bridging conduits ( bc ) also termed thick tunneling nanotubes ( tnt ) are cellular processes that sustain long distance intercellular transport of rna , organelles , and receptors . in immunocytes , they enable antigen presentation and pro - inflammatory immune responses.(5 ) supported by both actin filament and microtubules , macrophage bc extend > 100 m in length ( our unpublished observations).(1 ) viruses , prions and bacteria may utilize the conduits to escape immune surveillance and as such accelerate infection . for the human immunodeficiency virus type 1 ( hiv-1 ) , such activities likely occur in mononuclear phagocytes ( mp , monocytes , macrophages , dendritic cells ) and cd4 + t lymphocytes and are targets , reservoirs , and vehicles for microbial dissemination . indeed , direct cell - to - cell hiv-1 dissemination occurs 1000 faster than cell - free viral entry . this can occur through the abilities of hiv-1 to bypass the rate - limiting step of receptor - mediated fusion shielding it from humoral and adoptive immune surveillance.(6 ) the underlying mechanisms of hiv-1 intercellular transfer through conduits remained unknown as the process is complex and seemingly convoluted . first , hiv-1 enters cells through receptor - mediated endocytosis through clathrin - coated pits followed by fusion with endosomal intermediates where virus uncoating and content mixing can occur.(11)second , downstream fusion of clathrin - coated endosomes with a surface - accessible tubular network of the early endosome results in the accumulation of viral particulates in specific intracellular compartments.(12 ) in this manner , tubular endosome networks serve as intermediate station for downstream sorting of hiv-1 from early endosomes into multivesicular bodies ( mvb ) and rab9 late endosomes where virus can remain infectious for extended time periods.third , late endosomes can be further targeted to the trans - golgi network which functions as a sorting  hub  shuttling proteins to the plasma membrane.(15 ) sorting of hiv-1 from endosomes to the er and golgi can then only allow completion of viral life cycle.fourth , during productive infection hiv-1 constituents are shuttled to golgi and er and assemble into mature virions employing er membranes , golgi and post - golgi stacks and vesicles.lastly , prior to hiv-1 intercellular transfer through the bridging conduits ( our unpublished observations ) . in the present work we elucidated endocytic trafficking pathways that effect hiv-1 bc transport . endocytic trafficking through er and golgi networks was found necessary for hiv-1 cell - to - cell transfer . indeed , characterization of the proteome of isolated conduits revealed that these structures are highly enriched with endosome populations involved in hiv-1 endocytic entry and intracellular sorting to the er and golgi . imaging showed a distribution of fluorescently labeled hiv-1 , hiv-1 envelope ( env , gp120 , gp160 ) and gag ( pr55 , matrix , p24 ) with these endocytic compartments and er - golgi . disruption of endocytic transport from endosomes ( early and late ) to the er - golgi impaired hiv-1 intercellular transfer . these observations , taken together , provide new insights into intracellular viral processing routes that drive hiv-1 spread through bc serving as a potential mechanism for evasion of host immune surveillance . rabbit ab giantin , tgn38 , rab9 , and mouse ab to calnexin were purchased from cell signaling technologies . human and mouse abs to hiv-1 components including those to gag ( gag , p24 , p17 , matrix ) , gp120 , gp160 were obtained through the nih aids research and reference reagent program , division of aids , niaid , nih ( bethesda , md ) . chicken antirabbit , goat antimouse alexa 488 , 594 , 647 , donkey antisheep alexa 488 , cholera toxin subunit b ( ctx - b ) alexa 594-conjugated , click it metabolic labeling reagents for proteins imaging kit , -galactosidase ( -gal ) staining kit , 1,1dioctadecyl-3 , 3,3 , 3-tetramethylindodicarbocyanine perchlorate ( did ) , 3,3dioctadecyloxacarbocyanine perchlorate ( dio ) , er - tracker green ( bodipy fl glibenclamide ) , bodipy fl c5-ceramide ( golgi - tracker ) , prolong gold antifading solution with dapi all purchased from molecular probes . brefeldin a ( bfa ) was purchased from sigma - aldrich . experiments with human pbmc were performed in full compliance with the ethical guidelines of the national institutes of health and the university of nebraska medical center . monocytes from hiv-1 , 2 and hepatitis seronegative human donors were obtained by leukophoresis , and purified by countercurrent centrifugal elutriation.(21 ) human monocytes in suspension ( teflon flasks ) were cultured in dmem ( invitrogen ) supplemented with 10% heat - inactivated human serum , 1% glutamine , 10 g / ml ciprofloxacin ( sigma - aldrich ) , and 1000 u / ml highly purified recombinant macrophage colony stimulating factor ( mcsf ) , a generous gift by pfizer inc . , ( cambridge , ma ) . cells were allowed to differentiate to macrophages for 7 d at 37 c in a 5% co2 . at day 7 , mdm were exposed to the macrophage - tropic strain hiv-1ada at a multiplicity of infection ( moi ) of 0.5 infectious viral particles / cell for 24 h. prior to seeding in mixed cultures ( infected and uninfected mdm ) , hiv-1 infection was assessed by hiv-1 p24 immunostaining and flow cytometry . all confocal imaging was performed in monocyte - derived macrophages ( mdm ) mixed ( 1:1 uninfected and infected ratio ) cultures unless stated otherwise . mdm for imaging were grown in poly d - lysine - coated labtech ( bd biosciences ) chamber slides . labeling of er and golgi in living cells was performed using er - tracker green ( bodipy fl glibenclamide ) and bodipy fl c5-ceramide ( golgi - tracker ) per manufacturer s instructions . for retrograde tracing from early endosomes to goli - er using ctx - b , cells were incubated with 100 mm ctx - b in serum - free medium for 1 h. for disruption of endocytic sorting , mdm were treated with 0.5 mm bfa for 3 h. cells from all applications we washed three times with warm pbs and fixed with 4% paraformaldehyde ( pfa ) ph 7.4 for 30 min at room temperature . cells were treated with blocking / permeabilizing solution ( 0.1% triton , 5% bsa in pbs ) and quenched with 50 mm nh4cl for 15 min . cells were washed once with 0.1% triton in pbs and sequentially incubated with primary and secondary abs at room temperature . slides were covered in prolong gold antifading reagent with dapi and imaged using a 63 oil lens in a lsm 510 confocal microscope ( zeiss ) . cells were first maintained in teflon flasks and media changed every 2 d. cells were centrifuged at 300 g for 5 min and resuspended ( 2  10 cells / ml ) with minimal pipeting in growth media then seeded on a 9 mm thick polycarbonate filter ( neuroprobe ) containing 3 m in diameter pores separating the upper and the lower wells of a one - well boyden separation chamber ( neuroprobe ) . bc were collected by exposing them to the uninfected mdm - conditioned medium ( lower well ) . cell suspensions were added to the upper well and the chamber was placed in an enclosed humidifier box and incubated at 37 c and 5% co2 tissue culture incubator for 4 h. conduits were harvested in each filter which was immersed in 100% methanol for 15 s then placed firmly with migrated cell materials down ( attached to the undersurface of filters ) on a glass slide . cells were wiped from the filter top with kimwipes and the filter was then gently peeled off the slide with forceps . processes were solubilized in lysis buffer ph 8.5 [ 30 mm triscl , 7 m urea , 2 m thiourea , 4% ( w / v ) 3-(3-cholamidopropyl ) dimethylammonio)-1-propanesulfonic acid ( chaps ) , 20 mm dithiothreitol ( dtt ) and 1 protease inhibitor cocktail ( sigma ) ] by pipeting . the remainder of the lysate was allowed to crystallize on slides and harvested using razor blades . infected mdm to be examined by sem were seeded on a polycarbonate filter membrane of a boyden chemotaxis chamber for 4 h to allow for migration of their processes through the pores . filter membrane was removed washed in pbs and fixed in 2% glutaraldehyde and 2% pfa in 0.1 m sorensen s phosphate buffer . samples were critical point dried , mounted on specimen stubs , sputter- coated with 40 nm of gold / palladium and observed with a fei quanta 200 sem ( fei company ) operated at 25 kv . following bc isolation , proteins were solubilized in lysis buffer ph 8.5 [ 30 mm triscl ] , 7 m urea , 2 m thiourea , 4% weight / volume ( w / v ) chaps and 1 protease inhibitor cocktail ( sigma ) ] . proteins were precipitated using a 2d clean up kit and quantified by 2d quant ( ge healthcare ) per the manufacturer s instructions . samples underwent isoelectric focusing and second dimension electrophoresis as described.(22 ) proteins were fixed with 10% methanol , 7% acetic acid for 24 h and stained with syproruby at room temperature for 24 h. spots were automatically excised using an ettan spot picker ( ge healthcare ) followed by in - gel tryptic digestion ( 10 ng / spot of trypsin ( promega ) for 16 h at 37 c . peptide extraction and purification c18 ziptip ( millipore ) were performed on the proprep protein digestion and mass spec preparation systems ( genomic solutions ) . extracted peptides were fractionated on microcapillary rp - c18 column ( newobjectives ) and sequenced using esi ms / ms system ( proteomex system with ltq - orbitrap mass spectrometer , thermo scientific ) in a nanospray configuration . the acquired spectra were searched against the ncbi.fasta protein database narrowed to a subset of human proteins using sequest search engine ( bioworks 3.1sr software from thermo scientific ) . the turbosequest search parameters were set as follows : threshold dta generation at 10000 , precursor mass tolerance for the dta generation at 1.4 , dta search , peptide tolerance at 1.5 and fragment ions tolerance at 1.00 . proteins with two or more unique peptide sequences ( p < 0.05 ) were considered highly confident . functional connectivity maps were generated using string 8.3 software at http://string-db.org ( table 1 ) . metabolic labeling and detection of newly synthesized proteins in mdm were performed using click - it aha labeling kits per manufacturer s instructions ( invitrogen ) . metabolic labeling with aha was extended to 24 h. string database accession number ( accessible at http://string-db.org/ ) . accession numbers for uniprot ( accessible at http://www.uniprot.org/ ) . postulated subcellular localization ( accessible at http://locate.imb.uq.edu.au and http://www.uniprot.org/ ) as follows : plasma membrane ( pm ) ; secreted ( s ) ; endoplasmatic reticulum ( er ) ; ribosomes ( r ) ; cytoskeleton ( csk ) ; cytosol ( c ) ; mitochondria ( m ) ; endocytic vesicle ( ev ) ; lysosomes ( l ) ; golgi ( g ) ; hiv-1 ; not available ( n / a , in this group are included proteins with no postulated localization ) for immunoisolation of rab9 endosomes uninfected and infected mdm were seeded in mixed cultures and allowed to polarize ( > 90% of cell population connected by bc ) . cells were then washed three times with 1x pbs , scraped in tg buffer ( 10 mm hepes - koh , ph 7.2 , 250 mm sucrose , 1 mm edta , and 1 mm mg(oac)2 and disrupted by 15 strokes in a dunce homogenizer . nuclei and unbroken cells were removed by centrifugation at 800 g for 15 min at 4 c . supernatant was layered on a 20% sucrose cushion and centrifuged at 100  000 g for 1 h. the pellet was resuspended in pbs and incubated with protein a / g paramagnetic beads ( 20 l of slurry ; millipore ) conjugated to rab9 and isotype control abs ( 10 g of ab ) for 12 h at 4 c . endocytic compartments bound to beads were collected following three washes with 1 pbs on a magnetic separator ( invitrogen ) . the tzm - bl cells were cultured in high glucose dulbecco s modified medium ( dmem ) supplemented with 10% fetal bovine serum ( fbs ) , 100 units of penicillin and 100 g / ml of streptomycin . cells were allowed to reach 70% confluence then detached by 25 mm trypsin / edta for 5 min at 37 c . cells were then seeded onto 4 well poly - d - lysine labtech chamber slides for 2 d ( 40% confluence ) . protein a / g paramagnetic beads - bound to isotype control and rab9 abs were reconstituted in supplemented dmem ( 20 l of slurry/500 l of medium / well ) and added to tzm - bl . chamber slides were placed on a plate magnet for 24 h at 37 c and 5% co2 . untreated , hiv-1-exposed and isotype ab - exposed tzm - bl were used as controls . cells were maintained in culture medium for additional 48 h prior to -gal detection using -gal staining kit ( per manufacturer s instructions ) . quantification of immunostaining was performed with imagej software , utilizing jacop plugins ( http://rsb.info.nih.gov/ij/plugins/track/jacop.html ) to calculate pearson s colocalization coefficients . endosome size ( diameter ) was measured manually using lsm - browser software ( zeiss ) . scatter plots and bar graphs were generated using prism ( graphpad software inc . ) . one - tailed student s t - tests were used for all data and the error bars are shown as standard error of the mean ( s.e.m . ) unless noted otherwise . to assess whether hiv-1 infection affects bc formation , bc connected cells were counted at 3 h in adherent single ( uninfected or infected alone ) or mixed cultures ( uninfected and infected mdm at 1:1 ratio ) . bc frequency was increased in infected and mixed cultures compared to uninfected mdm in single culture ( figure 1a ) . infected mdm in single and mixed cultures acquired elongated morphologies and formed bc that extended > 100 m ( figure 1b ) . single culture uninfected mdm showed round ruffled appearances . however when these cells were mixed with virus - infected mdm at 1:1 ratio uninfected cells ( a ) mdm connected by bc in single ( uninfected or infected ) or mixed ( uninfected and infected at equal ratios ) cultures at 180 min in adherence ( * p < 0.05 ; * * p < 0.001 ; n = 250 cells / group , error bars + s.e.m ) . arrows indicate three long ( > 100 m ) tubular bc emanating from a single mdm toward neighboring cells ( scale bar , 20 m ) . ( c ) uninfected mdm labeled with dio lipophilic dye ( green ) in single culture . note lack of cell polarity ( bc ) and direct cell - to - cell contact . ( d , e ) confocal images ( including differential interference contrast , dic images ) of uninfected ( dio - labeled , green ) and infected ( did - labeled , red ) mdm in mixed culture . note both uninfected and infected mdm are capable of generating bc in mixed culture ( scale bar , 10 m ) . hiv-1-infected mdm were seeded on a filter membrane separating a boyden chemotaxis apparatus into an upper and a lower chamber . cells were allowed to polarize by extending their processes through the 3-m pores toward the conditioned medium from uninfected mdm ( lower chamber , figure 2a ) . sem was performed to monitor cell polarization and migration of cellular protrusions through the pores ( figure 2b ) . such bc extensions were detached from cell bodies ( figure 2c ) and collected from cells derived from five independent experiments . these are shown in representative images of 420% gradient 2d gels ( figure 2d ) . an average of 1100 spots were detected using decyder software and 275 proteins identified by mass spectrometry at high confidence ( p  0.05 and 2 or more unique peptides ; table 1 and s1 , supporting information ) . ingenuity pathway analysis ( ipa ) separated the bc proteome into 3 major functional groups . these included cellular morphology and migration ( actin cytoskeleton , cell adhesion and motion , leukocyte extravasation , tight junction and calcium signaling ; antigen and hiv-1 endocytic processing ( caveole- , clathrin- , and fc - dependent endocytosis , mechanisms of hiv-1 endocytic entry and exit , micropinocytosis ) ; metabolomic factors ( carbohydrate , protein and fatty acid synthesis ) . functional connectivity analysis using protein interaction networks ( http://www.string-db.org ) revealed high levels of interaction among these clusters as shown by bold lines in the connectivity map ( figure s1a , supporting information).(23 ) interestingly , removal of highly abundant cytosolic and nuclear proteins residing in the cell body , uncovered low abundance proteins that were not identified in the whole cell and membrane preparations.(22 ) moreover , proteomic analysis comparisons between uninfected and uninfected mdm bc showed no major differences in protein i d ( data not shown ) . seventy - nine proteins ( 30% of the overall proteome were organelles ) were 3% were hiv-1 components . the organelle markers included endolysosomal compartments ( 14% ) , er ( 9% ) and golgi ( 4% ; figure 2e ) . the excised spots corresponding to these proteins are shown in figure 4a and table 1 . further functional subclassification and connectivity analysis of endocytic , er and golgi proteins showed that major protein clusters interact as regulators of post - translational modifications such as n - glycosylation , protein folding , viral / antigen endocytic uptake and release and cell - to - cell communication ( figure 4b  d ) . the data show that the er and golgi are in the conduits and support their roles in viral / antigen processing . ( b ) sem image of a human macrophage cultured on the upper chamber of a boyden chemotaxis apparatus , extending its processes through the porous membrane . ( c ) confocal image of bc adhered to glass slide postdetachment from cell bodies , immunostained for microtubules with / tubulin - alexa 488 ab ( fluorescent green , pseudocolored white ) . ( d ) representative image of 2d electrophoresis of bc lysate pooled from five independent cultures ( donors ) . ( refer to table 1 and s1 , supporting information , for protein i d ) . major biological functions relevant to viral endocytic processing ( including post - translational modification ) and cytoskeletal rearrangements in bc . ( a ) classification by function of bc proteome based on higher statistical significance of protein groups [ log ( p value ) ; threshold set at 1.3 ] . identification of er , golgi and endosome proteins and their functional relevance in viral traffic through bc . ( a ) representative 2d electrophoresis of bc lysate . isolated bc from five separate cultures and 500 g of pooled precipitated protein was applied to a ph gradient ( 310 ) strip and separated on the first dimension by isoelectric focusing . the strip was loaded into a large format gradient gel ( 420% ) for second dimension separation of bc proteins . gel was fixed and poststained with deep purple dye for positive detection of proteins spots . encircled protein spots correspond to er ( red ) , golgi ( green ) , endosome ( black ) and hiv-1 ( green ) subproteome identified by nano - lc ( b , c ) ipa classification by biological and canonical function of er , golgi , endosome proteins with significant relevance [ log ( p value ) ] to hiv-1 bc trafficking . for classification by biological function only proteins above the set threshold ( 1.3 ) were grouped for analysis . ( d ) connectivity map of the bc subproteome displaying functional interaction between er , golgi , endosome and hiv-1 proteins . protein ids corresponding to abbreviations used in the connectivity map are included in table 1 and s1 , supporting information . mdm were immunostained for giantin ( golgi vesicle and membrane integral protein ) and calnexin ( er membrane protein ) . in nonpolarized cells er and golgi were located in the perinulcear region and displayed known ruffled and stacked morphologies ( figure 5a , arrows ) . in contrast , the golgi network acquired tubular morphologies ( figure 5a , b ; inset boxes 1 and 2 ) . interestingly , a single golgi was observed between two connected cells ( figure 5b , yellow arrows ) . confocal imaging showed gag and env compartments in bc that colocalized with giantin and calnexin ( figure 5a , b ) . env and gag were also observed in 1.6 m calnexin and 1.2 m ( in diameter ) giantin endocytic vesicles in conduits ( figure 5c ) . quantitation of fluorescence overlap using pearson s colocalization tests indicated 72 and 32% of env colocalized with er and golgi respectively ( figure 5d ) . gag distribution with calnexin and giantin was measured at 50 and 22% respectively ( figure 5e ) . distinct morphologies of er and golgi in bc - connected cells and their presence within the conduits . ( a and b ) confocal images of mdm in mixed culture connected by bc . immunostaining for giantin ( golgi , green ) and calnexin ( er , purple ) demonstrates organelle tubular morphologies and their codistribution with hiv-1 env ( red ) and gag ( red ) within the conduits ( inset box 1 , 2 ) . arrows in panel b indicate a single tubular golgi equidistant from two connected cells ( scale bars , 10 m ; inset boxes , 5 m ) . ( c ) size distribution of gag and env endocytic vesicles carrying er ( n = 35 vesicles ) and golgi ( n = 28 vesicles ) markers in the bc ( error bars  s.e.m ) . pearson s colocalization coefficients indicate percent overlap of env and gag with calnexin and giantin ( error bars  s.e.m ; n = 80 cells ) . having observed that hiv-1 env and gag are within the bc and are associated with the er and golgi , we next investigated whether these were newly synthesized viral proteins or shuttled to these organelles . to explore associations between newly synthesized viral proteins and the er and golgi , mdm were metabolically labeled with azidohomoalanine ( aha ; a methionine substitute ) in methionine - depleted medium . maximum overlap of viral and metabolically labeled proteins was observed at the plasma membrane ( likely reflecting processes of progeny assembly ) but not the conduits ( figure 6a , b ; inset boxes 1 and 2 ) . furthermore , minimal distribution of metabolically labeled protein with golgi was observed suggesting env and gag distributing with these organelles may be of endocytic origin ( figure 6c ) . live tracking of fluorescently labeled hiv-1 ( dio ) with er / golgi ( er- and golgi - tracker dyes ) revealed accumulation of hiv-1 in er and golgi tubules extending into the conduits ( figure 6d , arrows ) . mdm in mixed culture were labeled by metabolic incorporation of 50 m of azidohomoalanine ( aha ) into newly synthesized proteins for 24 h using methionine - free medium . ( a , b ) confocal imaging of metabolically labeled mdm immunostained for hiv-1 env and gag show overlap of these viral constituents with newly synthesized proteins at the plasma membrane but not bc . ( c ) co - immunostaining with giantin shows minimal distribution of aha - labeled proteins with the golgi tubule . ( d ) sequestration of endocytosed fluorescently labeled hiv-1 ( did dye , red ) into golgi and er ( golgi- and er - tracker dyes , green ) . mdm in mixed culture were first labeled with a combination of bodipy fl glibenclamide ( er - tracker ) and bodipy fl c5-ceramide ( golgi - tracker ) for 1 h then exposed hiv-1-did for 1 h. excess dye and noninternalized hiv-1 were removed by washing and cells were observed by time - lapse confocal imaging . arrows indicate distribution of endocytosed hiv-1 with er - golgi tubules in polarized mdm ( scale bars , 10 m ; inset boxes , 5 m ) . next , we investigated the pathways for hiv-1 env and gag trafficking to the golgi network . following endocytosis at the plasma membrane proteins destined for golgi can undergo retrograde sorting routes from early / recycling endosomes and late endosomes to the golgi network.(24 ) among the organelles of the secretory / recycling pathway trans - golgi has a central role as a site of protein sorting.(24 ) once proteins reach trans - golgi they face several sorting destinations : the extracellular space , different domains of the plasma membrane , and rab11 recycling endosomes.(24 ) therefore we questioned whether hiv-1 env and gag can undergo retrograde transport from early / recycling endosomes to the golgi network . co - distirbution of env and gag with fluorescently labeled cholera toxin subunit b ( ctx - b ) was tested , since ctx - b has been previously documented to undergo retrograde sorting from early / recycling - endosomes to the golgi.(25 ) mdm in mixed culture were exposed to the ctx - b - alexa 594 and then stained for env and gag . env / b - ctx and gag / b - ctx compartments were observed in bc ( figure 7a , b ) . env and gag colocalizations with b - ctx were measured at 39 and 41% , respectively ( figure 7c ) . these data suggested that a portion of the viral protein pool undergoes parallel sorting to ctx - b . hiv-1 env and gag undergo retrograde transport from early and late endosomes to the er - golgi . ( a - c ) env and gag endocytic sorting parallels that of cholera toxin subunit b ( ctx - b ) . mdm in mixed culture were exposed to ctx - b - alexa 594 conjugated ( red ) . cells were fixed and immunostained for env ( green ) and gag ( green ) and imaged by confocal microscopy . inset boxes show at a higher magnification bc - associated endocytic compartments transporting ctx - b , env and gag . pearson s colocalization coefficients are indicated as percent overlap of ctx - b with env and gag . ( d , e , g ) distribution of env and gag with rab9 late endosomes and the trans - golgi along the bc . mdm in mixed culture were immunostained for env ( red ) , gag ( red ) , tgn38 ( trans - golgi marker , green ) and rab9 ( late endosomes , purple ) . distribution by size ( diameter ) of rab9 and tgn38 compartments positive for env and gag ( n = 65 endocytic compartments / group , error bars  s.e.m ) . confocal images of mdm showing colocalization of env and gag with rab9 and tgn38 compartments in the bc ( inset boxes 1 ; scale bar , 10 m ) . ( f , h ) quantitation of colocalization ( fluorescence overlap ) of env and gag with rab9 and tgn38 compartments in the bc ( n = 80 cells / group , error bars  s.e.m ) . in addition to retrograde transport from early endosome to golgi , a crucial step in the hiv-1 replication cycle is rab9-mediated retrograde sorting to the trans - golgi . gene silencing of rab9 and other host factors ( tip47 , p40 ) that regulate late endosome to trans - golgi transport blocks hiv-1 replication.(16 ) since fluorescently labeled hiv-1 was detected in the er and golgi tubules , we tested the virulence of hiv-1 sequestered into rab9 endosomes and its distribution with the trans - golgi . confocal imaging showed env and gag distribution with in rab9 vesicles and tgn38 ( trans - golgi membrane protein ) in the conduits ( figure 7d , f ) . colocalization assays showed a 19 and 30% overlap of env with rab9 and tgn38 compartments . eighteen and 24% of gag overlapped with rab9 and tgn38 ( figure 7e , g ) . to test if hiv-1 present in rab9 endosomes were infectious , bc compartments were isolated by immunoaffinity chromatography . protein a / g paramagnetic microbeads conjugated to rab9 or isotype abs ( used as control of binding specificity ) were incubated with homogenized cell lysate clarified from cellular debris ( plasma membrane and nuclei ) and collected by magnetic separation . endocytic compartments bound to paramagnetic beads were then introduced to the tzm - bl cells using a magnet plate . the tzm - bl reporter cell line expressed ss - gal under the control of the hiv-1 promoter . at 72 h postexposure ss - gal ( blue ) bright field images in figure 8a show ss - gal expression ( blue ) in tzm - bl exposed to free hiv-1 ( positive control ) and rab9 endosomes ( arrows ) . quantitation of blue cells revealed that the contents of rab9 endosomes were infectious ( figure 8b ) . together these data indicate that hiv-1 destined for intercellular transfer undergoes retrograde sorting from early and late endosomes to the trans - golgi . rab9 endosomes carry infectious particles and disruption of er - golgi impairs hiv-1 transfer through the conduits . ( a ) rab9 endosomes were isolated by magnetic separation from mechanically ruptured polarized mdm using protein a / g paramagnetic beads conjugated to rab9 ab . bead - captured endosomes were washed and were forced into tzm - bl reporter cell line by subjecting the cells to magnetic fields . hiv-1 genome integration and expression in tzm - bl was measured at 72 h postexposure in the form of ss - gal production ( blue ) . representative bright field images of uninfected tzm - bl ( negative control ) , cells exposed to beads conjugated to isotype ab ( control for specificity of binding ) , cell - free hiv-1 ( positive control ) and rab9 immune - isolated endosomes at 72 h postexposure . ( b ) quantiation of overall endosome infectivity in tzm - bl ( scale bars 100 m ; error bars ,  s.e.m . , ( c , d ) confocal images of mdm connected by bc in mixed culture treated with fungal metabolite bfa and immunostained for hiv-1 env and gag . arrows indicate accumulation of viral proteins in large vacuoles in the cell body ( scale bar , 10 m ; inset boxes , 5 m ) . next we questioned whether disruption of recycling and the er - golgi network would affect transfer of hiv-1 through the conduits . incubation of cells with fungal metabolite bfa was found to inhibit er - to - golgi transport , resulting in the rapid redistribution of golgi markers into the er , collapsed trans - golgi and impair of rab11-mediated recycling.(26 ) exposure of polarized mdm to 0.5 mm of bfa resulted in the accumulation of env and gag in large tgn38 compartments ( figure 8c , d ; arrows ) . these data suggest that integrity of the er and golgi - trans - golgi networks is necessary for viral protein trafficking through the bc . employing functional proteomics and imaging assays we characterized the macrophage conduits and elucidated endocytic pathways that effect hiv-1 intercellular trafficking through bc . together with previous studies we show that following clathrin - dependent endocytosis , hiv-1 is sequestered into early and late endosomes and  hitchhikes  retrograde sorting pathways to reach the er and golgi . virus uses these organelles as a sorting station for hiv-1 prior to intercellular transfer . in parallel , hiv-1 induces cytoskeletal alterations enhancing the formation of a bc network.(32 ) the mechanisms of bc formation is incompletely understood . in t cells and mdm these protrusions are established as adjacent cells pull away from each - other following brief contact or by generation of the protrusion from one cell to another . since an increase in bc was observed in mdm mixed cultures . this may be due to an increase in the metabolic and secretory activities associated with viral infection.(9 ) the er and golgi networks commonly located in the perinuclear region of cells extend within the conduits in polarized mdm . what causes these to acquire such unique morphologies may be due to rearrangements of cytoskeletal and endocytic networks required for the formation and function of bridging conduits . it is likely that during conduit formation microtubule bundling may also lead to rearrangement from golgi and er stacks to tubules . alternatively , golgi and er may traffic between cells through conduits similar to mitochondria and lysosomes and their distribution within the bc may accelerate communication between connected cells . indeed , er and golgi cellular distribution and morphologies have been linked to the dynamics of microtubule rearrangements . these organelles attach to the growing plus end of the microtubules and travel on these tracks to divergent cellular locations . we posit that in addition to sustaining organelle travel between cells , conduits may accelerate intercellular exchange by acting as sites of mrna translation and protein synthesis . this may be similar to processes observed in the axon where synthesis and incorporation of neurotransmitters into presynaptic vesicles occurs in proximity of the synapse.(30 ) this kind of golgi / er rearrangements may accelerate hiv-1 intercellular spread as viral genome can be expressed in proximity to the uninfected target cell , rather than expressed in the cell body and then transported to the uninfected cell . elongation and translation factors , transporters of mrna , and cellular and viral rna were identified in the bc ( our unpublished observations).(31 ) furthermore , presence of enzymes regulating protein , carbohydrate , lipid and metabolism further support the role of bc as metabolically active structures . we have previously demonstrated that formation of the conduits precedes syncytium formation in hiv-1 infected mdm.(32 ) therefore , er and golgi extending within the conduits are likely to be shared by the connected cells functioning as a unicellular entity . indeed , in the current study we often observed single golgi tubules located halfway within the conduit between two cells . in multinucleated giant cells central accumulation of several golgi tubules surrounded by more than 50 nuclei and what drives transport of hiv-1 to the golgi network and er and more importantly how does this affect targeting of the viral constituents to their final destination ( i.e. , bc intercellular transfer ) remain open questions . the need for post - translational processing may target viral constituents to er and golgi . these in turn can also be used as address codes sending viral proteins to specific subcellular locations or bc for intercellular transfer . proper folding and glycosylation of envelope proteins is an important evolutionary process that impacts viral infectivity and selection.(33 ) indeed , mutations in the glycan shield of hiv-1 may account in part for viral immune evasion persistence to neutralizing antibodies.(34 ) hiv-1 gp120 undergoes heavy o- and n - glycosylation and mannose trimming in the er - golgi network . o - linked carbohydrates are attached to the hydroxyl group of serines and threonines residues of hiv-1 env after transport to golgi.(35 ) gag may also be targeted to the er following monoubiquitination , as a quality control measure.(36 ) ubiquitination seems to be responsible for viral budding within endocytic compartments such as mvb as well as gag maturation and infectivity.(37 ) regulators of the ubiquitin - proteosome pathway were readily identified in the bc proteome and it is likely that such processes may also drive intercellular transfer of active viral constituents . sorting of viral constituents through er and golgi may also be a survival mechanism hiv-1 adopts during infection - induced apoptosis or necrosis . at this time the virus survives by utilizing any available cellular membranes ( plasma and organelle ) for its assembly . indeed , previous ultrastructural studies have shown hiv-1 progeny assembling on golgi and er membranes at the late stages of macrophage infection . interestingly , recent studies have shown that immune cells such as natural killers ( nk ) use bridging conduits to transfer apoptosomes and other packed cellular fragments from a dying to healthy cells.(38 ) we have also often observed establishment of contact between multinucleated giant cells and single polarized macrophages at the late stages of infection ( our unpublished observations ) . as such , it is likely that presence of viral proteins in er and golgi compartments along the conduits may reflect the ability of hiv-1 to survive death of the host and use the bc - mediated debris clearing activity to accelerate infection . at the early stages of infection associated with minimal cell death sorting of hiv-1 constituents through er and golgi can also be explained by bc - mediated antigen presentation . it has been recently documented that antigen - presenting cells use bc in vivo to accelerate antigen presentation . we have previously detected major histocompatibility complex class ii ( mhc ii ) hla - dr in rab11 endosomes and mvb transporting hiv-1 constituents through the macrophage conduits ( our unpublished observations ) . hla - dr endocytic processing appears similar to hiv-1 as both undergo internalization and sorting through early endosomes to recycling endosomes and subsequently er , cis- and trans - golgi ( our unpublished observations).(39 ) therefore it is likely that a portion of the viral constituents parallels hla - dr intra- and intercellular traffic . this would explain the dual role of bfa in disrupting hiv-1 transport through the conduits and antigen presentation.(39 ) using proteomic and imaging technologies new insights into how hiv-1 uses basic immune cell functions to its advantage was explored . the virus  hitchhikes  from cell - to - cell using bc cytoskeletal and endocytic machineries to accelerate viral spread . such mechanisms are likely relevant for preventative vaccination strategies as they proves a means for the virus to evade immune surveillance .