J. Cell Sci. 130: doi: /jcs : Supplementary information. Supplementary figure. Journal of Cell Science Supplementary information

Transcription

1 Supplementary figure

2 Fig.S1 Normal rhabdomere biogenesis and structure during development in dpip4k 29 and dpip4k over expressing PRs. (A) dpip4k 29 PRs showing immunogold labeling for Rh1 in MVBs (i) and enlarged endosome-like compartments (enlarged image) (ii). R7 photoreceptor lacks Rh1, hence no immunogold labeling indicating the specificity of the antibody (iii). (B) TEM images showing TS of entire ommatidia, of control (i), dpip4k 29 (iv) PRs at 90% pd, reared in dark; control (ii), dpip4k 29 (v), Rh1>dPIP4K (iii) Rh1>dPIP4K; dpip4k 29 PRs (vi) under illumination. The rhabdomere/apm is maintained normally in all conditions wrt controls.

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4 Fig.S2 dpip4k 29 photoreceptors show altered levels of early endosomal markers (A) Western blot from head extracts of freshly eclosed Wild type (WT) and dpip4k 29 reared under illumination vs. dark, probed for Rh1. Loading control is Tubulin. (B) Western blot from retinal extracts of freshly eclosed control and dpip4k 29 flies reared under constant light vs. dark, probed with early endosome markers- EEA1 and Rab5 antibodies. Tubulin is used as the loading control. Levels of EEA1 and Rab5 are slightly but significantly increased in dpip4k 29 retinae reared under constant light with respect to controls. (C) TS of retinae at 78% pd, immunostained with Rh1 specific antibody represented (red) and phalloidin (green), of wild type reared in dark (i, i, i ) and under illumination (ii, ii, ii ), dpip4k 29 reared in dark (iii, iii, iii ) and under illumination (iv, iv, iv ). Arrow heads represents intracellular Rh1 loaded vesicles/rlvs in controls observed under light illumination. (D- E) TS (D) and LS (E)of PRs at eclosion, stained for Rh1 (red) and F- actin (green), for WT and dpip4k 29 under dark and illumination. Arrow heads indicate RLVs in each case. (F) LS of PRs stained for TRP (red) in WT and dpip4k 29 at 90% pd reared under illumination. (G) TS of ommatidia at 90% pd, showing proper localization of Na/K ATPase to the basolateral membrane (red) in both control and dpip4k 29 reared under constant illumination.

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6 Fig.S3 dpip4k 29 has no defect in vesicular exocytic transport from ER, Golgi pathway. (A-B) LS of retinae from WT (A) and dpip4k 29 (B) reared under illumination, showing KDEL staining for endoplasmic reticulum (green) (ii) and Rh1 (red) (i). In dpip4k 29 ER morphology is largely comparable to controls. (C) Golgi morphology marked by GM130 is comparable in controls (i, ii) and dpip4k 29 (iii, iv). (D) Myosin V staining at the base of the rhabdomere is comparable in controls (i, ii) and dpip4k 29 (iii, iv). (E) Spam/ Eys localizes to intra- rhabdomeric space in dpip4k 29 (iii, iv) just like in controls (i, ii). (F) Enlarged RLVs in dpip4k 29 colocalize with late endosomal markers LS of PRs showing the large and clustered RLVs (red) in dpip4k 29 colocalize with Rab7::GFP (i-iii) and Lamp1::GFP under illumination (iv-vi). dpip4k functions independent of TOR signaling to regulate endocytosis. (G) Rh1 staining (red) in Rh1>Rheb; dpip4k 29 (i), Rh1>TOR DN (ii), Rh1>TOR RNAi(iii) PRs at 90% pd under illumination. (I-J) Quantification shows no difference in fraction of large clustered RLVs and large RLVs in Rh1>Rheb; dpip4k 29 wrt dpip4k 29. (H) Rh1>Rheb; dpip4k 29 PRs, showed expanded endosomes and MVBs similar to dpip4k 29 (i-ii). Rh1>TOR DN PRs at 90% pd under illumination, show no expanded endomembranes and did not phenocopy dpip4k 29 (iii-iv).

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8 Fig.S4 (A) Alexa 568 Tf post 5 min pulse, in dpip4k (dsrnaii) knock down(i) and shi knock down (iii).surface TfR labeled by htfr antibody (OKT9) in dpip4k (dsrnaii) knock down (ii) and shi knock down (iv). (B)Relative depletion of dpip4k transcripts with dpip4k dsrna I and II in S2 cells. (C) dpip4k protein knocked down to undetectable levels with dpip4k ds RNA I wrt a control band (a non-specific band detected by dpip4k ab) (D) Population distribution of average cellular EEA1 intensities in mock KD and dpip4k KDs in a given experiment. (E) Population distribution of average lysotracker intensities in mock KD and dpip4k KDs in a given experiment. n> 50 cells for each experiment. (F) Western blot from retinal extracts of freshly eclosed control, dpip4k 29 and Rh1>dPIP4K; dpip4k 29 flies reared under constant light. Top blot probed with antibody for dpip4k, showing the 55kDa endogenous protein in WT and lacking in dpip4k 29 and Rh1>dPIP4K; dpip4k 29. Rh1>dPIP4K; dpip4k 29 shows the over expressed dpip4k::gfp band at ~80kDa, and the levels at which dpip4k::gfp is expressed is much lower than the endogenous protein levels but still sufficient to rescue the large and clustered RLVs formed in dpip4k 29 under illumination (Fig. 1, 2). The dpip4k::gfp is reconfirmed using an antibody for GFP. Actin is used as loading control. (G) PI(4,5)P 2 resynthesis dynamics during photoresponse assayed using the PH PLCδ probe florescence at the deep psuedopupil (dpp), in WT and dpip4k 29 are comparable. (H) Cellular PI(4,5)P 2 assayed using PH-PLCδ-GFP (green) and Rh1 (red) in WT and dpip4k 29. PI(4,5)P 2 is largely present in rhabdomere/apm in PRs and under light rearing conditions when expanded RLVs are formed in dpip4k 29, PH- PLCδ-GFP localization as well as the intensities do not change wrt. WT.