SUPPLEMENTARY INFORMATION

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Adele Simmons
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1 DOI:.38/n3263 Kinesin-II MKS-6 KAP-1::eGFP MKS-6::mCherry Kinesin-II OSM-3 Normlize intensity.6.2 B KAP-1 MKS-6 () Distne from se (µm) Avg. ilium intensity (.u.) Distne from se (µm) Distne from se (µm) Supplementry Figure 1 IFT-prtile suomplexes n IFT-motors show istint loliztion ptterns long ili (relte to Fig. 1). () Representtive two-olor time-verge fluoresene imge of kinesin-ii (green) n trnsition zone mrker MKS-6 (mgent) (top), n orresponing fluoresene-intensity profile (ottom) long the lower ilium (sme t s inset Fig. 1) from worms. Sle r, 1 µm. (,) Averge fluoreseneintensity profiles otine from time-verge fluoresene imges (22.5 s eh) of multiple ifferent phsmi ili. Line thikness represents 95% onfiene intervl of the men (CIM). () Kinesin-II poole from 15 worms, phsmi ili. () OSM-3 poole from 19 worms, 32 phsmi ili. (-) B Bse, Trnsition Zone, Proximl Segment n Distl Segment Mmilln Pulishers Limite. All rights reserve

2 e Trin veloity (µm/s) IFT-A nterogre IFT-B retrogre Kinesin-II OSM-3 retrogre Distne from se (µm) Trin veloity (µm/s) Trin veloity (µm/s) Distne from se (µm) CHE-11::mCherry Distne from se (µm) 1 Trin int. (no. of prtiles) Trin int. (no. of prtiles) 2 1 IFT-A retrogre IFT-B nterogre OSM-6::eGFP Distne from se (µm) Trin veloity (µm/s) CHE-11::mCherry Distne from se (µm) IFT-B retrogre 2 1 OSM-6::eGFP Distne from se (µm) 1 Trin int. (no. of prtiles) Supplementry Figure 2 Kinesin-2 motors ooperte to trnsport IFT-trins long ili (relte to Fig. 2). (,) Representtive nterogre () n retrogre () Fourier-filtere kymogrphs of IFT-A prtile suomplexes n orresponing trin veloity (lk) n trin intensity (ornge). () Representtive retrogre Fourier-filtere kymogrph of IFT-B prtile suomplexes n orresponing trin veloity. () Averge IFT-B intensity for (left) nterogre n (right) retrogre trins. (e) Veloities of retrogre trnsport of kinesin-ii (green) n OSM-3 (mgent). (-e) Minus (-) n plus (+) enote MT-polrity, horizontl sle rs, 1 µm; vertil sle rs, 2 s. Dshe lines represent men ± SD, line thikness represents 95% CIM. For numer of kymogrph trjetories inlue n numer of worms imge to otin representtive imges see Supplementry Fig Mmilln Pulishers Limite. All rights reserve

3 Kinesin-II A-to-R.15 s Kinesin-II R-to-A.6 s OSM-3 A-to-R.15 s OSM-3 R-to-A.15 s 3. s 1.35 s.75 s 1. s 4.35 s 3. s 1.8 s 1.8 s 4.8 s 9.6 s 2.55 s 2. s 5.25 s s 3. s 3. s e Time (s) IFT-B A-to-R Distne from se (µm) f.75 s 1.65 s IFT-B A-to-R 2.7 s 4. s 5.25 s Supplementry Figure 3 Single-motor turnrouns result in onfinement of kinesin-ii lose to the se n OSM-3 ner the istl segment (relte to Fig. 3). () All loliztions of the kinesin-ii nterogre-to-retrogre (A-to-R) trjetory of Figure 3 in min text; single kinesin-ii motor initilly moves in the nterogre iretion (green), suenly swithes iretion, to move in retrogre iretion (grey), eing trnsporte y IFTynein. () All loliztions of the kinesin-ii retrogre-to-nterogre (R-to-A) trjetory of Figure 3 in min text; single kinesin-ii motor first moves in the retrogre iretion (grey), trnsporte y IFT-ynein, wits t the se, n next moves in the opposite, nterogre iretion (green). () All loliztions of the OSM-3 A-to-R trjetory of Figure 3f in min text; single OSM-3 motor initilly moves in the nterogre iretion (mgent), suenly swithes iretion into the retrogre iretion (grey), trnsporte y IFT-ynein. () All loliztions of the OSM-3 R-to-A trjetory of Figure 3h in min text; single OSM-3 motor initilly moves in the retrogre iretion (grey), rrie y IFT-ynein, n suenly swithes iretion just efore the se, moving in the nterogre iretion (mgent). (e) One representtive single-prtile trjetory (left) n orresponing imges (right, inite y open symols in trjetories) of n IFT-B prtile suomplex A-to-R turnroun from worms, phsmi ili. (f) All loliztions of the IFT-B A-to-R trjetory of Supplementry Figure 3e; Sle rs,.5 µm Mmilln Pulishers Limite. All rights reserve

Avg. ilium intensity (.u.) 15 5 OSM-3 kp-1 OSM-3 WT -1 1 2 3 4 5 6 7 Distne from se (µm) SR-imge from SM-trjetories Σ Bse OSM-3 WT OSM-3 kp-1 OSM-3 kp-1 OSM-3 kp-1 nterogre e Bse Kinesin-II WT Trin int.

ilium intensity (.u.) 6 B Kinesin-II mksr-1 Kinesin-II WT -1 1 2 3 4 5 Distne from se (µm) h OSM-3 kp-1;mksr-1 Supplementry Figure 4 Mutnts with IFT-motor efets n trnsitionzone efets revel tht

() Fluoresene-intensity profiles otine from timeverge fluoresene imges (22.5 s eh) of multiple ifferent phsmi ili.

4 Avg. ilium intensity (.u.) 15 5 OSM-3 kp-1 OSM-3 WT Distne from se (µm) SR-imge from SM-trjetories Σ Bse OSM-3 WT OSM-3 kp-1 OSM-3 kp-1 OSM-3 kp-1 nterogre e Bse Kinesin-II WT Trin int. (no. of motors) B 6 OSM-3 kp-1 OSM-3 WT Distne from se (µm) Kinesin-II osm-3 f Normlize intensity.6.2 Kinesin-II MKS-6 mksr-1 B KAP-1::eGFP MKS-6::mCherry Kinesin-II mksr-1 MKS-6 mksr Distne from se (µm) g Avg. ilium intensity (.u.) 6 B Kinesin-II mksr-1 Kinesin-II WT Distne from se (µm) h OSM-3 kp-1;mksr-1 Supplementry Figure 4 Mutnts with IFT-motor efets n trnsitionzone efets revel tht kinesin-ii nvigtes IFT-trins into the proximl segment, where OSM-3 tkes over n rives fst, long-istne trnsport (relte to Fig. 5). () Fluoresene-intensity profiles otine from timeverge fluoresene imges (22.5 s eh) of multiple ifferent phsmi ili. Comprison of OSM-3 in kinesin-ii mutnt kgroun (kp-1; grey, poole 25 worms, 46 phsmi ili) n wil-type OSM-3 (mgent, poole from 19 worms, 32 phsmi ili, from Supplementry Figure 1) revels ~three-fol ifferene in OSM-3 intensity t the se. () Representtive super-resolution (SR) imges otine y umulting (S) loliztions (lue ots) of multiple single-moleule trjetories (grey lines) for OSM-3 (top right; from worms, 31 phsmi ili) n OSM-3 in kinesin-ii mutnt kgroun (kp-1; ottom right; from 11 worms, 18 phsmi ili), see lso Supplementry Figure 6i. () Representtive nterogre Fourier-filtere kymogrph of OSM-3 in kinesin-ii mutnt kgroun (kp-1). () Averge OSM-3 intensity for nterogre trins in C. elegns strins lking (kp-1; grey) n ontining (mgent) funtionl kinesin-ii. (e) Representtive super-resolution imges for kinesin-ii (top right; from 12 worms, 15 phsmi ili) n kinesinii in n OSM-3 mutnt kgroun (osm-3; ottom right; from worms, 34 phsmi ili, see lso Supplementry Figure 6j. (f) Representtive (from worms) two-olor timeverge fluoresene imge of kinesin-ii (green) n trnsition zone mrker MKS-6 (mgent) (top), n orresponing fluoresene-intensity profile (ottom) long the lower ilium. Sle r, 1 µm. The fluoresene-intensity profile of kinesin-ii is normlize to its mximum n tht of MKS-6 is sle reltive to MKS-6 in wil type (Supplementry Figure 1). (g) Averge fluoresene-intensity profiles otine from time-verge fluoresene imges (22.5 s eh) of multiple phsmi ili. Comprison of kinesin-ii in MKSR-1 mutnt kgroun (mksr-1; grey, poole from 22 worms, 42 phsmi ili) n wil-type kinesin-ii (green, poole from 15 worms, phsmi ili, from Supplementry Figure 1) revels erese of kinesin-ii in the ut n inrese in the. (h) Representtive nterogre Fourier-filtere kymogrph of OSM-3 in kinesin-ii n MKSR-1 oulemutnt kgroun (kp-1;mksr-1). (,e) Sle rs,.25 µm. (,h) Minus (-) n plus (+) enote MT-polrity, horizontl sle r, 1 µm; vertil sle r, 2 s. (,,g) Dshe lines re mens ± SD, line thikness represents 95% CIM. For numer of kymogrph trjetories inlue n numer of worms imge to otin representtive kymogrphs see Supplementry Fig. 6-g,i,j Mmilln Pulishers Limite. All rights reserve

5 Avg. ilium intensity (.u.) IFT-A WT IFT-A kp Distne iliry se (µm) Coeffiient of vrition (%) IFT-B WT IFT-B kp Distne from se (µm) Coeffiient of vrition (%) IFT-A WT IFT-A kp Distne from se (µm) Cumultive proility.6.2 IFT-B WT IFT-B kp Time (s) Supplementry Figure 5 IFT-prtile suomplex loing n istriution is ltere in the sene of kinesin-ii (relte to Fig. 6). () Averge fluoresene-intensity profiles otine from time-verge fluoresene imges (22.5 s eh) of multiple ifferent phsmi ili. IFT-A in kinesin-ii mutnt kgroun (kp-1; grey, poole from 22 worms, 44 phsmi ili), n wil-type IFT-A (ornge, poole from 25 worms, phsmi ili). () Coeffiient of vrition (stnr evition ivie y the men) of kymogrphs otine from imge stks of IFT-B in C. elegns strins lking (kp-1; grey, n = kymogrphs from 17 worms, phsmi ili) n ontining (lue, n = 52 kymogrphs from 19 worms, 52 phsmi ili) funtionl kinesin-ii show tht kymogrphs of C. elegns strins lking kinesin-ii re less regulr. () Coeffiient of vrition (stnr evition ivie y the men) of kymogrphs otine from imge stks of IFT-A in C. elegns strins lking (kp-1; grey, n = 36 kymogrphs from 22 worms, 36 phsmi ili) n ontining (ornge, n = kymogrphs from 25 worms, phsmi ili) funtionl kinesin-ii show tht kymogrphs of C. elegns strins lking kinesin-ii re less regulr. () Cumultive proility istriution of the urtions of single prtile suomplexes esping from the trnsition zone. Distriutions re otine y efining region of interest n mesuring the time it tkes for prtile suomplex to ross the ounry in nterogre iretion (inset). IFT-B prtile suomplexes espe the trnsition zone in.6 ±.1 s (men ± s.e.m., n = 45 IFT-B prtile suomplexes from worms, 19 phsmi ili) in the presene of kinesin-ii n in.6 ±.1 s (men ± s.e.m., n = 45 IFT-B prtile suomplexes from 25 worms, 38 phsmi ili) lking funtionl kinesin-ii (kp-1). Dshe lines re men ± SD, line thikness represents 95% CIM Mmilln Pulishers Limite. All rights reserve

6 Numer of trjetories Numer of trjetories e Numer of trjetories h Numer of trjetories l Averge intensity (.u.) IFT-B nterogre IFT-B retrogre IFT-A nterogre IFT-A retrogre (n = 19 worms, n = 52 phsmis) (n = 17 worms, n = 36 phsmis) (n = 25 worms, n = phsmis) (n = 24 worms, n = 42 phsmis) Distne from se (µm) Distne from se (µm) Distne from se (µm) Distne from se (µm) Distne from se (µm) Kinesin-II nterogre Kinesin-II retrogre OSM-3 nterogre OSM-3 retrogre (n = 15 worms, n = phsmis) (n = 15 worms, n = phsmis) (n = 19 worms, n = 32 phsmis) (n = 14 worms, n = phsmis) Distne from se (µm) f Distne from se (µm) Distne from se (µm) Distne from se (µm) OSM-3 kp-1 nterogre Kinesin-II mksr-1 nterogre Kinesin-II mksr-1 retrogre OSM-3 kp-1;mksr-1 nterogre (n = 25 worms, n = 46 phsmis) (n = 22 worms, n = 42 phsmis) (n = 22 worms, n = 41 phsmis) (n = 25 worms, n = 43 phsmis) 6 IFT-B kp-1 nterogre (n = 17 worms, n = phsmis) Distne from se (µm) KAP-1::eGFP KAP-1::mCherry Power (mw) Lolizttions (%) Distne from se (µm) Distne from se (µm) i j k 1.2 OSM-3 SR-imges Bse & OSM-3 WT OSM-3 kp-1 OSM-3 WT (lo. = 359) OSM-3 kp-1 (lo. = 682) Distne (µm) 1.2 Kinesin-II SR-imges Bse & Kinesin-II WT Kinesin-II osm-3 Kinesin-II WT (lo. = 362) Kinesin-II osm-3 (lo. = 471) Distne (µm) g Distne from se (µm) IFT-B WT IFT-B SR-imges Bse & IFT-B kp-1 IFT-B WT (lo. = 183) IFT-B kp-1 (lo. = 16) Distne (µm) Supplementry Figure 6 Histogrms of the mount of kymogrph trjetories use to nlyze trin ynmis n intensities, the istriution n numer of loliztions use to uil the super-resolution (SR) imges, n in vivo omprison of egfp n mcherry intensity (relte to multiple figures n experimentl proeures (see legen)). () IFT-B, (left) nterogre trins (orresponing to Figure 1; Tle 1; Figure 2,,e; Supplementry Figure 2; Figure 6,) n (right) retrogre trins (orresponing to Supplementry figure 2,). () IFT-A, (left) nterogre trins (orresponing to Figure 1; Tle 1; Supplementry Figure 2) n (right) retrogre trins (orresponing to Supplementry Figure 2). () Kinesin-II, (left) nterogre trins (orresponing to Figure 1; Tle 1; Figure 2,,e; Figure 5h (top),i) n (right) retrogre trins (orresponing to Figure 2f; Supplementry Figure 2e; Figure 5j). () OSM-3, (left) nterogre trins (orresponing to Figure 1e; Tle 1; Figure 2,,e; Figure 5; Supplementry Figure 4) n (right) retrogre trins (orresponing to Figure 2f; Supplementry Figure 2e). (e) Anterogre OSM-3 in kinesin-ii mutnt kgroun (kp-1; orresponing to Tle 1; Figure 5-,k; Supplementry Figure 4,). (f) Anterogre (left) (orresponing to Tle 1; Figure 5h (ottom),i) n retrogre (right) (orresponing to Figure 5j) kinesin-ii in n MKSR-1 mutnt kgroun (mksr-1). (g) Anterogre OSM-3 in kinesin-ii n MKSR-1 oule-mutnt kgroun (kp-1;mksr-1; orresponing to Figure 5k; Supplementry Figure 4h). (h) Anterogre IFT-B in kinesin-ii mutnt kgroun (kp-1; orresponing to Tle 1; Figure 6,). (i-k) SR-imges (top n ottom) n projetion of loliztions long long imge xis (mile, re for eh tre normlize), Relte to Figure 6 n Supplementry Figure 4. (i) OSM-3, from Supplementry Figure 4; (j) Kinesin-II, from Supplementry Figure 4e; (k) IFT-B, from Figure 6e. Sle r:.25 µm. (l) Fluoresene intensities of egfp-tgge (EJP13) n mcherry-tgge (EJP85) KAP-1 s funtion of exittion intensity. Intensities were otine y integrting ilium region of interest (~1.7 µm 2 ) over the first ten frmes of n imge series n verging over 12 to phsmi ili (6 to 14 worms for eh t point) for eh imging onition. Error rs represents s.e.m.. Liner fits re use to lulte mcherry to egfp fluoresene intensity orretion ftor of 2.2, Relte to "Quntifition of fluoresene intensities" in Methos Mmilln Pulishers Limite. All rights reserve

7 Strin Genotype Short nottion EJP13 kp-1(ok676) III; vusi1 [pbp; Pkp-1::kp-1::eGFP; -un-119(+)] IV Kinesin-II or Kinesin-II WT EJP16 vusi2 [pbp22; Posm-3::osm-3::mCherry; -un-119(+)] II; un-119(e3) III; osm-3(p82) IV OSM-3 or OSM-3 WT EJP18 vusi2 [pbp22; Posm-3::osm-3::mCherry; -un-119(+)] II; kp-1(ok676) III; vusi4 [pbp; Pkp-1::kp-1::pGFP; -un-119(+)] IV EJP22 vusi2 [pbp22; Posm-3::osm-3::mCherry; -un-119(+)] II; kp-1(ok676) III; osm-3(p82) IV OSM-3 kp-1 EJP34 vusi8 [pbp31; Posm-3::osm-3::pGFP; -un-119(+)] II; kp-1(ok676) III; vusi9 [pbp27; Pkp-1::kp-1::mCherry; -un-119(+)] IV EJP41 vusi [pbp33; Pkp-1::kp-1::eGFP; -un-119(+)] I; kp-1(ok676) III; osm-3(p82) IV Kinesin-II osm-3 EJP42 vusi [pbp33; Pkp-1::kp-1::eGFP; -un-119(+)] I; vusi2 [pbp22; Posm-3::osm-3::mCherry; -un-119(+)] II; kp-1(ok676) III; osm-3(p82) IV Kinesin-II OSM-3 EJP64 kp-1(ok676) III; vusi1 [pbp; Pkp-1::kp-1::eGFP; -un-119(+)] IV; mksr-1(ok92) X Kinesin-II mksr-1 EJP7 mks-6(gk674) I; vusi21 [pbp39; Pmks-6::mks-6::mCherry; -un-119(+)] II; kp-1(ok676) III; vusi1 [pbp; Pkp-1::kp-1::eGFP; -un-119(+)] IV Kinesin-II MKS-6 EJP71 mks-6(gk674) I; vusi21 [pbp39; Pmks-6::mks-6::mCherry; -un-119(+)] II; kp-1(ok676) III; vusi1 [pbp; Pkp-1::kp-1::eGFP; -un-119(+)] IV; mksr-1(ok92) X Kinesin-II MKS-6 mksr-1 EJP73 vusi2 [pbp22; Posm-3::osm-3::mCherry; -un-119(+)] II; kp-1(ok676) III; osm-3(p82) IV; mksr-1(ok92) X OSM-3 kp-1;mksr-1 EJP76 vusi15 [pbp36; Posm-6::osm-6::eGFP; -un-119(+)] I; un-119(e3) III; osm-6(p811) V IFT-B or IFT-B WT EJP77 vusi15 [pbp36; Posm-6::osm-6::eGFP; -un-119(+)] I; kp-1(ok676) III; osm-6(p811) V IFT-B kp-1 EJP81 vusi24 [pbp43; Phe-11::he-11::mCherry; -un-119(+)] II; un-119(e3) III; he-11(tm3433) V IFT-A or IFT-A WT EJP82 vusi24 [pbp43; Phe-11::he-11::mCherry; -un-119(+)] II; kp-1(ok676) III; he-11(tm3433) V IFT-A kp-1 EJP85 kp-1(ok676) III; vusi9 [pbp27; Pkp-1::kp-1::mCherry; -un-119(+)] IV Supplementry Tle. C. elegns strins use in this stuy. Short nottion is use throughout min text n figures to inrese reility Mmilln Pulishers Limite. All rights reserve

8 Supplementry Vieo Legens Supplementry Vieo 1 Dynmis of IFT-B in C. elegns strin EJP76. Vieo plys t twie rel time (time inite). Sle r, 2 μm. Relte to Figure 1; Figure 2; Supplementry Figure 2. Supplementry Vieo 2 Dynmis of kinesin-ii (green) n OSM-3 (mgent) in C. elegns strin EJP42. Vieo plys t twie rel time (time inite). Sle r, 2 μm. Relte to Figure 2,f. Supplementry Vieo 3 Single-moleule ynmis of kinesin-ii in C. elegns strin EJP13. Vieo plys t twie rel time (time inite). Sle r, 1 μm. Relte to Figure 3,. Supplementry Vieo 4 All single-moleule loliztions of Supplementry Vieo 3 of kinesin-ii in C. elegns strin EJP13. Vieo plys t twie rel time (time inite). Sle r, 1 μm. Relte to Figure 3,. Supplementry Vieo 5 Dynmis of OSM-3 in the sene of funtionl kinesin-ii in C. elegns strin EJP22. Vieo plys t twie rel time (time inite). Sle r, 2 μm. Relte to Figure 5-. Supplementry Vieo 6 Dynmis of kinesin-ii in the sene of OSM-3 in C. elegns strin EJP41. Vieo plys t twie rel time (time inite). Sle r, 2 μm. Relte to Figure 5,e. Supplementry Vieo 7 Dynmis of kinesin-ii in trnsition-zone mutnt C. elegns strin EJP64. Vieo plys t twie rel time (time inite). Sle r, 2 μm. Relte to Figure 5g-j Mmilln Pulishers Limite. All rights reserve

MOUNTING HOLES: 4X DIA 0.20 INCH 7.6 X 6.4 INCH PATTERN 42.50

MOUNTING HOLES: 4X DIA 0.20 INCH 7.6 X 6.4 INCH PATTERN 42.50 4 2 ISIONS ESRIPTION TE INITIL EVELOPMENT 206/04/29 UPTE ORERING INFORMTION TLE 206//28 256 02. NOTES:. ORERING INFORMTION: 205 290 248 42.5 6 42.50 MOUNTING HOLES: 4X I 0.20 INH 7.6 X 6.4 INH PTTERN PRT