Supplementary Materials for

Transcription

1 advances.sciencemag.org/cgi/content/full/3/6/e /dc1 Supplementary Materials for HIV virions sense plasma membrane heterogeneity for cell entry Sung-Tae Yang, Alex J. B. Kreutzberger, Volker Kiessling, Barbie K. Ganser-Pornillos, Judith M. White, Lukas K. Tamm The PDF file includes: Published 28 June 2017, Sci. Adv. 3, e (2017) DOI: /sciadv fig. S1. Association of CD4 and CCR5 with lipid rafts in CD4 + /CCR5 + HeLa cells with stably expressed CD4 and CCR5. fig. S2. Formation of GPMVs by treatment of CD4 + /CCR5 + HeLa cells with small amounts of formaldehyde and DTT. fig. S3. Partitioning of CD4 and CCR5 in GPMVs induced by NEM instead of formaldehyde and DTT from CD4 + /CCR5 + cells. fig. S4. HIV Env pseudovirus particles bind preferentially at boundaries between coexisting Lo and Ld domains in GPMVs. fig. S5. VSV-G pseudovirus particles bind to Ld membrane regions in GPMVs. fig. S6. Electron cryo-micrographs of HIV Env particles bound/fused to GPMVs. fig. S7. Modulation of lipid phases does not affect binding of HIV to GPMVs. fig. S8. Preparation of SPPMs with GPMVs. fig. S9. Lateral distribution of CD4/CCR5 in SPPMs. Legends for movies S1 to S9 Other Supplementary Material for this manuscript includes the following: (available at advances.sciencemag.org/cgi/content/full/3/6/e /dc1) movie S1 (.avi format). Growing GPMVs on cell surfaces. movie S2 (.avi format). Coexisting fluid phases of cell-attached GPMVs. movie S3 (.avi format). HIV binding to cell-attached GPMVs. movie S4 (.avi format). HIV binding to the Lo/Ld boundaries in cell-attached blebs. movie S5 (.avi format). HIV binding to the Lo/Ld boundaries in cell-detached GPMVs.

2 movie S6 (.avi format). Influence of MβCD on lipid phases of GPMVs. movie S7 (.avi format). Influence of lysosm on lipid phases of GPMVs. movie S8 (.avi format). Influence of lysosm on lipid phases of GUVs. movie S9 (.avi format). Fusion of HIV Env particles at Lo/Ld boundaries in an SPPM.

3 fig. S1. Association of CD4 and CCR5 with lipid rafts in CD4 + /CCR5 + HeLa cells with stably expressed CD4 and CCR5. CD4 and CCR5 are visualized at room temperature by the immunostaining and examined for colocalization with the raft-resident ganglioside GM1. CD4 + /CCR5 + HeLa cells were stained with 10 μg/ml Alexa Fluor 555-conjugated CtxB, which specifically binds to GM1, at 4C for 60 min, followed by treatment with 10 μg/ml Alexa Fluor 488-labeled anti-cd4 (first row) antibody or with Alexa Fluor 647-labeled anti-ccr5 (second row) antibody at 4C for 60 min. Scale bars are 10 m.

4 fig. S2. Formation of GPMVs by treatment of CD4 + /CCR5 + HeLa cells with small amounts of formaldehyde and DTT. (A) Bright-field micrographs showing the growth of GPMVs on the surface of CD4 + /CCR5 + cells. These are snapshots of the movie S1. (B) Example of cell-detached GPMVs. GPMVs were detached from the cells by gentle shaking at 37 C. (C and D) Fluorescence micrographs of phase separated GPMVs. Cell-attached blebs (C) or cell-detached GPMVs (D) were labeled with the Ld-phase marking lipid probe DiI and visualized by epifluorescence microscopy. See also movie S2. (E) Temperature-dependence of Lo/Ld phase separation of GPMVs. Most GPMVs show large scale phase separation below 25C. The fraction of phase-separated GPMVs (n=30) was quantified at various temperatures. Data are mean s.e.m. (n = 3). Scale bars are 10 μm.

5 fig. S3. Partitioning of CD4 and CCR5 in GPMVs induced by NEM instead of formaldehyde and DTT from CD4 + /CCR5 + cells. GMPVs were stained with DiI-C12 at 4C for 60 min and subsequently labeled with fluorescent CtxB (Alexa Fluor 488), anti-cd4 (Alexa Fluor 488) or anti-ccr5 (Alexa Fluor 647) antibodies at 4C for 60 min. Epifluorescence images taken at 10 C show large-scale fluid/fluid phase coexistence. The overlay images show that CD4 is localized predominantly in Lo domains, whereas CCR5 accumulates at Lo/Ld phase boundaries. Scale bars are 10 m.

6 fig. S4. HIV Env pseudovirus particles bind preferentially at boundaries between coexisting Lo and Ld domains in GPMVs. (A) A cell-attached GPMV visualized by bright-field microscopy. (B) A spherical GPMV imaged in top and equatorial views. The GPMVs (left panels) and HIV Env particles (center panels) were stained with DiO or contained mko-gag, respectively, and were visualized by epifluorescence microscopy. Merged images (right panels) show virions bound to Lo/Ld domain boundaries. Images were taken after 60 min of incubation at 4C. Note that cell-attached GPMVs were immobilized on glass to facilitate monitoring of HIV binding, but show not as much contrast due to the much higher fluorescence of the cell bodies. See also movie S4. Scale bars are 10 μm.

7 fig. S5. VSV-G pseudovirus particles bind to Ld membrane regions in GPMVs. (A) VSV-G particle binding to cell-attached GPMVs. R18-labeled pseudotyped VSV-G virions were incubated with GPMVs that were stained with the fluorescent lipid dye DiO. Images were taken after 60 min of incubation at 4C. (B) VSV-G particle binding to cell-detached GPMVs. A spherical GPMV imaged in top and equatorial views. Merged images (right panel) show most VSV-G particles bound to Ld regions in GPMVs. (C) Quantification of VSV-G particles bound to three different regions (Lo, Ld, and Lo/Ld boundary) of the GPMVs. Data are mean s.d. of triplicates. A total of 152 particles were analyzed for their distribution between the three compartments. The largest fraction of VSV-G particles was found in the Ld regions. Scale bars are 10 m.

8 fig. S6. Electron cryo-micrographs of HIV Env particles bound/fused to GPMVs. Contact and/or fusion intermediates (A and B) and full fusion (C) were obtained after incubation for 60 sec at room temperature before the samples were rapidly frozen. V and G indicate the virus and GPMV, respectively. Note that virus particles with a core or capsid are distinguishable from GPMVs. Scale bars are 100 nm.

9 fig. S7. Modulation of lipid phases does not affect binding of HIV to GPMVs. (A) Schematic diagram of the bulk assay for binding of HIV Env particles to GPMVs. To quantify HIV binding to GPMVs, R18- labeled virions were incubated with GPMVs stained with DiO for 60 min at 4C. GPMVs (with or without bound virions) are isolated from unbound virions by low speed centrifugation. Pellets were resuspended in buffer containing 0.1% Triton X-100 and the fractions bound were estimated from the fluorescence intensity ratios of R18 (HIV) to DiO (GPMV). (B) Fractions of bound HIV Env particles using GPMVs with and without receptors and with and without cholesterol. (C) Fractions of bound HIV Env particles using GPMVs with and without lysosm, lysopc, SCDase, and PLA2. Data are mean s.e.m. (n = 3).

10 fig. S8. Preparation of SPPMs with GPMVs. (A and B) Representative epifluorescence images of phase-separated SPPMs stained with DiI (A) and Alexa 555 CtxB (B). Polymer-supported monolayers were transferred from the water-air interface of a Langmuir trough to substrates and GPMVs were added to the monolayers to build supported planar plasma membranes. (C and D) Lateral mobility of DiI and CtxB in SPPMs. The images show the fluorescence recovery with time after photobleaching of DiI (C) and CtxB (D) in the membranes. The FRAP kinetics were used to determine the diffusion coefficients (D) and mobile fractions (mf) indicated below the images. Data are mean s.d. (n = 3). Scale bars are 10 m.

11 fig. S9. Lateral distribution of CD4/CCR5 in SPPMs. (A) The SPPMs were stained with the fluorescent lipid dye DiI (left panels) and incubated with fluorescent labeled anti-cd4 (Alexa Fluor 488) or anti-ccr5 (Alexa Fluor 647) antibodies (middle panels) at 4C for 60 min. The overlay images (right panels) show that CD4 favors Lo domains, whereas CCR5 preferentially localizes to Lo/Ld domain boundaries. Scale bars are 10 m. (B) Relative partitioning of receptors and coreceptors in phaseseparated membranes is analyzed by the ratio of fluorescence intensity in the Lo, Lo/Ld boundary, and Ld phases. Data are mean s.d. of triplicates.

12 movie S1. Growing GPMVs on cell surfaces. GPMVs were formed by treatment of formaldehyde and DTT from CD4 + /CCR5 + HeLa cells and imaged by light microscopy. Images were acquired every 5 sec for 670 sec. Each frame is 50 x 50 m 2. movie S2. Coexisting fluid phases of cell-attached GPMVs. GPMVs were induced from CD4 + /CCR5 + cells labeled with DiI and visualized in the z direction by epifluorescence microscopy. Images were acquired every 0.2 sec for 18.6 sec. Each frame is 50 x 50 m 2. movie S3. HIV binding to cell-attached GPMVs. Cell-attached GPMVs (left) were incubated with HIV Env particles labeled with mko-gag (center) and visualized by bright field (still image) and epifluorescence microscopy, respectively. Image overlay (right) shows that virions are sitting on the GPMV surface. Images were acquired every 1 sec for 168 sec. Each frame is 3 times (42 x 42) m 2. movie S4. HIV binding to the Lo/Ld boundaries in cell-attached blebs. Blebs (left) and HIV Env particles (right) were labeled with DiO and mko-gag, respectively, and monitored by epifluorescence microscopy. Bound virions are seen moving along and with the Lo/Ld phase boundary on the bleb surface. Images were acquired every 0.2 sec for 7.6 sec. Each frame is 2 times (25 x 25) m 2. movie S5. HIV binding to the Lo/Ld boundaries in cell-detached GPMVs. GPMVs and HIV particles were labeled with DiI and mko-gag, respectively, and observed by epifluorescence microscopy. Bound virions are seen to moving along and with the Lo/Ld phase boundary on the GPMV surface. Images were acquired every 0.1 sec for 53.7 sec. Each frame is 20 x 20 m 2. movie S6. Influence of MβCD on lipid phases of GPMVs. 5 mm MβCD was added to GPMVs stained with DiI. Images were acquired every 0.1 sec for 75.6 sec. Each frame is 50 x 50 m 2. movie S7. Influence of lysosm on lipid phases of GPMVs. 5 μm lysosm was added to GPMVs stained with DiI. Images were acquired every 0.2 sec for 95.8 sec. Each frame is 50 x 50 m 2. movie S8. Influence of lysosm on lipid phases of GUVs. 5 μm lysosm was added to GUVs composed of bsm/bpc/bps/cholesterol (2:1:1:1). Images were acquired every 0.2 sec for sec. Each frame is 62 x 62 m 2. movie S9. Fusion of HIV Env particles at Lo/Ld boundaries in an SPPM. Lipid phase separation in the SPPM labeled with DiI (left) was visualized by epifluorescence microscopy and fusion events of individual HIV particles labeled with the lipid dye DiD (center) were monitored by TIRF microscopy. Image overlay (right) shows that virions (green) fuse with SPPM (red) at the Lo/Ld boundaries. Images were acquired every 2 sec for 476 sec. Each frame is 3 times (45 x 40) m 2.