Journal of Cell Science rfikn ftefbols rwhfco eetr(Hoepfner receptor factor Rab4-dependent growth as fibroblast such the superfamily of specific trafficking exploit the that of identified been members have pathways Other 2008). 1 Hunt D. Sylvie organization domain by functional sorting maintaining endosomal mediate motors Article Research h rgnlwr spoel ie n l ute itiuin ftewr raatto r ujc to subject are adaptation or work terms. the License of distributions Commons further Creative Attribution all same and Commons that cited the provided Creative properly medium is any the work in of original reproduction the and which terms distribution use, the (http://creativecommons.org/licenses/by-nc-sa/3.0/), non-commercial under unrestricted License permits distributed Alike article Share Access Non-Commercial Open an is This early whereas late (Loube at kinesin-1 Loube and and and involves 2005; to 1993) motility al., traffic al., et in et (Aniento implicated dynein (Brown example, 2011). for many Stephens, kinesin-2 in steps; and implicated these trafficking been Hunt Many have of discrete motors 2006; opposing system. in Schliwa, of Combinations implicated endosomal and (Soldati been the steps have of motors function different and degradation. avoid organization to nutrient systems of these use bacterial recycling various that to by pathogens and co-opted viral relates also and uptake are it pathways the These as receptors. and positioning sensing, and These organelle metabolic factor functions growth signalling, 2012). cellular downregulation essential morphogen Hoflack, and of array interpretation traverse the and diverse including to a which route (Anitei govern exist degradative pathways compartments a pathways via recycling distinct or either Multiple surface pathways cell several . the through to traffic recycling can cargoes destinations. multiple of Endocytic to flux constant trafficking a membrane with driving dynamic membrane highly is network endosomal The Introduction underpins that endosomes of layout mosaic the words: define Key to by help of governed motors are segregation dynamics microtubule the endosome Thus, to specific of motors. addition features of microtubule sorting. in kinesin key these depletion opposing membrane and cargo that of well-characterized SNX-coated dynein with show set of each data used coupled combinations of same our have microscopy together, motility discrete the Taken and to light We subdomains. couple structure endosomal high-resolution segregation. the SNX8 functional Motor using and govern distinct cargo SNX4 combinations dynein. coupling affect SNX1, specific motor motor with These and microtubule many elucidate labelled motors. morphology domains minus-end to to their Endosomal the addition family motors. can dictate to in proteins SNX microtubule motility, These dynein indirectly the endosomes. their or include of of drive function motors directly members and endosomes These couple organization the on and membranes. to endosomal membranes acting central to endosomal are (SNXs) couple shape nexins to actively Sorting members. shown family been kinesin have different motors microtubule Many Summary ( correspondence for *Author 2 o:10.1242/jcs.122317 doi: 2493–2501 126, ß Science Cell of Journal 2013 March 11 Accepted er eloeItgae inligLbrtre,Sho fBohmsr,Uiest fBitl rso S T,UK UK 1TD, 1TD, BS8 BS8 Bristol Bristol Bristol, Walk, of University University Bristol, Biochemistry, of of University School Building, Laboratories, Sciences Signalling Medical Integrated Biochemistry, Wellcome of Henry School Laboratories, Biology Cell 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. irtbl-ae oo rtisaeitga othe to integral are proteins motor Microtubule-based irtbl oo,Edsm,Crosrig Tubulation sorting, Cargo Endosome, motor, Microtubule 1 naK Townley K. Anna , [email protected] ´ 1 ye l,20)are 2008) al., et ry hi .Danson M. Chris , ) ´ ye al., et ry 2 ee .Cullen J. Peter , hnRb n ial a1.Teefntoa nt are units including functional contents These their Rab11. by maintained finally and and generated Rab5, Rab7 with first endosomal coated then of endosomes) series ultimately a traffic (and via domains that sub-domains occurs shown network into has endosomal work organelles the Elegant through single 2001). McBride, of and (Zerial organization in functional LIC2 the than being 2011). more despite al., LIC1 et 2011) (Tan al., implicate function et al., endocytic data (Tynan motors Localization et dynein 2000). of (Tan components exclusive traffic Indeed (DYNC1LI1) mutually functions 2010a). redundant endomembrane LIC1 al., have LICs et two both in (Horgan the that LIC2 for suggested and light have roles others 2010b) 2009b) al., contains shown al., et that have (Horgan et (Palmer recycling others steady-state dynein (DYNC1LI2) while LIC2 endocytic that on 2 chain dependent showed transferrin-positive intermediate was we the early compartment work 2007). the of at al., previous events localization sorting et and In motility (Driskell in dynein endosome. sorting involved the is of cargo that composition the motor and surrounding exist 1995), data 1993; Conflicting al., al., major et et (Aniento localization a movement organelle clearly Oda vesicle in 1997), is roles al., et with DYNC1H1) (Burkhardt function chain endocytic in heavy player the around (built UNC-104/ fungus example filamentous for in membrane, kinesin-3 notably Drosophila systems plasma important, in several be the KIF1A to In to shown 2011). been al., recycling have et members Ueno family 2005; al., et uho h opeiyo h nooa ytmaie from arises system endosomal the of complexity the of Much Dictyostelium Hcaae l,21) n iei- nthe in kinesin-3 and 2011), al., et (Huckaba 2 n ai .Stephens J. David and Ustilago Spiae l,20) iei-3in kinesin-73 2009), al., et (Soppina Shse ta. 01) Dynein 2011b). al., et (Schuster 1, * 2493 Journal of Cell Science eurdfrbt h omto n iso ftblrcarriers. tubular is of coupling fission This and 2009). formation al., the et both Wassmer for 2009; required al., et (Hong dynactin o osteSXdpnettblto fteemembranes motors? microtubule these by force of of tubulation application the SNX-dependent to motor relate the same does Third, endosomes? the how motor of this architecture domain to does affect Second, directly tubulation? couple coupling for as subdomain motility the for endosomal First, complexes questions. study following each the answer to to does to domains order membrane to chose in SNX-coated motors linked different these We each of coupling function. are functional that motor endosomes microtubule-based of organization functional 2012) risk disease. alzheimer’s greater al., onset a late to et of leading (Rosenthal polymorphisms nucleotide toxicity single beta-amyloid some as with identified for been also has modifier SNX8 al., a 2012a). et al., Weering et from (van Weering distinct van complex 2012b; is that endosome-to-TGN SNX1-containing pathway the a of regulates through that 2009) al., SNX8 et retromer- endosome (Dyve transport. of transport in terms in and SNX1 function to organization 2012a) comparator to al., interesting an et it understood tubular Weering making (van generating is traffic of endosome-to-TGN capable It independent also carriers. is of it member endosomal a subfamily As SNX-BAR 2009). al., the transport et retrograde the (Dyve on trafficking effect endosome-to-TGN direct a has suppression al., Its et 2012a). Weering (van complex retromer the with colocalizes partially (Ska clathrin and al., 2003) et amphiphysin- al., with et (Hettema interact (Leprince to 2 pathway reported been recycling also has the the SNX4 Snx42p 2003). on and are in Snx41p defect SNX30 involved Snx4p, a complexes and the to SNX7 of orthologues SNX4, leads mammalian sorting. expression receptor SNX4 transferrin of al., Suppression et (Traer and 2007). complex spatially SNX1-retromer the are to which different elements functionally tubular specific with associated N5SX ope otedni oo ope hog an through complex 2011). Cullen, motor and dynein McGough the p150 2007; with interaction to al., Carlton 2004; et couples al., Wassmer et SNX5/SNX6 (TGN) (Carlton or 2005; SNX6 SNX1 network al., or includes SNX5 et trans-Golgi retromer with mammalian concert the in The SNX2 to 2004). al., back et (Carlton endosomes early from sorting. coupling cargo SNX–motor in 2010), involved al., be et also geometric Weering could in (van proteins and/or SNX sorting of tubulation Associations cargo role help 2011). their might Given Stephens, fission. proteins (van membrane and motor network cargo- and Hunt SNXs microtubule 2010; couple between the al., to et to dynein Weering subdomains with tubule to complexes shown enriched been have multi-protein proteins and SNX form (Hunt the of defined several been 2011), have Stephens, membranes endosomal and motors 2012a). al., et Weering SNX- correlating (van the by tubulation architecture dependent a endosomal produces of of Rab11, view and unified Rab7, localization)more Rab5, maturation and to SNX4, relative SNX1, SNX- by defining localization work the SNX8 This defined 2012a). the al., be et with Weering correlated can (van (which events have system endosomal data tubulation Recent dependent and 2012). 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(SNX) nexin Korswagen, sorting and Rab the of members 2494 hs N1 N4 n N8poietremresfrthe for markers three provide SNX8 and SNX4, SNX1, Thus, and 2009) al., et (Dyve endosome early to localizes SNX8 is it pathway; recycling endosomal the in involved is SNX4 N1i opnn ftertoe ope,mdaigtraffic mediating complex, retromer the of component a is SNX1 molecular between coupling of mechanisms several While ora fCl cec 2 (11) 126 Science Cell of Journal Glued uui ftedni cesr complex accessory dynein the of subunit a , nade l,2009). al., et ˚nland ncnrlcls F-N1lble nooe r moving are endosomes GFP-SNX1-labelled frames), cells, few tracks. a multi-coloured control as within appear In occurs slowly more motility moving those a all while of Immobile (where paths This linear dynamics. colour show time). rapidly single over organelle moving those coding sequence of white, colour appear material each objects this visualization supplementary of of simple (see example frames allows an frame for by 300 7 frame all Movie coded which colour in are projections time nrae h inlt-os ai fordt aiiaigmore facilitating data our of greatly with coverslip ratio the coupled signal-to-noise to close the cells, objects increases these those only of of imaging morphology pigment (TIRF) TIRF flat fluorescence retinal reflection The internal immortalized microscopy. total and telomerase and hTERT- cells) spatial use (human epithelial to better expressed) chose cells obtain we transiently to traffic RPE1 endocytic order of SNX4 In resolution SNX8). temporal transiently expressed, or or SNX4, stably (SNX1, stably and (SNX1 were by cells proteins SNX8 of monitored SNX protein-tagged imaging was fluorescent with coupled expressing fluorescence depletion We with S1). experiments Two shown of Fig. this are sorting. material all Efficacy results (supplementary endosomal siRNA. immunoblotting in the in one clarity LIC2 used from for and outcomes; were LIC1 indistinguishable siRNAs of relating role data independent clear conflicting rationalize the to 2009a). which out al., to set et for also Palmer we 2008; subunits we addition, al., which In et and (Gupta those evident siRNAs is validated motility selected endosomal have in We role a for of 2009b). evidence Palmer 2008; expression al., al., et suppress (Gupta et duplexes characterized previously oligonucleotides to have using we RNAs that subunits protein interfering motor small specific used We Results oipoeoealsailadtmoa eouinthrough resolution temporal ratio. and noise to spatial signal but image many the membrane overall to enhancing plasma lost; TIRF the improve use beneath is to to happening not events intensity was those rationale detected only illumination Our and depth. the excited that the therefore beyond system were of are structures the with 30% endosomes depth fluorescent-tagged the On represents which S3, of nm) SNX-coating. 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Indeed object trafficking endosomes and specific of couple. to cohort imaging they linked the TIRF been by which have defined trafficking to be different motors can that hypothesis endosomes microtubule the early test from to routes SNX8 sought and first SNX4, SNX1, We to coupling motor Microtubule 9 –F 9 hwclu oe aiu intensity maximum coded colour show Journal of Cell Science perwie n oigvsce perwt oordtak.Saebr:5 bars: Scale tracks. coloured with appear vesicles moving and white, appear N4culst yencnann I2adknsn2 and kinesin-2, and LIC2 containing dynein to ( couples whether GFP-SNX8 SNX4 motility to range and according ( motility long endosomes GFP-SNX4 underwent of using they are motility then We the endosomes approach kinesin-1. quantifying GFP-SNX1-labelled by this and that LIC2 repeated containing show dynein-1 together, 2 by Taken driven 2008). and al., et 1 and (Spence Figs fitting eye checked Gaussian was by lengths 2D displacement determined using of were assignment this Distances of immobile. accuracy then were long were that moving objects sets or of data numbers ( the These define distances track). to 2) green a of (Fig. the are quantified panel (see coded colour colour the in single observed tracks the thus rapidly (A (B–F). subunits motor microtubule different for depleted domains. cells endosomal in SNX-labelled and of (A) motility cells driving control motors Microtubule 1. Fig. , . 0 m,o eeimbl.Fg Bsosta GFP- that shows 2B Fig. immobile. were or nm), 500 0 m,sotdsacs( distances short nm), 500 , . 0 mbut nm 500 0 m,sotrange short nm), 500 . 0 nm) 200 irtbl oosdiigedsm yais2495 dynamics endosome driving motors Microtubule m m. oanculst itntchr fmtr htdietheir drive that motors of cohort endosomal distinct each a Thus, motility. to to kinesin-1. couple couples and endosomes domain LIC1 GFP-SNX8-labelled containing that dynein shows 2C Fig. hi egh emlile hs aaeest eiea define to might of there where analysis parameters situations facilitates account into these then taking parameters, which both multiplied 3B) (Fig. We index’’ and ‘‘tubulation cell of per length. tubules tubulation of the numbers their both quantified measuring 3A. by we Fig. domains sequences in these shown image are analyzing depletion of By motor Examples was on motors. endosomes tubulation microtubule SNX-coated endosomal of of depletion structure following the altered that noted also structure We endosomal govern motors Microtubule ( A–F 9 –F rmsdpcigtemvmn fSX-otdedsms in endosomes, SNX1-coated of movement the depicting Frames ) 9 oorcddrpeetto fmvmn:imbl vesicles immobile movement: of representation Colour-coded ) Journal of Cell Science fseii irtbl oo uuisipiigbt motility both to impairing respect subunits depletion as with motor The 1). microtubule coded outcome (Fig. specific colour motility the of of have that and that mirrors we tubules, show exactly of tubulation which data length index These the tubulation shows indicated. the of 3D These proportion Fig. 3E the on. case, Fig. shows so 3C each and Fig. in 3C–E. longer tubules Fig. but in more presented tubules, are data shorted but more be controls indicate GL2 with Asterisks (** comparing study. test present Mann–Whitney from the significance throughout statistical repeated consistent were is treatments This Non-effective twice. s.d. times show three bars that repeated error Note were (independently); kinesin-1. outcomes and defined LIC1 with GFP-SNX8 containing experiments and dynein-1 kinesin-2, to and couple endosomes LIC2 endosomes GFP-SNX4 containing kinesin-1, dynein-1 and to LIC2 couple LIC2 containing endosomes and dynein-1 GFP-SNX1 (LIC1 that to Note 2 couple SNXs. and of 1 motility the chains impaired intermediate respectively) kinesin-2 light depleted, (KIF5B), dynein When kinesin-1 and study. DYNC1H1), (KAP3) was present (DHC1, 2001) the chain al., nm. throughout heavy et 500 cells dynein-1 (Elbashir than control less luciferase transfect range firefly to short the used nm, targeting 500 GL2 are than siRNA movements greater The range range Long a motility. by their characterized to according structures domains. decorated endosome SNX-labelled of motility ( of Quantification 2. 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H. Pelham, and W. M. Black, J., M. Lewis, H., E. Hettema, J. D. Stephens, and A. Hudson, P., Spence, J., K. Palmer, V., Gupta, lahr .M,Hrot,J,Lnekl . acn . ee,K n Tuschl, and K. Weber, A., Yalcin, W., Lendeckel, J., Harborth, K. M., S. Sandvig, Elbashir, and A. Utskarpen, J., Bergan, B., A. Dyve, ogn .P,Hncm .R,Jly .S,Fte,C .adMcCaffrey, and E. C. Futter, S., R. Jolly, R., S. Hanscom, P., C. Horgan, rpPdPim l mgswr rprdfrpbiainwt htso CS Photoshop with publication for prepared were images (Adobe). All Prism. GraphPad rsel .J,Mrnv . la,V .adWomn .G. P. Woodman, and J. V. Allan, A., Mironov, J., O. Driskell, og . ag . hn,C,Nu . i . ho .adLu .J. J. Liu, and X. Zhao, K., Li, Y., Niu, C., Zhang, Y., Yang, Z., Hong, uln .J n osae,H C. H. Korswagen, and J. P. Cullen, ly . asn .G,Bra,K,Rc,S .adGlad .I. V. Gelfand, and E. S. Rice, K., Barlan, G., A. Larson, S., Ally, J. V. Allan, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.122317/-/DC1 online available material Supplementary release. [grant immediate Council for Research PMC Medical in UK Deposited the G0801848]. by number funded was work This Funding paper. designed the wrote D.J.S. analyzed and and and data P.J.C. to analyzed reagents reagents; research, contributed new new the contributed and contributed C.M.D. A.K.T. data data; paper; analyzed the research, writing the performed S.D.H. training. contributions Author with Medical help their the for Mark Leard and Foundation, Alan Facility, and and Bioimaging establishing Wolfson Bristol Jepson for of Bristol the University of the thank University maintaining the to and Council, like Research would We Acknowledgements hn .A n kym,H. Okayama, and A. C. Chen, Arkell, A., Rutherford, M., V. Oorschot, J., B. Peter, V., M. Bujny, G., J. Carlton, B. R. Vallee, and T. Nilsson, J., C. Echeverri, K., J. Burkhardt, J. Gruenberg, and G. Griffiths, N., Emans, F., Aniento, alo,J,Bjy . ee,B . osht .M,Rtefr,A,Mlo,H., Mellor, A., Rutherford, M., V. Oorschot, J., B. Peter, M., Bujny, J., Carlton, Vernos, L., Wordeman, M., L. Ginkel, T., Stauber, C., K. Maier, L., C. Brown, B. Hoflack, and M. Anitei, tnak .adZra,M. Zerial, and H. Stenmark, h otn eisSx/14 eit itntrtivlptwy rmyeast from pathways retrieval distinct mediate Snx4/41/42 nexins endosomes. sorting efficient and the sites exit ER of motility bidirectional transport. for ER-to-Golgi required is (uKHC/KIF5B) 1 cells. mammalian T. 114. transport. endosome-to-Golgi regulates vrxrsinfamnsteGlicomplex. 387-392. Golgi the fragments overexpression W. M. srqie o eetrsrigadtemrhgnsso al endosomes. early of morphogenesis the and sorting receptor Biol. Cell for Nat. required is ermrcmoetSX neat ihdnci 10Gud n mediates and p150(Glued) dynactin with interacts transport. SNX6 endosome-to-TGN component retromer ermrdpnettafcigevents. trafficking retromer-dependent erdto yteedsmlknsnKIF16B. kinesin endosomal the by degradation ihyefcettaseto ytmfrsal rnfrigclswt lsi DNA. plasmid with cells transforming for stably essential for Biotechniques not system is transfection efficient but highly endosome, early the of transport. J. elements endosome-to-TGN P. tubular retromer-mediated Cullen, and with T. associated H. is McMahon, J., Klumperman, S., R. endosomes. late to early from transport vesicular dependent eitstblredsm-oTNtasottruhcicdnesnigo high- J. of P. sensing 3-phosphoinositides. coincidence Cullen, and through membranes and transport curvature T. endosome-to-TGN H. tubular mediates McMahon, J., disrupts Klumperman, complex dynactin the of distribution. subunit organelle 469-484. membrane (p50) of dynamitin maintenance dynein-dependent the of Overexpression Traffic A. T. 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