: Control and U18666A treated cells were analyzed by electron microscopy. B: The diameters of 132 late endosomes from control cells and 189 from U18666A treated cells was measured and the % of profiles for different size ranges were determined and plotted.</p>", "links"=>[], "tags"=>["leads", "enlargement"], "article_id"=>616155, "categories"=>["Microbiology", "Cell Biology"], "users"=>["Komla Sobo", "Isabelle Le Blanc", "Pierre-Philippe Luyet", "Marc Fivaz", "Charles Ferguson", "Robert G. Parton", "Jean Gruenberg", "F. Gisou van der Goot"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0000851.g002", "stats"=>{"downloads"=>0, "page_views"=>26, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_U18666A_leads_to_an_enlargement_of_late_endosomes_/616155", "title"=>"U18666A leads to an enlargement of late endosomes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 12:00:40"}
  • {"files"=>["https://ndownloader.figshare.com/files/946097"], "description"=>"<p>AB: BHK cells were treated with or not with U18666A for 8 hrs, followed by 9 hrs treatment, still in the presence of the drug, with the mutant aerolysin toxin ASSP (500 ng/ml). B. For each conditions, the number of vacuolated cells, out of a total of n = 240, was counted. C: BHK cells were treated with trypsin nicked ASSP (500 ng/ml) either for 1 h at 4°C or for 24 h at 37°C. For cells treated at 4°C, 20 µg of total cell extracts were analyzed for the presence of monomeric and heptameric aerolysin by non-reducing SDS-PAGE and Western blotting. Cells treated at 37°C, were submitted to subcellular fractionation to isolate late endosomes, which were analyzed for the presence of monomeric and heptameric aerolysin by non-reducing SDS-PAGE and western blotting. Monomeric ASSP underwent differential migration when reduced in late endosomes vs. non-reduced at the cell surface as previously observed <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000851#pone.0000851-Fivaz1\" target=\"_blank\">[22]</a>. D: BHK cells were treated with U18666A for 8 hrs. Alexa-ASSP was subsequently added and allowed to be internalized for 6 hrs. Cells were fixed and processed for fluorescence microscopy to detect flotillin-1 in late endosomes.</p>", "links"=>[], "tags"=>["protects", "cells"], "article_id"=>616460, "categories"=>["Microbiology", "Cell Biology"], "users"=>["Komla Sobo", "Isabelle Le Blanc", "Pierre-Philippe Luyet", "Marc Fivaz", "Charles Ferguson", "Robert G. Parton", "Jean Gruenberg", "F. Gisou van der Goot"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0000851.g008", "stats"=>{"downloads"=>1, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_U18666A_treatment_protects_cells_against_the_action_of_ASSP_/616460", "title"=>"U18666A treatment protects cells against the action of ASSP.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 12:02:32"}
  • {"files"=>["https://ndownloader.figshare.com/files/945846"], "description"=>"<p>The % of cytoplasmic volume occupied by late endosomes was determined by quantitative electron microscopy on U18666A treated late endosomes, NPC1 deficient CHO cells and fibroblast from an NPC1 patient <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000851#pone.0000851-Kobayashi1\" target=\"_blank\">[24]</a> in comparison to their respective controls.</p>", "links"=>[], "tags"=>["deficiency", "leads", "enlargement"], "article_id"=>616213, "categories"=>["Microbiology", "Cell Biology"], "users"=>["Komla Sobo", "Isabelle Le Blanc", "Pierre-Philippe Luyet", "Marc Fivaz", "Charles Ferguson", "Robert G. Parton", "Jean Gruenberg", "F. Gisou van der Goot"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0000851.g003", "stats"=>{"downloads"=>1, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_NPC1_deficiency_leads_to_the_enlargement_of_late_endosomes_/616213", "title"=>"NPC1 deficiency leads to the enlargement of late endosomes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 12:01:00"}
  • {"files"=>["https://ndownloader.figshare.com/files/945927"], "description"=>"<p>DRMs were prepared from late endosomes (BHK cells +/− U18666A) by flotation on an OptiPrep gradient. Six fractions of 400 µl were collected. A: Lipids were extracted and the amount of cholesterol determined by 1D TLC. Cholesterol contents were expressed as a percentage of the total cholesterol on the gradient. Error bars represent the standard deviation (n = 3). B: After pooling fractions 1 and 2 from the top of the Optiprep gradient (corresponding to the DRMs), lipids were extracted and analyzed by 2D TLC (shown TLC corresponds to the U18666A condition). I: unidentified lipid, PI: phosphatidyl inositol, PS: phosphatidyl serine, SM: sphingomyelins, PE: phosphatidyl ethanolamine, PC: phosphatidyl choline, LBPA: Lysobisphosphatidic acid. C–D: The amount of each phospholipid in fractions 1+2 (C) and fraction 6 (D) were quantified by densitometry. Each phospholipid is expressed as percentage of the total amount of phospholipids on the TLC plate. Error bars represent the standard deviation (n = 3). E: In order to evaluate the distribution of SM, PS, PC, LBPA and I4 through out the Optiprep gradient upon fractionation of late endosomes form U18666A treated cells, the content of these lipids in fractions 1+2 and in fraction 5+6 was determined. For each lipid, the distribution between these two pools was plotted.</p>", "links"=>[], "tags"=>["endosomal", "drms", "u18666a", "treated"], "article_id"=>616294, "categories"=>["Microbiology", "Cell Biology"], "users"=>["Komla Sobo", "Isabelle Le Blanc", "Pierre-Philippe Luyet", "Marc Fivaz", "Charles Ferguson", "Robert G. Parton", "Jean Gruenberg", "F. Gisou van der Goot"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0000851.g005", "stats"=>{"downloads"=>1, "page_views"=>18, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Lipid_analysis_of_late_endosomal_DRMs_from_U18666A_treated_cells_/616294", "title"=>"Lipid analysis of late endosomal DRMs from U18666A treated cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 12:01:31"}
  • {"files"=>["https://ndownloader.figshare.com/files/946044"], "description"=>"<p>A: DRMs were prepared from late endosomes (BHK cells +/− U18666A) by flotation on an OptiPrep gradient. Six fractions were collected from the top and analyzed by immunoblotting for the presence of flotillin-1 (Flot-1), Rab7, Lamp-1, annexin II (AnnII) and MLN64. B: BHK cells treated or not with U18666A. Total cells or purified late endosomes were solubilized in 1% Triton ×100 at 4°C (as in A and all other figures) or 37°C. The lysats were submitted to flotation on Optiprep gradients, 6 fractions collected and analyzed for the distribution of GPI-anchored proteins by aerolysin overlay. C: For the 4 conditions illustrated in B and for 3 independent experiments, the total GPI content of each fraction was quantified by densitometry and normalized to the total GPI-content of the gradient. Error bars represent the standard deviation (n = 3). * represent statistically significant differences (p<0.05).</p>", "links"=>[], "tags"=>["alters", "detergent", "solubility", "gpi-anchored", "proteins"], "article_id"=>616401, "categories"=>["Microbiology", "Cell Biology"], "users"=>["Komla Sobo", "Isabelle Le Blanc", "Pierre-Philippe Luyet", "Marc Fivaz", "Charles Ferguson", "Robert G. Parton", "Jean Gruenberg", "F. Gisou van der Goot"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0000851.g007", "stats"=>{"downloads"=>1, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_U18666A_alters_the_detergent_solubility_of_GPI_anchored_proteins_in_late_endosomes_/616401", "title"=>"U18666A alters the detergent solubility of GPI-anchored proteins in late endosomes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 12:02:08"}
  • {"files"=>["https://ndownloader.figshare.com/files/945888"], "description"=>"<p>A: Late endosomes were purified from control and U18666A treated cells. An equivalent amount of protein (100 µg) from each late endosomal fraction was submitted to lipid extraction. The samples were run on two dimensional thin layer chromatography (TLC) plates for phospholipids analysis. The phospholipids were quantified by densitometry and each one expressed as percentage of the total amount of phospholipids on the plate. Error bars represent the standard deviation (n = 3). I: unidentified lipid, PI: phosphatidyl inositol, PS: phosphatidyl serine, PE: phosphatidyl ethanolamine, PC: phosphatidyl choline, LBPA: Lysobisphosphatidic acid. B: The content in total PC and PE, corresponding to an equivalent amount of protein was compared between control and U18666A treated cells.</p>", "links"=>[], "tags"=>["endosomes", "u18666a", "treated"], "article_id"=>616253, "categories"=>["Microbiology", "Cell Biology"], "users"=>["Komla Sobo", "Isabelle Le Blanc", "Pierre-Philippe Luyet", "Marc Fivaz", "Charles Ferguson", "Robert G. Parton", "Jean Gruenberg", "F. Gisou van der Goot"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0000851.g004", "stats"=>{"downloads"=>2, "page_views"=>19, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Lipid_composition_of_late_endosomes_from_control_and_U18666A_treated_cells_/616253", "title"=>"Lipid composition of late endosomes from control and U18666A treated cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 12:01:15"}
  • {"files"=>["https://ndownloader.figshare.com/files/945743"], "description"=>"<p>BHK cells were treated or not with U18666A at 3 µg/ml for 18 hours. A: Cells were labeled with filipin (to detect cholesterol) and anti-LBPA antibodies. B: Cells were submitted to subcellular fractionation, as described in experimental procedures, to separate late from early endosomes. 10 µg of protein from each fraction were separated in 12.5% SDS-PAGE and blotted for the presence of the small GTPases rab7 (late endosomal marker) and rab5 (early endosomal marker), calnexin (endoplasmic reticulum marker), caveolin-1 (caveolar marker) and ß-galactosyl transferase (Golgi enzyme). An equivalent proportion (C) or an equal amount of protein (D) of the late endosomal fraction from BHK cells treated or not with U18666A (3 µg/ml for 18 hours) was submitted to lipid extraction and 1D TLC for cholesterol determination. The cholesterol was quantified by densitometry. Error bars represent the standard deviation (n = 3). E: The protein content of equivalent volume of the late endosomal fraction from BHK cells treated or not with U18666A was determined. Error bars represent the standard deviation (n = 3).</p>", "links"=>[], "tags"=>["endosomes", "u18666a", "treated-bhk"], "article_id"=>616106, "categories"=>["Microbiology", "Cell Biology"], "users"=>["Komla Sobo", "Isabelle Le Blanc", "Pierre-Philippe Luyet", "Marc Fivaz", "Charles Ferguson", "Robert G. Parton", "Jean Gruenberg", "F. Gisou van der Goot"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0000851.g001", "stats"=>{"downloads"=>3, "page_views"=>24, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Purification_of_late_endosomes_from_control_and_U18666A_treated_BHK_cells_/616106", "title"=>"Purification of late endosomes from control and U18666A treated-BHK cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 12:00:19"}
  • {"files"=>["https://ndownloader.figshare.com/files/946161"], "description"=>"<p>A: BHK cells treated or not with 3 µg/ml U18666A were incubated 1 h at 4°C with 500 ng/ml anthrax protective antigen (PA) and 100 ng/ml lethal factor (LF) <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000851#pone.0000851-Abrami2\" target=\"_blank\">[34]</a>. The cells were then transferred to 37°C for different periods of time in a toxin-free medium. 40 µg protein of the PNS was analyzed by western blotting against PA to detect the heptameric channel form and against MEK1, using an anti N-terminal antibody. B: BHK cells were pretreated or not during 18 hrs with U18666A and then incubated with Dil labeled VSV (0.3 MOI) at 4°C for 10 min. The temperature was then increased to 37°C. The number of cells containing fused viruses was counted 45 min post-infection. C:BHK cells were pretreated or not during 9 hrs with U18666A and then infected with VSV at an MOI of 1. After 3 hrs of VSV infect, cells were analyzed by immunofluorescence microscopy using antibodies against VSV-G. Typically, ≈70% of the cells were infected under control conditions and the number of infected cells was expressed as a percentage of the untreated control.</p>", "links"=>[], "tags"=>["protects", "cells", "anthrax", "toxin", "vesicular", "stomatitis"], "article_id"=>616516, "categories"=>["Microbiology", "Cell Biology"], "users"=>["Komla Sobo", "Isabelle Le Blanc", "Pierre-Philippe Luyet", "Marc Fivaz", "Charles Ferguson", "Robert G. Parton", "Jean Gruenberg", "F. Gisou van der Goot"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0000851.g009", "stats"=>{"downloads"=>0, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_U18666A_treatment_protects_cells_against_the_action_of_anthrax_toxin_and_from_infection_by_the_Vesicular_Stomatitis_Virus_/616516", "title"=>"U18666A treatment protects cells against the action of anthrax toxin and from infection by the Vesicular Stomatitis Virus.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 12:02:51"}

PMC Usage Stats | Further Information

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Relative Metric

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