Kristiina Kanerva, Riikka-Liisa Uronen, Tomas Blom, Shiqian Li, Robert Bittman, Pekka Lappalainen, Johan Peränen, Graça Raposo, and Elina Ikonen

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1 Developmental Cell, Volume 27 Supplemental Information LDL Cholesterol Recycles to the Plasma Membrane via a Rab8a-Myosin5b-Actin- Dependent Membrane Transport Route Kristiina Kanerva, Riikka-Liisa Uronen, Tomas Blom, Shiqian Li, Robert Bittman, Pekka Lappalainen, Johan Peränen, Graça Raposo, and Elina Ikonen Inventory of Supplemental Materials Figure S1, related to Figure 1. Assessment of sterol hydrolysis or localization in LE/LY or at the PM. Figure S2, related to Figure 2. Effects of Rab silencing on BC LN-LDL uptake, delivery to LE/LY, and cholesterol deposition. Figure S3, related to Figure 3. Rab8 recruitment to LE. Figure S4, related to Figure 4. Rab8 regulated motility of LE. Figure S5, related to Figure 7. Effects of LDL on FA and cell migration. Table S1, related to Figure 4. Characteristics of BC vesicles. Movie S1, related to Figure 4A. Time-lapse images of a ctrl sirna-transfected (part 1, starting time :::) and Rab8-depleted (part 2, starting time ::7:73) A431 cells overexpressing LifeAct-RFP, pulse-labeled with BC LN-LDL, and chased for 2 h. The images were obtained every 1.5 s for 3 min. Movie S2, related to Figure 4F. A431 cells stably overexpressing CD63-Cherry were pulse-labeled with BC LN-LDL and chased for 2 h. Shown are 2D projections of time-lapse z-stacks recorded at 137 ms intervals for ~1 min. Movie S3, related to Figure 5D. Time-lapse TIRFM images of an A431 cell pulse-labeled with BC LN- LDL and chased for 2 h. Acquisition, 1 fps for 2 min. Movie S4, related to Figure 6B. Time-lapse series of BC-vesicle transport in an A431 cell overexpressing zyxin-cherry, pulse-labeled with BC LN-LDL, and chased for 3 h. The sequence was acquired at 1 fps for 2 min 45 s. Movie S5, related to Figure 6F and G. Time-lapse series of the dynamics of overexpressed GFPpaxillin in A431 cells incubated with LDL for 2 h (part 1, starting time :::) or without LDL

2 (part 2, starting time ::1:67).The cells were imaged after 3 h of chase, and the images were obtained every 2.5 min for 1 h. Supplemental Experimental Procedures Target sequences of sirnas used in the study. Supplementary References

3 Figure S1. A % hydrolyzed [3H]-CL BC LN * time (h) B ctrl nocodazole C sirna: ctrl - oxidase + oxidase D % [3H] chol oxidized sirna: ctrl NPC1 Rab8 NPC1 h 4h *** *** *** E Fraction of BC in CD63/dextran/ Lamp1-positive organelles,8,6,4,2 2 4 Chase time (h) BC-CD63 BC-dextran BC-Lamp1 *** *** F sirna: Rab8 +Myo5C Myo5C BC dextran merge

4 Figure S1. Assessment of sterol hydrolysis or localization in LE/LY or at the PM. Related to Figure 1. (A) In vitro hydrolysis of BC LN-LDL and [ 3 H]-CL-LDL in A431 homogenate. The fraction of steroid hydrolyzed was analyzed after incubation of post-nuclear homogenate with labeled LDL at 37 o C for h, 24 h, or 48 h at ph 4.3 (n = 3 independent experiments, *P <.5, t-test, mean ± s.e.m.). (B) Confocal images of A431 cells pulse-labeled with BC LN-LDL and chased for 4 h with or without nocodazole. Scale bar, 2 m. (C) Epifluorescent images of filipin staining in A431 cells transfected with the indicated sirnas and treated with or without cholesterol oxidase. Arrowheads indicate filipin fluorescence at the PM in the absence of oxidase. Note the loss of fluorescence after oxidase treatment. Scale bar, 2 m. (D) Analysis of the PM arrival of LDL-derived [ 3 H]-CL as determined by cholesterol oxidation (n = 6, ***P <.1, t-test, mean ± s.e.m., compared to ctrl 4 h chase). (E) Quantification of the fraction of BC fluorescence residing in CD63, dextran-, or Lamp1 positive organelles after 2 h and 4 h of chase in A431 cells. The cells were pulse-labeled with BC LN-LDL after transfection with either CD63-Cherry or Lamp1-RFP (n = cells, ***P <.1, t-test, mean ± s.e.m., compared to BC-CD63 at 4 h). (F) Confocal images of A431 cells transfected with the indicated sirnas prior to pulse-labeling with BC LN-LDL. The cells were imaged after 4 h of chase. Scale bar, 2 m.

5 Figure S2. A 2 Uptake of BC-LDL relative to control sirna 1 sirna: * *** B Fraction of BC in dextranpositive organelles at h chase 1,5 1,5 sirna: *** C Rab7 BC dextran E Filipin intensity in Lamp1- positive organelles sirna: D sirna: filipin ctrl Rab9 Rab8 + Rab9 Rab8

6 Figure S2. Effects of Rab silencing on BC LN-LDL uptake, delivery to LE/LY, and cholesterol deposition. Related to Figure 2. (A) Quantification of the uptake of BC LN-LDL after 2 h of pulse-labeling in cells transfected with the indicated sirnas (n = 9-3 cells, ***P <.1, *P <.5, t-test, mean ± s.e.m., compared to ctrl). (B) Quantification of the fraction of BC fluorescence residing in dextran-positive organelles after h of chase. Cells were transfected with the indicated sirnas and imaged immediately after pulse-labeling with BC LN-LDL (n = cells, ***P <.1, mean ± s.e.m., compared to ctrl). (C) Overlayed confocal images of BC and dextran in A431 cells depleted of Rab7. The cells were pulse-labeled with BC LN-LDL and chased for 4h prior to imaging. Note that most BC-positive vesicles do not colocalize with dextran. Scale bar, 1 μm. (D) Epifluorescent images of filipin staining in A431 cells transfected with the indicated sirnas. Scale bar, 2 μm. (E) Quantification of filipin fluorescence intensity in Lamp1-positive organelles. Note the increased accumulation of cholesterol in Lamp1-positive organelles in cells depleted of both Rab8 and Rab9 (n = cells, mean ± s.e.m.).

7 Figure S3. BC merge zoom B RFP-Rab8 BC merge zoom NPC1 sirna A Cherry-Rab8 C -LDL +LDL D Rab8 CD63 Figure S3. Rab8 recruitment to LE. Related to Figure 3. (A) Confocal images of A431 cells overexpressing Cherry-Rab8 after pulse-labeling with BC LN-LDL. Images were captured after a 3 h chase. Right panel shows a magnification of the area indicated. Scale bar, 1 μm. (B) Confocal images of A431 cells transfected with NPC1 sirna prior to overexpression of RFP-Rab8 and pulse-labeling with BC LN-LDL. Images were captured after a 2 h chase. Right panel shows a magnification of the area indicated. Scale bar, 1 μm. (C) Epifluorescence images of endogenous Rab8 immunostaining in A431 cells incubated with (+LDL) or without LDL (LDL). Arrows indicate Rab8-positive tubules/punctae that appear upon LDL-loading. Scale bar, 1 μm. (D) 3D reconstruction of endogenous Rab8- and CD63-positive organelles in A431 cells transfected with ctrl sirna and labeled with LDL prior to immunostaining. Z-stacks were imaged with a confocal microscope. Inset at the top-right corner shows a magnification of the organelle indicated, and the panel on the right represents a 3D reconstruction of the same organelle. Scale bar, 1 μm (confocal image); 1 μm (3D reconstruction).

8 Figure S4. B Maximal distance from origin ( m) A mcherry 16 min 15 min 3 min Rab8-Cherry RFP RFP-Rab Dextran fluorescence intensity (AU) Figure S4 S4. Rab8 regulated motility of LE. LE. Related to Figure 4. (A) Quantification of tthe distance travelled by the vesicles plotted against their dextran fluorescence intensity. A431 ccells ells transfected with the indicated cdnas were pulse pulse-labeled labeled with BC LNLN-LDL and time-lapse lapse confocal images were obtained at 1.63 s intervals for 1.5 min after a 2 h chase (n n = cells). (B B) Confocal images from a movie of A431 cells ells transfected with Rab8 sirna, pulse-labeled pulse with BC LN LN-LDL, LDL, and chased in the presence of cytochalasin D for the times indicated. Images were captured every 2 s for 3 min. Scale bar, 2 m.

9 Figure S5. A -LDL +LDL Migration distance relative to -LDL (mm) B 2 *** 1 - LDL + LDL + mitomycin C Figure S5. Effects of LDL on FA and cell migration. Related to Figure 7. (A) Epifluorescence images of vinculin immunostaining in scratch-wounded A431 incubated 6 h in medium with (+LDL) or without LDL (-LDL). Scale bar, 2 μm. (B) Quantification of wound healing in A431 cells in the presence of mitomycin C. The monolayer was scratched and the medium was supplemented with mitomycin C with (+LDL) or without LDL (-LDL). After 24 h, the extent of wound closure was analyzed (n = 5; ***P <.1, t-test, mean ± s.e.m., compared to LDL). The results were normalized to -LDL.

10 Table S1. Characteristics of BC vesicles. Related to Figure 4. Table summarizing the characteristics of BC-containing vesicle morphology, velocity, interactions, and markers. morphology vesicles mostly spherical or oval, sometimes tubular, diameter m velocity maximal average 2.76 ± m s ±.5 m s -1 direction of movement interactions between vesicles LE/LY-PM bidirectional, also lateral along the PM fission, fusion (both homotypic and heterotypic between BC- and CD63-positive vesicles), tubulation markers at perinuclear region at cell periphery CD63, Rab8, NPC1, Lamp1, dextran CD63, Rab8 (negative for Lamp1 and dextran)

11 Supplemental Experimental Materials Table S2. Target sequences of sirnas used in the study.

12 Supplemental Experimental Procedures Cell culture, transient transfections, and stable cell lines. A431 cells, obtained from ATCC, were cultured in DMEM supplemented with 1% fetal bovine serum, 2 mm L-glutamine (all from Gibco), 1 IU ml -1 penicillin, and 1 µg ml -1 streptomycin (Lonza). The human primary fibroblasts AG8498 and GM321 (healthy controls) were purchased from Coriell cell repository, and Wolman disease patient fibroblasts have been described (Wang et al., 28). HT18 fibrosarcoma cells were cultured as described (Hattula et al., 26). To generate stable cell lines, CD63-mCherry and GFP-Rab8a plasmids were transfected into A431 cells using Lipofectamine LTX (Invitrogen). Transfected cells were selected with.6 mg ml 1 G418 (Gibco) for two weeks, and single-cell clones were obtained with limiting dilution and screened by fluorescence microcopy and immunoblotting. sirnas (see supplementary experimental procedures) were transfected with HiPerFect for 72 h and cdna constructs with Effectene (both Qiagen) for 24 h. In plasmid overexpression experiments, the cells were co-tansfected with LDL-receptor (LDLR) to increase LDL uptake. Plasmids. The following human cdna constructs were used in the study: LifeAct-RFP (a gift from Dr. Maria Vartiainen, Institute of Biotechnology, Helsinki, Finland), CD63-Cherry (a gift from Prof. Gillian Griffiths, Oxford University, UK), and Lamp1-RFP (a gift from Prof. Ana Maria Cuervo, Albert Einstein College of Medicine, Bronx, NY, USA). LDLR-pCB6 and pegfp-rab8a-wt described in (Hattula and Peranen, 2; Hunziker et al., 1991), respectively. The pmcherry-rab8a-wt was constructed by cloning Rab8a-wt into the EcoRI/HindIII sites of pmcherry-c1 (Clontech). mrfp1-rab8a was created by moving the mrfp1 open reading frame (ORF) from mrfp-rab7 (Johansson et al., 27) into NheI/BglII sites of pegfp-rab8a-wt. The pmcherry-rabin8 was created by cloning Rabin8 into the BglII/SalI sites of pmcherry-c1. The ORF of TBC1D3 was amplified from human brain cdna and cloned into the BglII/HindIII sites of pmcherry-c1, and verified by sequencing. The zyxin ORF was moved from pecfp-zyxin (Hotulainen and Lappalainen, 26) into the EcoRI/BamHI sites of pmcherry-n1 (Clontech) to create pmcherry-zyxin. pegfp-paxillin was created by cloning the human paxillin ORF in frame with EGFP into the BglII/EcoRI sites of pegfp-n1, and verified by sequencing. Supplementary References Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (21). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 6836,

13 Ganley, I.G., Carroll, K., Bittova, L., and Pfeffer, S. (24). Rab9 GTPase regulates late endosome size and requires effector interaction for its stability. Mol. Biol. Cell 12, Hattula, K., and Peranen, J. (2). FIP-2, a coiled-coil protein, links Huntingtin to Rab8 and modulates cellular morphogenesis. Curr. Biol. 24, Hotulainen, P., and Lappalainen, P. (26). Stress fibers are generated by two distinct actin assembly mechanisms in motile cells. J. Cell Biol. 3, Hunziker, W., Harter, C., Matter, K., and Mellman, I. (1991). Basolateral sorting in MDCK cells requires a distinct cytoplasmic domain determinant. Cell 5, Johansson, M., Rocha, N., Zwart, W., Jordens, I., Janssen, L., Kuijl, C., Olkkonen, V.M., and Neefjes, J. (27). Activation of endosomal dynein motors by stepwise assembly of Rab7-RILP-p15Glued, ORP1L, and the receptor betalll spectrin. J. Cell Biol. 4, Wang, F., Wang, W., Wahala, K., Adlercreutz, H., Ikonen, E., and Tikkanen, M.J. (28). Role of lysosomal acid lipase in the intracellular metabolism of LDL-transported dehydroepiandrosteronefatty acyl esters. Am. J. Physiol. Endocrinol. Metab. 6, E