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Cited by 51Cellular transport and secretionAutophagy in Disease and TherapyCalcium signaling and nucleotide metabolism

A novel live-cell imaging assay reveals regulation of endosome maturation

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Podinovskaia, Maria, Prescianotto-Baschong, Cristina, Buser, Dominik P, Spang, Anne·2021·eLife
DOI 10.7554/elife.70982PMC PMC8635980

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Abstract

Cell-cell communication is an essential process in life, with endosomes acting as key organelles for regulating uptake and secretion of signaling molecules. Endocytosed material is accepted by the sorting endosome where it either is sorted for recycling or remains in the endosome as it matures to be degraded in the lysosome. Investigation of the endosome maturation process has been hampered by the small size and rapid movement of endosomes in most cellular systems. Here, we report an easy versatile live-cell imaging assay to monitor endosome maturation kinetics, which can be applied to a variety of mammalian cell types. Acute ionophore treatment led to enlarged early endosomal compartments that matured into late endosomes and fused with lysosomes to form endolysosomes. Rab5-to-Rab7 conversion and PI(3)P formation and turn over were recapitulated with this assay and could be observed with a standard widefield microscope. We used this approach to show that Snx1 and Rab11-positive recycling endosome recruitment occurred throughout endosome maturation and was uncoupled from Rab conversion. In contrast, efficient endosomal acidification was dependent on Rab conversion. The assay provides a powerful tool to further unravel various aspects of endosome maturation.

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  • Lipid droplet accumulation is considered a marker of inflammation in microglia during aging and Alzheimer's disease.

    “the accumulation of which is considered a marker of inflammation in microglia during aging and Alzheimer’s disease32–35”

    From INPP5D/SHIP1 regulates endo-lysosomal function and cargo-selective phagocytosis in human microglia (2025) · cites this paper as [32]

    The Results passages on nigericin-induced endosome maturation and the Discussion passage on Golgi membranes do not address lipid droplets or microglial inflammation. Searches for lipid droplet accumulation, microglia, aging, and Alzheimer’s disease found no passage supporting the attributed claim. No page numbers are available in our copy. (LLM, conf=0.76)

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Research works printed but not linked: 51

  1. Balderhaar HJ Ungermann C 2013 CORVET and HOPS tethering complexes - coordinators of endosome and lysosome fusion Journal of Cell Science 126 1307 1316 10.1242/jcs.107805 23645161 · DOI
  2. Beuret N Hasler F Prescianotto-Baschong C Birk J Rutishauser J Spiess M 2017 Amyloid-like aggregation of provasopressin in diabetes insipidus and secretory granule sorting BMC Biology 15 5 10.1186/s12915-017-0347-9 28122547 PMC5267430 · DOI
  3. Bucci C Thomsen P Nicoziani P McCarthy J van Deurs B 2000 Rab7: a key to lysosome biogenesis Molecular Biology of the Cell 11 467 480 10.1091/mbc.11.2.467 10679007 PMC14786 · DOI
  4. Burgstaller S Bischof H Gensch T Stryeck S Gottschalk B Ramadani-Muja J Eroglu E Rost R Balfanz S Baumann A Waldeck-Weiermair M Hay JC Madl T Graier WF Malli R 2019 pH-Lemon, a Fluorescent Protein-Based pH Reporter for Acidic Compartments ACS Sensors 4 883 891 10.1021/acssensors.8b01599 30864782 PMC6488996 · DOI
  5. Buser DP Schleicher KD Prescianotto-Baschong C Spiess M 2018 A versatile nanobody-based toolkit to analyze retrograde transport from the cell surface PNAS 115 E6227 E6236 10.1073/pnas.1801865115 29915061 PMC6142194 · DOI
  6. Casey JR Grinstein S Orlowski J 2010 Sensors and regulators of intracellular pH Nature Reviews. Molecular Cell Biology 11 50 61 10.1038/nrm2820 19997129 · DOI
  7. Cezanne A Lauer J Solomatina A Sbalzarini IF Zerial M 2020 A non-linear system patterns Rab5 GTPase on the membrane eLife 9 e54434 10.7554/eLife.54434 32510320 PMC7279886 · DOI
  8. Choudhury A Dominguez M Puri V Sharma DK Narita K Wheatley CL Marks DL Pagano RE 2002 Rab proteins mediate Golgi transport of caveola-internalized glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells The Journal of Clinical Investigation 109 1541 1550 10.1172/JCI15420 12070301 PMC151017 · DOI
  9. Compton LM Ikonomov OC Sbrissa D Garg P Shisheva A 2016 Active vacuolar H+ ATPase and functional cycle of Rab5 are required for the vacuolation defect triggered by PtdIns(3,5)P2 loss under PIKfyve or Vps34 deficiency American Journal of Physiology. Cell Physiology 311 C366 C377 10.1152/ajpcell.00104.2016 27335171 PMC5129759 · DOI
  10. De Luca M Bucci C 2014 A new V-ATPase regulatory mechanism mediated by the Rab interacting lysosomal protein (RILP) Communicative & Integrative Biology 7 29616 10.4161/cib.29616 26843904 PMC4594554 · DOI
  11. De Luca M Cogli L Progida C Nisi V Pascolutti R Sigismund S Di Fiore PP Bucci C 2014 RILP regulates vacuolar ATPase through interaction with the V1G1 subunit Journal of Cell Science 127 2697 2708 10.1242/jcs.142604 24762812 · DOI
  12. Del Conte-Zerial P Brusch L Rink JC Collinet C Kalaidzidis Y Zerial M Deutsch A 2008 Membrane identity and GTPase cascades regulated by toggle and cut-out switches Molecular Systems Biology 4 206 10.1038/msb.2008.45 18628746 PMC2516367 · DOI
  13. Dove SK Dong K Kobayashi T Williams FK Michell RH 2009 Phosphatidylinositol 3,5-bisphosphate and Fab1p/PIKfyve underPPIn endo-lysosome function The Biochemical Journal 419 1 13 10.1042/BJ20081950 19272020 · DOI
  14. Falcón-Pérez JM Nazarian R Sabatti C Dell’Angelica EC 2005 Distribution and dynamics of Lamp1-containing endocytic organelles in fibroblasts deficient in BLOC-3 Journal of Cell Science 118 5243 5255 10.1242/jcs.02633 16249233 · DOI
  15. Gillooly DJ Morrow IC Lindsay M Gould R Bryant NJ Gaullier JM Parton RG Stenmark H 2000 Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells The EMBO Journal 19 4577 4588 10.1093/emboj/19.17.4577 10970851 PMC302054 · DOI
  16. Guerra F Bucci C 2016 Multiple Roles of the Small GTPase Rab7 Cells 5 E34 10.3390/cells5030034 27548222 PMC5040976 · DOI
  17. Gullapalli A Garrett TA Paing MM Griffin CT Yang Y Trejo J 2004 A role for sorting nexin 2 in epidermal growth factor receptor down-regulation: evidence for distinct functions of sorting nexin 1 and 2 in protein trafficking Molecular Biology of the Cell 15 2143 2155 10.1091/mbc.e03-09-0711 14978220 PMC404011 · DOI
  18. Hsu F Hu F Mao Y 2015 Spatiotemporal control of phosphatidylinositol 4-phosphate by Sac2 regulates endocytic recycling The Journal of Cell Biology 209 97 110 10.1083/jcb.201408027 25869669 PMC4395482 · DOI
  19. Huotari J Helenius A 2011 Endosome maturation The EMBO Journal 30 3481 3500 10.1038/emboj.2011.286 21878991 PMC3181477 · DOI
  20. Ledger PW Uchida N Tanzer ML 1980 Immunocytochemical localization of procollagen and fibronectin in human fibroblasts: effects of the monovalent ionophore, monensin The Journal of Cell Biology 87 663 671 10.1083/jcb.87.3.663 7007394 PMC2110792 · DOI
  21. Ma L Ouyang Q Werthmann GC Thompson HM Morrow EM 2017 Live-cell Microscopy and Fluorescence-based Measurement of Luminal pH in Intracellular Organelles Frontiers in Cell and Developmental Biology 5 71 10.3389/fcell.2017.00071 28871281 PMC5566985 · DOI
  22. Mari M Bujny MV Zeuschner D Geerts WJC Griffith J Petersen CM Cullen PJ Klumperman J Geuze HJ 2008 SNX1 defines an early endosomal recycling exit for sortilin and mannose 6-phosphate receptors Traffic 9 380 393 10.1111/j.1600-0854.2007.00686.x 18088323 · DOI
  23. McDermott H Kim K 2015 Molecular dynamics at the endocytic portal and regulations of endocytic and recycling traffics European Journal of Cell Biology 94 235 248 10.1016/j.ejcb.2015.04.003 25921524 · DOI
  24. Merion M Sly WS 1983 The role of intermediate vesicles in the adsorptive endocytosis and transport of ligand to lysosomes by human fibroblasts The Journal of Cell Biology 96 644 650 10.1083/jcb.96.3.644 6220018 PMC2112385 · DOI
  25. Morré DJ Boss WF Grimes H Mollenhauer HH 1983 Kinetics of Golgi apparatus membrane flux following monensin treatment of embryogenic carrot cells European Journal of Cell Biology 30 25 32 6852059
  26. Nagano M Toshima JY Siekhaus DE Toshima J 2019 Rab5-mediated endosome formation is regulated at the trans-Golgi network Communications Biology 2 419 10.1038/s42003-019-0670-5 31754649 PMC6858330 · DOI
  27. Naufer A Hipolito VEB Ganesan S Prashar A Zaremberg V Botelho RJ Terebiznik MR 2018 pH of endophagosomes controls association of their membranes with Vps34 and PtdIns(3)P levels The Journal of Cell Biology 217 329 346 10.1083/jcb.201702179 29089378 PMC5748975 · DOI
  28. Nordmann M Cabrera M Perz A Bröcker C Ostrowicz C Engelbrecht-Vandré S Ungermann C 2010 The Mon1-Ccz1 complex is the GEF of the late endosomal Rab7 homolog Ypt7 Current Biology 20 1654 1659 10.1016/j.cub.2010.08.002 20797862 · DOI
  29. Peter BJ Kent HM Mills IG Vallis Y Butler PJG Evans PR McMahon HT 2004 BAR domains as sensors of membrane curvature: the amphiphysin BAR structure Science 303 495 499 10.1126/science.1092586 14645856 · DOI
  30. Podinovskaia M Lee W Caldwell S Russell DG 2013 Infection of macrophages with Mycobacterium tuberculosis induces global modifications to phagosomal function Cellular Microbiology 15 843 859 10.1111/cmi.12092 23253353 PMC3620910 · DOI
  31. Podinovskaia M Spang A 2018 The Endosomal Network: Mediators and Regulators of Endosome Maturation Progress in Molecular and Subcellular Biology 57 1 38 10.1007/978-3-319-96704-2_1 30097770 · DOI
  32. Poteryaev D Fares H Bowerman B Spang A 2007 Caenorhabditis elegans SAND-1 is essential for RAB-7 function in endosomal traffic The EMBO Journal 26 301 312 10.1038/sj.emboj.7601498 17203072 PMC1783445 · DOI
  33. Poteryaev D Datta S Ackema K Zerial M Spang A 2010 Identification of the switch in early-to-late endosome transition Cell 141 497 508 10.1016/j.cell.2010.03.011 20434987 · DOI · Paper page
  34. Ran FA Hsu PD Wright J Agarwala V Scott DA Zhang F 2013 Genome engineering using the CRISPR-Cas9 system Nature Protocols 8 2281 2308 10.1038/nprot.2013.143 24157548 PMC3969860 · DOI · Paper page
  35. Rink J Ghigo E Kalaidzidis Y Zerial M 2005 Rab conversion as a mechanism of progression from early to late endosomes Cell 122 735 749 10.1016/j.cell.2005.06.043 16143105 · DOI
  36. Rojas R van Vlijmen T Mardones GA Prabhu Y Rojas AL Mohammed S Heck AJR Raposo G van der Sluijs P Bonifacino JS 2008 Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7 The Journal of Cell Biology 183 513 526 10.1083/jcb.200804048 18981234 PMC2575791 · DOI
  37. Schink KO Raiborg C Stenmark H 2013 Phosphatidylinositol 3-phosphate, a lipid that regulates membrane dynamics, protein sorting and cell signalling BioEssays 35 900 912 10.1002/bies.201300064 23881848 · DOI
  38. Simonetti B Danson CM Heesom KJ Cullen PJ 2017 Sequence-dependent cargo recognition by SNX-BARs mediates retromer-independent transport of CI-MPR The Journal of Cell Biology 216 3695 3712 10.1083/jcb.201703015 28935633 PMC5674890 · DOI
  39. Solinger JA Spang A 2013 Tethering complexes in the endocytic pathway: CORVET and HOPS The FEBS Journal 280 2743 2757 10.1111/febs.12151 23351085 · DOI
  40. Solinger JA Rashid HO Prescianotto-Baschong C Spang A 2020 FERARI is required for Rab11-dependent endocytic recycling Nature Cell Biology 22 213 224 10.1038/s41556-019-0456-5 31988382 PMC7616953 · DOI
  41. Spang A 2016 Membrane Tethering Complexes in the Endosomal System Frontiers in Cell and Developmental Biology 4 35 10.3389/fcell.2016.00035 27243003 PMC4860415 · DOI
  42. Spang A 2021 FERARI and Cargo Adaptors Coordinate Cargo Flow through Sorting Endosomes Europepmc.org 10.21203/rs.3.rs-701696/v1 PMC9359993 35941155 · DOI
  43. Subach OM Cranfill PJ Davidson MW Verkhusha VV 2011 An enhanced monomeric blue fluorescent protein with the high chemical stability of the chromophore PLOS ONE 6 e28674 10.1371/journal.pone.0028674 22174863 PMC3234270 · DOI
  44. Tartakoff AM Vassalli P 1977 Plasma cell immunoglobulin secretion: arrest is accompanied by alterations of the golgi complex The Journal of Experimental Medicine 146 1332 1345 10.1084/jem.146.5.1332 925606 PMC2180962 · DOI
  45. van den Boomen DJH Sienkiewicz A Berlin I Jongsma MLM van Elsland DM Luzio JP Neefjes JJC Lehner PJ 2020 A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export Nature Communications 11 5559 10.1038/s41467-020-19032-0 33144569 PMC7642327 · DOI
  46. van der Schaar HM Rust MJ Chen C van der Ende-Metselaar H Wilschut J Zhuang X Smit JM 2008 Dissecting the cell entry pathway of dengue virus by single-particle tracking in living cells PLOS Pathogens 4 e1000244 10.1371/journal.ppat.1000244 19096510 PMC2592694 · DOI
  47. van Weering JRT Verkade P Cullen PJ 2012 SNX-BAR-mediated endosome tubulation is co-ordinated with endosome maturation Traffic 13 94 107 10.1111/j.1600-0854.2011.01297.x 21973056 · DOI
  48. Vladutiu GD 1984 Immunocytochemical localization of beta-hexosaminidase and electron-microscopic characterization of human fibroblasts following treatment with monensin and nigericin Bioscience Reports 4 1079 1088 10.1007/BF01116702 6241482 · DOI
  49. Wu Y Boulogne C Carle S Podinovskaia M Barth H Spang A Cintrat JC Gillet D Barbier J 2020 Regulation of endo-lysosomal pathway and autophagic flux by broad-spectrum antipathogen inhibitor ABMA The FEBS Journal 287 3184 3199 10.1111/febs.15201 31901207 · DOI
  50. Zerial M McBride H 2001 Rab proteins as membrane organizers Nature Reviews. Molecular Cell Biology 2 107 117 10.1038/35052055 11252952 · DOI · Paper page
  51. Zhong Q Watson MJ Lazar CS Hounslow AM Waltho JP Gill GN 2005 Determinants of the endosomal localization of sorting nexin 1 Molecular Biology of the Cell 16 2049 2057 10.1091/mbc.e04-06-0504 15673616 PMC1073682 · DOI

Cited by

Sorted by citations

Authors
Podinovskaia, M., Prescianotto-Baschong, C., Buser, D. P., & Spang, A.
Affiliations
University of Basel (CH)
Corresponding author
Spang, Anne
ORCID
Venue
eLife
Publisher
eLife Sciences Publications, Ltd
Published
2021-11-30
Volume
10
ISSN
2050-084X (ISSN-L)
DOI
10.7554/elife.70982
PMC
PMC8635980
OpenAlex
W3175290268
Cited by
51
References
1
Citations by year
2026: 11 · 2025: 14 · 2024: 7 · 2023: 11 · 2022: 7 · 2021: 1
FWCI
3.39
Type
journal article
Language
en
Open access
Licence
Locations
  • eLife (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • edoc (University of Basel) (acceptedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (CRSII3_141956, 310030_197779, 310030_185127)
  • Universität Basel
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Health Sciences › Medicine › Epidemiology › Autophagy in Disease and Therapy
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Physiology › Calcium signaling and nucleotide metabolism
Keywords
endosome maturation, early endosomes, late endosomes, endolysosomes, lysosome fusion, Rab5, Rab7, phosphatidylinositol 3-phosphate, endosomal acidification, recycling endosomes, SNX1, Rab11, intercellular communication
MeSH terms
HeLa Cells, Humans, Lysosomes, Microscopy, Fluorescence, Endosomes

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