d Lamp1-GFP Starvation 2h Starvation 2h Starvation 2h + ML-SI3
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1 DOI: /ncb3324 a b c Lamp1-GFP Lamp1-GFP Lamp1-GFP Lamp1-GFP WT ibroblast Lysotrackr WT ibroblast xtran Starvation 2h WT ibroblast γ-tubulin Starvation 2h γ-tubulin GFP-LC3 HLa LC3-GFP LC3-RFP g Starvation Starvation 2h Starvation 2h + ML-SI3 Supplmntary Figur 1 Lysosoms unrgo prinuclar migration uring acut starvation to acilitat lysosom-autophagosom usion. (a) WT ibroblasts showing th co-localization btwn Lamp1-GFP an Lyso- Trackr. (b) Lamp1-transct WT ibroblasts wr loa with xtran-r, with (right) or without (lt) starvation. (c, ) WT ibroblasts wr transct with Lamp1-GFP an lt untrat (c), or starv or 2 h (), thn ix an immunolabll or γ-tubulin to visualiz cntrosoms (MTOC, whit arrows). () HLa clls stably xprssing GFP-RFP-LC3 wr subjct to starvation or 2 h (lowr panl). Th GFP channl shows puncta that labl autophagosoms spciically, but not autolysosoms. Ths rsults show Li t al., Suppl. Fig. 1 that both lysosoms an autophagosoms ar prinuclarly localiz unr acut starvation. () HLa clls stably xprssing GFP-RFP-LC3 wr lt untrat (uppr), starv or 2 h (mil), or starv or 2 h in th prsnc o 25 μm ML-SI3 (bottom). (g) Quantiication o th numbr o puncta that wr GFP- an RFP-positiv (autophagosoms, AP) ovr th numbr o puncta that wr only RFP-positiv (autolysosoms, AL) or groups shown in (). ucli ar labl with. Graph ata ar prsnt as mans ± SEM, th numbrs o clls (n) us or quantiication wr pool across at last thr inpnnt xprimnts an ar shown in th parnths.s *p <.05, **p <.01 in AOVA. Scal bar = 10 μm Macmillan Publishrs Limit. All rights rsrv.
2 a b c Torin-1 Starvation Ammonia Ringr ps6k S6K Tubulin Supplmntary Figur 2 Ect o cytosolic ph on TRPML1 channl activity an lysosom positioning. (a) ML-SA1 (10 μm)-activat whollysosom TRPML1 currnts wr inhibit potntly by th TRPML1 inhibitor ML-SI3 (2.5 mm). (b) PI(3,5)P 2 -activat whol-nolysosom TRPML1 currnts wr moulat by cytosolic ph. (c, ) Lamp1- mchrry-transct WT ibroblasts wr trat with 30 min ammonia Ringr s solution in th prsnc () or absnc (c) o 25 μm ML-SI3. () Quantiication o groups shown in (c) an (). () Wstrn blot showing th phosphorylat orm o S6K (ps6k) in HLa clls starv or 2 h, trat with 1 μm Torin-1 or 2 h, or trat with ammonia Ringr s or 30 min. R lins outlin cll bounaris an nucli ar mark with a r. Graph ata ar prsnt as mans ± SEM, th numbrs o clls (n) us or quantiication wr pool across at last thr inpnnt xprimnts an ar shown in th parnthss.*p <.05, **p <.01 in AOVA. Scal bars = 10 μm. Uncropp wstrn blot imags ar shown in Supplmntary Figur Macmillan Publishrs Limit. All rights rsrv.
3 a ML-SA3 25 µm ML-SA5 1 µm b WT ML1 o/ ML1 o/ + ML-SA1 c ML2 o/ ML3 o/ ML1 o/ +ML-SI3 1µM +ML-SI3 25µM g h Lamp1 Mitotrackr j i Lamp1 Mitotrackr + ML-SA1 Supplmntary Figur 3 Spciic rgulation o lysosom istribution by TRPML1. (a) WT ibroblasts trat with ML-SA3 (25 μm) or ML-SA5 (1 μm), two structurally-inpnnt TRPML1 agonists, or 1 h. (b) In TRPML1-ovrxprssing clls, th majority o lysosoms wr localiz in th prinuclar rgion in th prsnc or absnc o 25 μm ML-SA1. (c) Rprsntativ imags showing WT ibroblasts ovrxprssing TRPML2 or TRPML3. () Quantiications o groups shown in (b). () Quantiication o groups shown in (c). () Th prinuclar localization o lysosoms inuc by TRPML1 ovrxprssion (lt) was rvrs by ithr a low (1 μm, mil) or high (25 μm, right) os o ML-SI3. (g) Quantiication o groups shown in (). (h, i) Rprsntativ imags o Lamp1-GFPtransct ibroblasts stain with MitoTrackr (100 nm) or 1 h (h), or 1 h o MitoTrackr in th prsnc o 25 μm ML-SA1 (i). (j) Quantiication o groups shown in (h) an (i). Upon ML-SA1 tratmnt, lysosoms bcam mor prinuclar, whil th istribution o mitochonria was not altr. R lins outlin cll bounaris an nucli ar mark with a r. Graph ata ar prsnt as mans ± SEM, th numbrs o clls (n) us or quantiication wr pool across at last thr inpnnt xprimnts an ar shown in th parnthss *p <.05, **p <.01 in AOVA. Scal bars = 10 μm Macmillan Publishrs Limit. All rights rsrv.
4 a DMSO 18h b ML-SI1 18h ML-SI3 18h c Lamp1 Rab7-T22 ML-SI3 18h Lamp1 Filipin ML1KO DynIC2-D-GFP Filipin ML1KO Supplmntary Figur 4 Chronic loss o TRPML1 activity causs cholstrol accumulation an prinuclar localization o lysosoms in ibroblasts. (a) Lysosom istribution in WT ibroblasts trat with vhicl (0.1% DMSO) (uppr right), ML-SI1 25 μm (bottom lt), or ML-SI3 25 μm (bottom right) or 18 h in complt mium. (b) Quantiication o th groups shown in (a). (c) Tim-pnnc o TRPML1 inhibition o lysosom istribution in WT ibroblasts in complt mium. () Rprsntativ imags showing lysosom istribution in Rab7-T22-xprssing clls in th prsnc o ML-SI3 (25 μm) Li t al., Suppl. Fig. 4 or 18 h. () Rprsntativ imags showing ML1 KO ibroblasts transct with Lamp1-mChrry, an stain with ilipin. Intracllular puncta ilipin staining co-localiz wll with Lamp1. () Rprsntativ imags showing ML1 KO ibroblasts transct with DynIC2-D-GFP, an stain with ilipin. Graph ata ar prsnt as mans ± SEM, th numbrs o clls (n) us or quantiication wr pool across at last thr inpnnt xprimnts an ar shown in th parnthss. *p <.05, **p <.01 in AOVA. Scal bars = 10 μm or (a) an (), an = 50 μm or () an () Macmillan Publishrs Limit. All rights rsrv.
5 a b c Apilimo 1µM Starvation Torin1 WT ML1 o/ YM µM Starvation + Apilimo 1µM Torin1 + Apilimo 1µM ML1-7Q o/ YM µM ML-SA1 25µM Starvation + YM µM Torin1 + YM µM ML1-7Q o/ ML-SA1 25µM 1h g i j k l WT Fibroblast WT Fibroblast Starvation ML-SA1 h + Ki5B-D + DynIC2-D + DynIC2-D + DynIC2-D m ML1 o/ ML1 o/ n ML-SA1 ML-SA1 o Ciliobrvin D Ciliobrvin D p q ps6k ML-SA1(µM) S6K Supplmntary Figur 5 Rgulation o lysosoms motility by PI(3,5)P 2 an ynin motors. (a) Lysosom istribution in WT ibroblasts trat with 1 μm apilimo or 1 h (uppr), 1 μm YM or 1 h (mil), or 1 μm YM plus 25 μm ML-SA1 or 1 h (bottom). (b) Lysosom istribution in WT ibroblasts starv or 1 h (uppr), or starv or 1 h in th prsnc o 1 μm apilimo (mil) or YM (bottom). (c) Lysosom istribution in WT ibroblasts trat with 1 μm Torin-1 or 1 h in th prsnc o 1 μm apilimo (mil) or YM (bottom). () Lysosom istribution in TRPML1-7Q-transct clls in th prsnc (bottom) or absnc (mil) o 25 μm ML-SA1 or 1 h. () Quantiication o groups shown in (a). () Quantiication o groups shown in (b). (g) Quantiication o groups shown in (c). (h) Quantiication o groups shown in (), compar to clls xprssing Lamp1 alon. (i) Lysosom (labl with Lamp1-EGFP) istribution in WT ibroblasts transct with mchrry-tagg ominant-ngativ Ki5B (Ki5B-D). (j-l) Lysosom (labl with Lamp1-mChrry) istribution in WT ibroblasts transct with GFP-tagg ominant-ngativ cytoplasmic ynin intrmiat chain 2 (DynIC2-D), thn lt untrat (j), starv or 2 h (k), or trat with 25 μm ML-SA1 or 2 h (l). (m) Ect o ynin inhibitor ciliobrvin D (20 μm, 2 h) on lysosom istribution in TRPML1- xprssing ibroblasts. (n) WT ibroblasts trat with ML-SA1 (25 μm) or togthr with ciliobrvin D (20 μm) or 2 h. (o) Quantiication o lysosom istribution in xprimntal groups shown in (i) an (j). R lins outlin cll bounaris; marks nucli. (p) Whol-lysosom TRPML1 currnts wr not activat by Torin-1 in TRPML1-xprssing Cos1 clls. ML-SA1 raily activat whol-lysosom TRPML1 currnts. (q) Application o ML- SA1 (1, 5, 10 mm) in HEK293T clls or 3 h i not la to a signiicant chang in th lvl o phophorylat S6K, a major mrotc1 targt. Graph ata ar prsnt as mans ± SEM, th numbrs o clls (n) us or quantiication wr pool across at last thr inpnnt xprimnts an ar shown in th parnthss). *p <.05, **p <.01 in AOVA. Scal bars = 10 μm. Uncropp wstrn blot imags ar shown in Supplmntary Figur Macmillan Publishrs Limit. All rights rsrv.
6 a Lamp1 Lamp1 ALG-2 WT ibroblast SytVII ML-SI3 ML-SA1 b Lamp1 g Lamp1 ORP1L WT ibroblast () c Li t al., Suppl. Fig. 6 Supplmntary Figur 6 Ects o Syt VII, ALG-2, an ORP1L ovrxprssion on lysosom istribution. (a, b) WT ibroblasts ovrxprssing Lamp1-mChrry with (a) or without (b) Syt VII co-xprssion. (c) Quantiication o groups shown in (a, b). (-) WT ibroblasts co-transct with ALG-2-GFP an Lamp1-mChrry, thn lt without tratmnt (), trat with 25 μm ML-SI3 or 2 h (), or trat with 25 μm ML-SA1 or 2 h (). Som prinuclar bright ots o ALG-2 not co-localiz with Lamp1 wr sn in all tratmnt conitions. Tratmnt o ML-SI3 an ML-SA1 rsult in lss (ML-SI3) or mor (ML-SA1) co-localization with Lamp1 compar to non-trat control clls, rspctivly. (g) ORP1L ovrxprssion inuc lysosom clustring as wll as nlargmnt in WT ibroblasts. R lins outlin cll bounaris an nucli ar mark with r. Graph ata ar prsnt as mans ± SEM, th numbr o clls (n) us or quantiication wr pool across at last thr inpnnt xprimnts an ar shown in th parnthss. *p <.05, **p <.01 in AOVA. Scal bars = 10 μm Macmillan Publishrs Limit. All rights rsrv.
7 a b c ML1-GFP Filipin ML1KO ML1KO ML1-R44A-GFP Filipin ML1KO+ML1 o/ ML-SI3 ML1KO ML-SI3 h IP: mchrry-alg-2 mchrry input + + Dynamitin + + g IB: mchrry * mchrry-alg-2 mchrry i IP: mchrry-alg-2 mchrry IB: Dynamitin IP: mchrry-alg-2 mchrry IB: mchrry input + + mchrry + + mchrry + + GFP input + _ + + _ + IgG HC Dynamitin mchrry-alg-2 mchrry j IP: IB: GFP input GFP mchrry (5X loaing) Dynamitin IgG HC Cos1: GFP-Dynamitin + mchrry-alg-2 Supplmntary Figur 7 ALG-2 miats th intraction o TRPML1 an ynactin. (a-c) Rprsntativ whol-lysosom currnts in clls ovrxprssing WT TRPML1 (a), TRPML1-R 44 -A (b), or TRPML1-R 44 LK- AAA (c). Whol-lysosom currnts wr licit by th nognous agonist PI(3,5)P 2 or th synthtic agonist ML-SA1. (, ) Filipin staining o ML1 KO ibroblasts ovrxprssing TRPML1 () or TRPML1-R 44 -A (). Purpl arrows inicat clls with ovrxprssion. () Ovrxprssion o TRPML1 in ML1 KO ibroblasts causs prinuclar accumulation o lysosoms, but contrary to ML1 KO ibroblasts without TRPML1 xprssion, this was rvrsibl through application o 25 μm ML-SI3 or 2 h. (g) Quantiication o groups shown in (). (h) Clls xprssing mchrry or mchrry-alg-2 wr subjct to Co-IP with ithr anti-mchrry or anti-dynamitin, thn blott against mchrry (top) or Dynamitin (bottom). Astrisk inicats a non-spciic ban sn with HEK293 pull-own. (i) Cos1 clls xprssing ithr mchrry or mchrry-alg-2 wr subjct to Co-IP with ithr anti-mchrry or anti- GFP antibois, thn blott against mchrry. (j) Cos1 clls co-xprssing GFP-ynamitin an mchrry-alg-2 wr subjct to Co-IP with ithr antimchrry or anti-gfp antibois, thn blott against GFP. R lins outlin cll bounaris an nucli ar mark with a r. Graph ata ar prsnt as mans ± SEM, th numbrs o clls (n) us or quantiication wr pool across at last thr inpnnt xprimnts an ar shown in th parnthss. *p <.05, **p <.01 in AOVA. Scal bars = 10 μm or (), an 50 μm or (, ). Uncropp wstrn blot imags ar shown in Supplmntary Figur Macmillan Publishrs Limit. All rights rsrv.
8 a b.t. + ML-SI3 + ML-SI1 c RK Supplmntary Figur 8 Moulation o lysosom tubulation by TRPML1, motor protins, an ALG-2. (a) Quantiication o lysosom tubulation in CV-1 clls. Dominant-ngativ constructs o Ki5B an DynIC2 liminat spontanous tubulation in Lamp1-GFP-xprssing CV1 clls almost compltly. (b) Rprsntativ imags o Lamp1-mChrry-transct RK clls starv or 16 h (lt), or starv or 16 h with th last 2 h in th prsnc o 25 μm ML-SI3 (mil) or ML-SI1 (right). (c) Quantiication o groups shown in (b). () Quantiication o lysosom tubulation in WT ibroblasts transct with Lamp1-GFP, or Lamp1-GFP plus ALG-2- mchrry, with or without starvation or 24 h. ALG-2 xprssion inhibit lysosom tubulation strongly in starv clls. (, ) Sam rgion o a Lamp1-GFP-transct ibroblast starv or 24 h unr convntional () or STED supr-rsolution () conocal imaging. Th supr-rsolution imag was takn 6 s atr th convntional conocal imag. Yllow arrows point to svral small lysosoms that ar lin-up to hav a tubular apparanc unr th convntional conocal imags; r arrows point to a gnuin tubul. Graph ata ar prsnt as mans ± SEM, th numbr o clls (n) us or quantiication wr pool across at last thr inpnnt xprimnts an ar shown in th parnthss *p <.05, **p <.01. Scal bars = 10 μm or (b), an = 2 μm or (, ) Macmillan Publishrs Limit. All rights rsrv.
9 blot or Figur 5j blot or Suppl. Figur 5q blot or Suppl. Fig. 7h (uppr) blot or Figur 6b blot or Suppl. Fig. 7h (lowr) blot or Figur 6i blot or Suppl. Fig. 7i 97 blot or Suppl. Fig blot or Suppl. Fig. 7j 97 Supplmntary Figur 9 Unprocss scans o original wstrn blots us in th main an supplmntary igurs. For ach imag, black boxs inicat roughly th positions o ach original blotting mmbrans, an th r ott boxs inicat th rgions us in th igurs. Each imag is labl on top with th panl in th igur thy appar Macmillan Publishrs Limit. All rights rsrv.
10 Supplmntary vio lgns Supplmntary Vio 1 FRAP analysis o irctional movmnt o lysosoms unr normal conitions. Vio (1 ram/s) o a Lamp1-mChrry-transct WT ibroblast. Photoblaching was conuct at th 5 th ram (T = 0 s). Scal bar = 10 μm. Supplmntary Vio 2 FRAP analysis o irctional movmnt o lysosoms unr acut starvation. A Lamp1-mChrry-transct WT ibroblast imag 15~30 min atr starvation. Scal bar = 10 μm. Supplmntary Vio 3 FRAP analysis o irctional movmnt o lysosoms in th prsnc o th Ca 2+ chlator BAPTA-AM. Imaging o a Lamp1-mChrrytransct WT ibroblast atr it was incubat with 10 μm BAPTA-AM or 1 h. Scal bar = 10 μm. Supplmntary Vio 4 FRAP analysis o irctional movmnt o lysosoms unr starvation conition in th prsnc o ML-SI3. A Lamp1-mChrrytransct WT ibroblast imag 15~30 min atr starvation in th prsnc o ML-SI3 (25 μm). Scal bar = 10 μm. Supplmntary Vio 5 FRAP analysis o irctional movmnt o lysosoms in th prsnc o ML-SA1. A Lamp1-mChrry-transct WT ibroblast imag 15~30 min atr application o ML-SA1 (25 μm). Scal bar = 10 μm. Supplmntary Vio 6 Tim-laps imaging o lysosom migration unr normal conitions. A Lamp1-mChrry-transct WT ibroblast was imag 1 ram/10 s or 250 rams. Th vio plays 300 ral-tim sp. Scal bar = 10 μm. Supplmntary Vio 7 Tim-laps imaging o lysosom migration upon acut application o ML-SA1. A Lamp1-mChrry-transct WT ibroblast was imag at 1 ram/10 s or 250 rams in th prsnc o 25 μm ML-SA1, which was appli 3 min atr th start o th imaging (i.. ram 18). Th vio plays 300 ral-tim sp. Scal bar = 10 μm. Supplmntary Vio 8 Tim-laps imaging o lysosom migration in TRPML1-xprssing clls upon acut application o ML-SI3. TRPML1-GFP-xprssing WT ibroblast was imag at 1 ram/10 s or 250 rams in th prsnc o 25 μm ML-SI3, which was appli 3 min atr th start o th imaging (i.. ram 18). Th vio plays 300 ral-tim sp. Scal bar = 10 μm Macmillan Publishrs Limit. All rights rsrv.
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