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• W2883513762 abstract "The endocytic pathway plays an instrumental role in recycling internalized molecules back to the plasma membrane or in directing them to lysosomes for degradation. We recently reported a new role of endosomes–the delivery of components from extracellular vesicles (EVs) to the nucleoplasm of recipient cells. Using indirect immunofluorescence, FRET, immunoisolation techniques, and RNAi, we report here a tripartite protein complex (referred to as the VOR complex) that is essential for the nuclear transfer of EV-derived components by orchestrating the specific localization of late endosomes into nucleoplasmic reticulum. We found that the VOR complex contains the endoplasmic reticulum–localized vesicle-associated membrane protein (VAMP)-associated protein A (VAP-A), the cytoplasmic oxysterol-binding protein–related protein 3 (ORP3), and late endosome–associated small GTPase Rab7. The silencing of VAP-A or ORP3 abrogated the association of Rab7-positive late endosomes with nuclear envelope invaginations and, hence, the transport of endocytosed EV-derived components to the nucleoplasm of recipient cells. We conclude that the VOR complex can be targeted to inhibit EV-mediated intercellular communication, which can have therapeutic potential for managing cancer in which the release of EVs is dysregulated. The endocytic pathway plays an instrumental role in recycling internalized molecules back to the plasma membrane or in directing them to lysosomes for degradation. We recently reported a new role of endosomes–the delivery of components from extracellular vesicles (EVs) to the nucleoplasm of recipient cells. Using indirect immunofluorescence, FRET, immunoisolation techniques, and RNAi, we report here a tripartite protein complex (referred to as the VOR complex) that is essential for the nuclear transfer of EV-derived components by orchestrating the specific localization of late endosomes into nucleoplasmic reticulum. We found that the VOR complex contains the endoplasmic reticulum–localized vesicle-associated membrane protein (VAMP)-associated protein A (VAP-A), the cytoplasmic oxysterol-binding protein–related protein 3 (ORP3), and late endosome–associated small GTPase Rab7. The silencing of VAP-A or ORP3 abrogated the association of Rab7-positive late endosomes with nuclear envelope invaginations and, hence, the transport of endocytosed EV-derived components to the nucleoplasm of recipient cells. We conclude that the VOR complex can be targeted to inhibit EV-mediated intercellular communication, which can have therapeutic potential for managing cancer in which the release of EVs is dysregulated. Extracellular vesicles (EVs), 3The abbreviations used are: EVextracellular vesicleNEInuclear envelope invaginationPFAparaformaldehydeOSBPoxysterol-binding proteinBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolCLSMconfocal laser-scanning microscopyRFPred fluorescent proteinRFIrelative fluorescence intensityERendoplasmic reticulumFFATdiphenylalanine in an acidic tractONMouter nuclear membraneINMinner nuclear membraneshRNAsmall hairpin RNATRITCtetramethylrhodamineROIregion of interestDiI1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorateAbantibody. such as exosomes and microvesicles, play diverse roles as shuttles between cells in numerous physiological processes, notably cellular differentiation, inflammation, and immunity among others (1Tkach M. Théry C. Communication by extracellular vesicles: where we are and where we need to go.Cell. 2016; 164 (26967288): 1226-123210.1016/j.cell.2016.01.043Abstract Full Text Full Text PDF PubMed Scopus (2005) Google Scholar). Their functional impact on target cells is related to the delivered components, i.e. proteins, lipids, and nucleic acids, which reflect the status and source of donor cells (2Raposo G. Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends.J. Cell Biol. 2013; 200 (23420871): 373-38310.1083/jcb.201211138Crossref PubMed Scopus (5145) Google Scholar). The heterogeneity of EVs in terms of surface antigens also suggests different cargoes and hence distinct biological roles associated with their action (3Kowal J. Arras G. Colombo M. Jouve M. Morath J.P. Primdal-Bengtson B. Dingli F. Loew D. Tkach M. Théry C. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes.Proc. Natl. Acad. Sci. U.S.A. 2016; 113 (26858453): E968-E97710.1073/pnas.1521230113Crossref PubMed Scopus (1958) Google Scholar). Molecular mechanisms regulating EV biogenesis, their release, and subsequent uptake by target cells have emerged during the last 2 decades (4Kowal J. Tkach M. Théry C. Biogenesis and secretion of exosomes.Curr. Opin. Cell Biol. 2014; 29 (24959705): 116-12510.1016/j.ceb.2014.05.004Crossref PubMed Scopus (1149) Google Scholar). How their cargo molecules are selectively delivered to the intracellular sites of action, including the intranuclear compartment (5Nunukova A. Neradil J. Skoda J. Jaros J. Hampl A. Sterba J. Veselska R. Atypical nuclear localization of CD133 plasma membrane glycoprotein in rhabdomyosarcoma cell lines.Int. J. Mol. Med. 2015; 36 (25977066): 65-7210.3892/ijmm.2015.2210Crossref PubMed Scopus (14) Google Scholar6Waldenström A. Gennebäck N. Hellman U. Ronquist G. Cardiomyocyte microvesicles contain DNA/RNA and convey biological messages to target cells.PLoS ONE. 2012; 7 (22506041): e3465310.1371/journal.pone.0034653Crossref PubMed Scopus (295) Google Scholar, 7Dovrat S. Caspi M. Zilberberg A. Lahav L. Firsow A. Gur H. Rosin-Arbesfeld R. 14-3-3 and β-catenin are secreted on extracellular vesicles to activate the oncogenic Wnt pathway.Mol. Oncol. 2014; 8 (24721736): 894-91110.1016/j.molonc.2014.03.011Crossref PubMed Scopus (36) Google Scholar8Cai J. Han Y. Ren H. Chen C. He D. Zhou L. Eisner G.M. Asico L.D. Jose P.A. Zeng C. Extracellular vesicle-mediated transfer of donor genomic DNA to recipient cells is a novel mechanism for genetic influence between cells.J. Mol. Cell. Biol. 2013; 5 (23580760): 227-23810.1093/jmcb/mjt011Crossref PubMed Scopus (179) Google Scholar), is still obscure (9Kanada M. Bachmann M.H. Hardy J.W. Frimannson D.O. Bronsart L. Wang A. Sylvester M.D. Schmidt T.L. Kaspar R.L. Butte M.J. Matin A.C. Contag C.H. Differential fates of biomolecules delivered to target cells via extracellular vesicles.Proc. Natl. Acad. Sci. U.S.A. 2015; 112 (25713383): E1433-E1442Crossref PubMed Scopus (302) Google Scholar). This issue is particularly important given that the biogenesis and functionality of EVs are dysregulated under pathological conditions (10Desrochers L.M. Antonyak M.A. Cerione R.A. Extracellular vesicles: satellites of information transfer in cancer and stem cell biology.Dev. Cell. 2016; 37 (27219060): 301-30910.1016/j.devcel.2016.04.019Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). EVs have received great attention as noninvasive biomarkers in biological fluids for diagnostic use, given that their amount is significantly increased in various cancerous tissues (11Soung Y.H. Ford S. Zhang V. Chung J. Exosomes in cancer diagnostics.Cancers. 2017; 9: 810.3390/cancers9010008Crossref Scopus (229) Google Scholar). Furthermore, EVs might be of therapeutic relevance as they could act as nanosized drug delivery vehicles in oncology and possibly in regenerative medicine (12Fais S. O'Driscoll L. Borras F.E. Buzas E. Camussi G. Cappello F. Carvalho J. Cordeiro da Silva A. Del Portillo H. El Andaloussi S. Ficko Trček T. Furlan R. Hendrix A. Gursel I. Kralj-Iglic V. et al.Evidence-based clinical use of nanoscale extracellular vesicles in nanomedicine.ACS Nano. 2016; 10 (26978483): 3886-389910.1021/acsnano.5b08015Crossref PubMed Scopus (323) Google Scholar). Altogether, interference with the release of EVs, their uptake by recipient cells, and identification of new intracellular routes and/or mechanisms, including the molecular players that regulate the delivery of the endocytosed EV-derived components in host cells, are potential new targets for innovative cancer treatments (13Sarko D.K. McKinney C.E. Exosomes: origins and therapeutic potential for neurodegenerative disease.Front. Neurosci. 2017; 11 (28289371): 8210.3389/fnins.2017.00082Crossref PubMed Scopus (96) Google Scholar, 14Ying W. Riopel M. Bandyopadhyay G. Dong Y. Birmingham A. Seo J.B. Ofrecio J.M. Wollam J. Hernandez-Carretero A. Fu W. Li P. Olefsky J.M. Adipose tissue macrophage-derived exosomalmiRNAs can modulate in vivo and in vitro insulin sensitivity.Cell. 2017; 171 (28942920): 372-384.e1210.1016/j.cell.2017.08.035Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar). extracellular vesicle nuclear envelope invagination paraformaldehyde oxysterol-binding protein 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol confocal laser-scanning microscopy red fluorescent protein relative fluorescence intensity endoplasmic reticulum diphenylalanine in an acidic tract outer nuclear membrane inner nuclear membrane small hairpin RNA tetramethylrhodamine region of interest 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate antibody. Recently, we described a novel subnuclear compartment that is created by the entry of small GTPase Rab7-containing late endosomes in the nucleoplasmic reticulum (15Rappa G. Santos M.F. Green T.M. Karbanová J. Hassler J. Bai Y. Barsky S.H. Corbeil D. Lorico A. Nuclear transport of cancer extracellular vesicle-derived biomaterials through nuclear envelope invagination-associated late endosomes.Oncotarget. 2017; 8 (28129640): 14443-14461Crossref PubMed Scopus (41) Google Scholar). The latter is shaped by superficial and deep nuclear envelope invaginations (NEI) penetrating into the nucleoplasm. Although the function of nucleoplasmic reticulum remains somehow enigmatic, various membrane-bound organelles and cytoskeletal components were identified therein (16Malhas A. Goulbourne C. Vaux D.J. The nucleoplasmic reticulum: form and function.Trends Cell Biol. 2011; 21 (21514163): 362-37310.1016/j.tcb.2011.03.008Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 17Jorgens D.M. Inman J.L. Wojcik M. Robertson C. Palsdottir H. Tsai W.T. Huang H. Bruni-Cardoso A. López C.S. Bissell M.J. Xu K. Auer M. Deep nuclear invaginations are linked to cytoskeletal filaments- integrated bioimaging of epithelial cells in 3D culture.J. Cell Sci. 2017; 130 (27505896): 177-18910.1242/jcs.190967Crossref PubMed Scopus (54) Google Scholar). Likewise, adeno-associated virus particles were also observed in nuclear invaginations following infection (18Lux K. Goerlitz N. Schlemminger S. Perabo L. Goldnau D. Endell J. Leike K. Kofler D.M. Finke S. Hallek M. Büning H. Green fluorescent protein-tagged adeno-associated virus particles allow the study of cytosolic and nuclear trafficking.J. Virol. 2005; 79 (16140755): 11776-1178710.1128/JVI.79.18.11776-11787.2005Crossref PubMed Scopus (107) Google Scholar). Given that late endosomes in NEI had often an elongated appearance and looked as a sword in its scabbard, we proposed to name this dual-structure “spathasome” from the Greek/Latin words “spathi/spatha” for sword (15Rappa G. Santos M.F. Green T.M. Karbanová J. Hassler J. Bai Y. Barsky S.H. Corbeil D. Lorico A. Nuclear transport of cancer extracellular vesicle-derived biomaterials through nuclear envelope invagination-associated late endosomes.Oncotarget. 2017; 8 (28129640): 14443-14461Crossref PubMed Scopus (41) Google Scholar). This structure appears to act as an intermediate compartment for the delivery of the content of endocytosed EVs (e.g. CD9–CD133 protein complexes) to the nucleoplasm of their host cell (15Rappa G. Santos M.F. Green T.M. Karbanová J. Hassler J. Bai Y. Barsky S.H. Corbeil D. Lorico A. Nuclear transport of cancer extracellular vesicle-derived biomaterials through nuclear envelope invagination-associated late endosomes.Oncotarget. 2017; 8 (28129640): 14443-14461Crossref PubMed Scopus (41) Google Scholar). The NEI-associated late endosomes and nuclear localization of EV-derived proteins were observed in cancer cells and mesenchymal stromal cells in cultures and in breast cancer patient biopsies (15Rappa G. Santos M.F. Green T.M. Karbanová J. Hassler J. Bai Y. Barsky S.H. Corbeil D. Lorico A. Nuclear transport of cancer extracellular vesicle-derived biomaterials through nuclear envelope invagination-associated late endosomes.Oncotarget. 2017; 8 (28129640): 14443-14461Crossref PubMed Scopus (41) Google Scholar). Although the nuclear transfer of components derived from EVs represented solely a minute fraction of total endocytosed EV proteins, they nonetheless modified the gene expression profile (15Rappa G. Santos M.F. Green T.M. Karbanová J. Hassler J. Bai Y. Barsky S.H. Corbeil D. Lorico A. Nuclear transport of cancer extracellular vesicle-derived biomaterials through nuclear envelope invagination-associated late endosomes.Oncotarget. 2017; 8 (28129640): 14443-14461Crossref PubMed Scopus (41) Google Scholar). Little is known about the biogenesis and dynamics of NEI-associated late endosomes. Being composed of two distinct organelles (i.e. nucleus and late endosome), they can be regulated by several mechanisms involving intrinsic and extrinsic factors. For instance, factors influencing the formation and/or stabilization of NEI can control their organization (16Malhas A. Goulbourne C. Vaux D.J. The nucleoplasmic reticulum: form and function.Trends Cell Biol. 2011; 21 (21514163): 362-37310.1016/j.tcb.2011.03.008Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 19Gehrig K. Cornell R.B. Ridgway N.D. Expansion of the nucleoplasmic reticulum requires the coordinated activity of lamins and CTP:phosphocholine cytidylyltransferase α.Mol. Biol. Cell. 2008; 19 (17959832): 237-24710.1091/mbc.e07-02-0179Crossref PubMed Scopus (58) Google Scholar, 20Drozdz M.M. Vaux D.J. Shared mechanisms in physiological and pathological nucleoplasmic reticulum formation.Nucleus. 2017; 8 (27797635): 34-4510.1080/19491034.2016.1252893Crossref PubMed Scopus (19) Google Scholar). In transformed cells that contain a hyper-developed nucleoplasmic reticulum (16Malhas A. Goulbourne C. Vaux D.J. The nucleoplasmic reticulum: form and function.Trends Cell Biol. 2011; 21 (21514163): 362-37310.1016/j.tcb.2011.03.008Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), nuclear delivery of EV components via NEI-associated late endosomes may contribute to cancer progression. Extracellular factors and regulators of endocytosis may also modulate their dynamics. The amount of EVs added to the recipient cells has been shown to influence their frequency, and hence, the nuclear delivery of EV components (15Rappa G. Santos M.F. Green T.M. Karbanová J. Hassler J. Bai Y. Barsky S.H. Corbeil D. Lorico A. Nuclear transport of cancer extracellular vesicle-derived biomaterials through nuclear envelope invagination-associated late endosomes.Oncotarget. 2017; 8 (28129640): 14443-14461Crossref PubMed Scopus (41) Google Scholar). The membrane contact sites of organelles with the endoplasmic reticulum (ER) are recognized in various physiological processes (e.g. lipid transport and membrane traffic) and are conserved in all eukaryotic cells (21Raiborg C. Wenzel E.M. Stenmark H. ER-endosome contact sites: molecular compositions and functions.EMBO J. 2015; 34 (26041457): 1848-185810.15252/embj.201591481Crossref PubMed Scopus (119) Google Scholar, 22Phillips M.J. Voeltz G.K. Structure and function of ER membrane contact sites with other organelles.Nat. Rev. Mol. Cell Biol. 2016; 17 (26627931): 69-8210.1038/nrm.2015.8Crossref PubMed Scopus (550) Google Scholar). The ER network makes close contact with mitochondria, Golgi apparatus, endosomes, and plasma membrane. Tethering complexes that maintain close proximity between the apposing ER and organelle-specific membranes stabilize contact sites. The ER contacts could regulate the maturation of endosomes and their fission and transport along microtubules (23Johansson M. Rocha N. Zwart W. Jordens I. Janssen L. Kuijl C. Olkkonen V.M. Neefjes J. Activation of endosomal dynein motors by stepwise assembly of Rab7-RILP-p150Glued, ORP1L, and the receptor βlll spectrin.J. Cell Biol. 2007; 176 (17283181): 459-47110.1083/jcb.200606077Crossref PubMed Scopus (352) Google Scholar24Rowland A.A. Chitwood P.J. Phillips M.J. Voeltz G.K. ER contact sites define the position and timing of endosome fission.Cell. 2014; 159 (25416943): 1027-104110.1016/j.cell.2014.10.023Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 25Raiborg C. Wenzel E.M. Pedersen N.M. Olsvik H. Schink K.O. Schultz S.W. Vietri M. Nisi V. Bucci C. Brech A. Johansen T. Stenmark H. Repeated ER-endosome contacts promote endosome translocation and neurite outgrowth.Nature. 2015; 520 (25855459): 234-23810.1038/nature14359Crossref PubMed Scopus (269) Google Scholar26Dong R. Saheki Y. Swarup S. Lucast L. Harper J.W. De Camilli P. Endosome-ER contacts control actin nucleation and retromer function through VAP-dependent regulation of PI4P.Cell. 2016; 166 (27419871): 408-42310.1016/j.cell.2016.06.037Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). One major player in these contact zones is the integral ER protein VAP-A (vesicle-associated membrane protein (VAMP)-associated protein-A) (27Lev S. Ben Halevy D. Peretti D. Dahan N. The VAP protein family: from cellular functions to motor neuron disease.Trends Cell Biol. 2008; 18 (18468439): 282-29010.1016/j.tcb.2008.03.006Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). VAP-A interacts in trans with the peripheral late endosomal multidomain oxysterol-binding protein (OSBP)–related protein 1L (ORP1L) via the FFAT (diphenylalanine in an acidic tract) domain, whereas the N-terminal ankyrin repeat region of ORP1L binds to small GTPase Rab7 (28Rocha N. Kuijl C. van der Kant R. Janssen L. Houben D. Janssen H. Zwart W. Neefjes J. Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7-RILP-p150 glued and late endosome positioning.J. Cell Biol. 2009; 185 (19564404): 1209-122510.1083/jcb.200811005Crossref PubMed Scopus (457) Google Scholar, 29Zerial M. McBride H. Rab proteins as membrane organizers.Nat. Rev. Mol. Cell Biol. 2001; 2 (11252952): 107-11710.1038/35052055Crossref PubMed Scopus (2701) Google Scholar). The contact site of the ER-late endosome is regulated by cholesterol level in endosomes, which is sensed by ORP1L or other members of the highly conserved OSBP family (30Suchanek M. Hynynen R. Wohlfahrt G. Lehto M. Johansson M. Saarinen H. Radzikowska A. Thiele C. Olkkonen V.M. The mammalian oxysterol-binding protein-related proteins (ORPs) bind 25-hydroxycholesterol in an evolutionarily conserved pocket.Biochem. J. 2007; 405 (17428193): 473-48010.1042/BJ20070176Crossref PubMed Scopus (119) Google Scholar, 31Wyles J.P. McMaster C.R. Ridgway N.D. Vesicle-associated membrane protein-associated protein-A (VAP-A) interacts with the oxysterol-binding protein to modify export from the endoplasmic reticulum.J. Biol. Chem. 2002; 277 (12023275): 29908-2991810.1074/jbc.M201191200Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Sterol binding leads to conformational changes of ORP1L, which modulate their interaction with VAP-A (28Rocha N. Kuijl C. van der Kant R. Janssen L. Houben D. Janssen H. Zwart W. Neefjes J. Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7-RILP-p150 glued and late endosome positioning.J. Cell Biol. 2009; 185 (19564404): 1209-122510.1083/jcb.200811005Crossref PubMed Scopus (457) Google Scholar). Two other late endosomal proteins, STARD3 (MLN64) and STARD3 N-terminal like, which contain the FFAT motif, are also able to bind VAP-A and sense sterol at ER–endosome contact sites (32Alpy F. Rousseau A. Schwab Y. Legueux F. Stoll I. Wendling C. Spiegelhalter C. Kessler P. Mathelin C. Rio M.C. Levine T.P. Tomasetto C. STARD3 or STARD3NL and VAP form a novel molecular tether between late endosomes and the ER.J. Cell Sci. 2013; 126 (24105263): 5500-551210.1242/jcs.139295Crossref PubMed Scopus (169) Google Scholar). In addition, complexes of VAP-A and OSBP or OSBP-related protein 3 (ORP3) are implicated in the tethers of ER with Golgi apparatus and plasma membrane, respectively (33Mesmin B. Bigay J. Moser von Filseck J. Lacas-Gervais S. Drin G. Antonny B. A four-step cycle driven by PI(4)P hydrolysis directs sterol/PI(4)P exchange by the ER-Golgi tether OSBP.Cell. 2013; 155 (24209621): 830-84310.1016/j.cell.2013.09.056Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, 34Weber-Boyvat M. Kentala H. Lilja J. Vihervaara T. Hanninen R. Zhou Y. Peränen J. Nyman T.A. Ivaska J. Olkkonen V.M. OSBP-related protein 3 (ORP3) coupling with VAMP-associated protein A regulates R-Ras activity.Exp. Cell Res. 2015; 331 (25447204): 278-29110.1016/j.yexcr.2014.10.019Crossref PubMed Scopus (55) Google Scholar). The VAP-A homolog, VAP-B, is involved in the close physical association of ER with mitochondria (35Stoica R. De Vos K.J. Paillusson S. Mueller S. Sancho R.M. Lau K.F. Vizcay-Barrena G. Lin W.L. Xu Y.F. Lewis J. Dickson D.W. Petrucelli L. Mitchell J.C. Shaw C.E. Miller C.C. ER-mitochondria associations are regulated by the VAPB-PTPIP51 interaction and are disrupted by ALS/FTD-associated TDP-43.Nat. Commun. 2014; 5 (24893131): 399610.1038/ncomms4996Crossref PubMed Scopus (356) Google Scholar). Given the membrane continuity between ER and outer nuclear membrane (ONM), we sought whether VAP proteins are implicated in tethering late endosomes within NEI. Here, we report that VAP-A, ORP3, and Rab7 form a tripartite complex essential for the presence of late endosomes in the nucleoplasmic reticulum and the nuclear transfer of EV-derived components. The discovery of the VOR complex (an acronym of VAP-A, ORP3, and Rab7) may lead to therapeutic applications by interfering with the mechanism of intercellular communication. We recently described in various cell types the presence of Rab7-positive (hereafter Rab7+) late endosomes in NEI (15Rappa G. Santos M.F. Green T.M. Karbanová J. Hassler J. Bai Y. Barsky S.H. Corbeil D. Lorico A. Nuclear transport of cancer extracellular vesicle-derived biomaterials through nuclear envelope invagination-associated late endosomes.Oncotarget. 2017; 8 (28129640): 14443-14461Crossref PubMed Scopus (41) Google Scholar). Given that the ONM is a continuation of the ER membrane, we investigated whether the ER-associated protein VAP-A, a protein involved in the tethering of ER to late endosomes distributed in the cytoplasm, was present in NEI. We chose to use human FEMX-I melanoma cells as a primary model since we discovered NEI-associated Rab7+ late endosomes from that (15Rappa G. Santos M.F. Green T.M. Karbanová J. Hassler J. Bai Y. Barsky S.H. Corbeil D. Lorico A. Nuclear transport of cancer extracellular vesicle-derived biomaterials through nuclear envelope invagination-associated late endosomes.Oncotarget. 2017; 8 (28129640): 14443-14461Crossref PubMed Scopus (41) Google Scholar). VAP-A was visualized by expressing a fusion of this protein with green fluorescence protein (VAP-A–GFP), and NEI were observed with an antibody to the inner nuclear membrane (INM) protein SUN domain-containing protein 2 (SUN2). We found VAP-A–GFP in SUN2+ NEI (Fig. 1A), but not all of them, were positive (see below). The presence of late endosomes in VAP-A+ NEI was then investigated. Rab7 as a fusion protein with the red fluorescence protein (Rab7–RFP) was used to highlight late endosomes in VAP-A–GFP-transfected FEMX-I cells. As displayed in Fig. 1B, Rab7–RFP+ late endosomes were found in VAP-A–GFP+ NEI. Their dynamic entry therein was also observed live by time-lapse video microscopy (Fig. 1C). Similar data were obtained by applying anti-Rab7 and VAP-A antibodies instead of fluorescent fusion proteins, and the expression level of VAP-A–GFP was comparable with the endogenous protein (see below). Two types of NEI were reported (16Malhas A. Goulbourne C. Vaux D.J. The nucleoplasmic reticulum: form and function.Trends Cell Biol. 2011; 21 (21514163): 362-37310.1016/j.tcb.2011.03.008Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The first one contains solely the INM that is penetrating into the nucleoplasm, and the second involves both INM and ONM (Fig. S1A). Consequently, only type II NEI can host cytoplasmic organelles, notably late endosomes. To confirm the presence of VAP-A in type II NEI and to determine the proportion of type I versus type II NEI, we expressed a chimeric ER protein (i.e. GFP containing the ER signal sequence of calreticulin with a KDEL retention signal sequence) in FEMX-I cells and immunolabeled them for VAP-A and SUN2. Both types of NEI were detected (Fig. 1D and Fig. S1B and Video S1). Nonetheless, more than 70% of SUN2+ NEI were positive for chimeric ER protein and/or VAP-A, suggesting that the majority of NEI is of type II (Fig. 1E, data not shown). Originating from the nuclear surface, type II NEI can reach deep regions of the nucleoplasm, as observed by confocal laser-scanning microscopy (CLSM) or EM (Fig. 1, C and F). VAP-A–GFP proteins were also detected in deep NEI by immunogold EM (Fig. 1G), whereas Rab7+ late endosomes were observed in both NEI and cytoplasm (Fig. 1H). Of note, the entry of late endosomes in VAP-A+ NEI seems to be selective. Early endosomes or Golgi apparatus as highlighted by the expression of Rab5a and Golgi-resident enzyme N-acetylgalactosaminyltransferase 2 (GALNT2) fused in-frame to RFP, respectively, were excluded from there (Fig. S2, A and B). Altogether, these data indicate that VAP-A is found in NEI that contain late endosomes. To assess the requirement of VAP-A for the presence of late endosomes in NEI, its expression was silenced in melanoma cells using small hairpin (sh) RNAs (for technical details see under “Experimental procedures”). Scrambled shRNA was used as control. The overall expression of VAP-A was reduced by 65% as evaluated by immunoblotting (Fig. 2, A and B) and >95% at the single cell level by immunochemistry (Fig. S3A). No morphological alterations in VAP-A–deficient cells were observed by scanning EM (Fig. S3B). However, silencing VAP-A expression resulted in the absence of late endosomes in NEI (Fig. 2, C and D, and Videos S2 and S3), without affecting their distribution throughout the cytoplasm (see below). VAP-A has a homolog, VAP-B. Although the VAP-A silencing was not compensated by an up-regulation of VAP-B (Fig. 2, A and B), and Rab7+ late endosomes were still absent in VAP-B+ NEI of shVAP-A cells (Fig. S3C), we evaluated whether VAP-B substitutes VAP-A function by silencing its expression with shRNA. The overall VAP-B expression was reduced by 65% as observed by immunoblotting (Fig. 2, E and F), and by up to 95% at the single-cell level as detected by CLSM (Fig. 2G, data not shown). Silencing VAP-B did not affect VAP-A expression (Fig. 2, E and F) and had no effect on the entry of late endosomes in NEI (Fig. 2, G and H), indicating that VAP-A, but not VAP-B, is required for the presence of NEI-associated late endosomes. Given the absence of late endosomes in NEI of cells specifically deficient in VAP-A, we evaluated whether their entry therein was also impaired. Scrambled shRNA and shVAP-A FEMX-I cells expressing the ER–GFP marker and Rab7–RFP were analyzed by time-lapse video microscopy. Although the movement of Rab7–RFP+ late endosomes in ER–GFP+ NEI was observed in control cells (Fig. 2I, top panels), none of them were observed going there upon VAP-A silencing (Fig. 2I, bottom panels). It is noteworthy that the traffic of late endosomes within the cytoplasm also seemed perturbed (Fig. 2I, data not shown). Altogether, these data suggest that the entry and retention of late endosomes in NEI requires VAP-A. Besides the data acquired with FEMX-I cells, a similar set of observations was made with another cell line, i.e. HeLa cells (Fig. S4), indicating that the current phenomenon is not restricted to melanoma cells. To find out how VAP-A regulates the presence of late endosomes in NEI, we investigated whether known interacting partners of VAP-A (i.e. OSBP, ORP1L, STARD3, and ORP3) are expressed in FEMX-I cells and if they localize in NEI. Immunoblots of FEMX-I cell lysates revealed that they are all expressed, and the VAP-A silencing did not influence their expression level (Figs. S2C and S5, A and B). Indirect immunofluorescence analysis demonstrated that OSBP, ORP1L, or STARD3 was not associated with NEI (Fig. S2, C and D), whereas ORP3, a protein implicated in the tether of ER and plasma membrane, was (Fig. 3A). Moreover, ORP3 co-localized with VAP-A (Fig. S5C), and silencing VAP-A reduced the number of SUN2+ NEI containing ORP3, suggesting that VAP-A expression is necessary for the presence of ORP3 in NEI (Fig. 3, A and B). The latter observation prompted us to test whether ORP3 is also required for the localization and/or retention of late endosomes in NEI. Again, its expression was silenced using shRNA. Under these conditions, the expression level of VAP-A was not significantly changed as observed by immunoblotting (Fig. S6, A and B, p = 0.1717 (scrambled shRNA control) or 0.1243 (untransfected control)), and its localization in NEI was maintained as detected by CLSM (data not shown). Upon Rab7–RFP expression, Rab7 fluorescence was found associated with NEI (ORP3+/SUN2+) in scrambled shRNA cells, but not in NEI (ORP3−/SUN2+) of shORP3 cells (Fig. 3C, top and bottom panels, respectively, and Video S4), indicating that ORP3 is required for the presence of late endosomes in NEI (Fig. 3D). The dual co-localizations of VAP-A/Rab7, VAP-A/ORP3, and ORP3/Rab7 in NEI indirectly suggest an interaction among all of them. This issue was substantiated by double-immunolabeling for ORP3 and Rab7 of FEMX-I" @default.
• W2883513762 created "2018-08-03" @default.
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• W2883513762 date "2018-09-01" @default.
• W2883513762 modified "2023-10-18" @default.
• W2883513762 title "VAMP-associated protein-A and oxysterol-binding protein–related protein 3 promote the entry of late endosomes into the nucleoplasmic reticulum" @default.
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• W2883513762 doi "https://doi.org/10.1074/jbc.ra118.003725" @default.
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