Supplemental Information. A Versatile Tool for Live-Cell Imaging. and Super-Resolution Nanoscopy Studies. of HIV-1 Env Distribution and Mobility

Transcription

1 Cell Chemical Biology, Volume 24 Supplemental Information A Versatile Tool for Live-Cell Imaging and Super-Resolution Nanoscopy Studies of HIV-1 Env Distribution and Mobility Volkan Sakin, Janina Hanne, Jessica Dunder, Maria Anders-Össwein, Vibor Laketa, Ivana Nikic, Hans-Georg Kräusslich, Edward A. Lemke, and Barbara Müller

2 Supplementary data Figure S1: Amber suppression in HEK293T cells using an optimized test construct. Extended data to main Figure 1. Cells were transfected with pmcherryegfp Y39TAG. Where indicated (+), ptrna pyl /pylrs AF was co-transfected and 250 µm BCN-Lys was added to the growth medium. At 40 h.p.t. cell lysates were harvested and analyzed by quantitative immunoblotting using polyclonal rabbit antiserum raised against recombinant egfp. Band intensities were quantitated; numbers given below the lanes indicate the proportion of full length protein band intensity determined relative to the total intensity of antibody reactive bands.

3 Figure S2: Quantitative comparison of Env (WT) and Env407 ncaa amounts on the plasma membrane by confocal microscopy. Extended data to main Figures 2 and 5. (A) The experiment was performed as in Figure 5C; cells were grown in the presence of BCN-Lys. Images were recorded by SDCM. Identical settings were used for recording of all images and image representation to allow for visual comparison of

4 staining intensities. Shown are three examples each for Env407 ncaa (rows 1-3) and Env(wt) (rows 4-6) expressing cells, representing the observed range of staining intensities on the plasma membrane on individual cells in the respective samples. Cyan, Hoechst staining; magenta, AlexaFluor488; yellow, H-Tet-Cy5. Scale bars: 10 m. (B) Quantification of the intensity of Env(wt) and Env407 ncaa immunostaining on the plasma membrane. Each dot represents a mean fluorescence intensity of the Alexa488 signal for all cells in a microscopic field of view; the magenta line shows the median fluorescence intensity for all fields analyzed. To measure the mean fluorescence intensity per field of view, the systematic camera offset (offset=2100) was subtracted, followed by automated segmentation of the Alexa488 signal using Niblack algorithm (radius = 10px, parameter 1 = 0, parameter 2 = -40) and FIJI image analysis software. Large unspecific clusters of Alexa488 not associated with cells were filtered out manually. A minimum of 20 images comprising >200 cells in total were analysed per condition. Statistical significance was assessed using a Mann- Whitney U-test (p-value < ).

5 Figure S3: Quantitative comparison of Env (WT) and Env407 ncaa amounts on the plasma membrane analyzed by flow cytometry. Extended data to main figure 2. HEK293T cells were co-transfected with pcdna3.1, penv(wt) or penv407 TAG together with ptrna pyl /pylrs AF and perf1 (E55D) in the presence or absence of 250 M BCN-Lys. At 40 h.p.t., cells were resuspended in PBS, fixed briefly (PFA, 10 min at RT) and immunostained using gp120 antibody 2G12 followed by AlexaFluor488- coupled secondary antibody. Cell-associated fluorescence was measured for

6 cells in each sample by flow cytometry using a BD FACSVerse TM instrument, and data were analyzed using FlowJo (FlowJo, LLC, USA) software. AlexaFluor488 staining was detected in the FITC-A channel. The figure shows dot plots from one representative experiment; the percentages and the mean fluorescence intensity (MFI) values of the gated populations are highlighted. Three independent experiments yielded mean MFI values of 7718+/-1564 and 5642+/-1745 for Env WT and Env 407 ncaa, respectively.

7 Figure S4: HIV-1 particles carrying engineered Env BCN-Lys retain single-round infectivity on TZM-bl cells (A) HEK293T cells were co-transfected with the proviral plasmid pnl4-3 Env-), the indicated Env TAG expression plasmids together with ptrna pyl /pylrs AF (+) or empty vector (-) as indicated and grown in the presence or absence of 250 µm BCN-Lys. At 40 h.p.t., virus particles were concentrated from the tissue culture supernatant by ultracentrifugation through a 20% (w/v) sucrose cushion. Relative infectivity was analyzed by titration on TZM-bl indicator cells as described in STAR methods. Values were normalized to the amount of CA protein determined by quantitative immunoblotting. The graph shows mean values and SEM from three independent experiments. (B-C) Immunoblot analysis of samples. At 40 h.p.t. cell lysates were harvested and viral particles were pelleted from the tissue culture supernatant by ultracentrifugation through a sucrose pellet. Immunoblot analysis of cell lysates (B) and virus particles (C) was performed using the indicated

8 polyclonal antisera. The volumes of cell lysate loaded in B were adjusted to account for different expression levels (variant samples 1-6: wt samples 7-10 = 2.5: 1). Numbers above the lanes refer to the experimental conditions shown in A. See main Figure 4 for an experiment carried out in the presence of erf1 (E55D)

9 Figure S5: Structures of non-canonical amino acids and tetrazinefunctionalized dyes used in this study. (A) Non-canonical amino acids: endo Bicyclo [6.1.0] nonyne-l-lysine (BCN-Lys), strained cyclooctyne-l-lysine (SCO-Lys) and axial trans-cyclooct-2-ene-l-lysine (TCO*-Lys). (B) Tetrazine-functionalized dyes: 3-(p-Benzylamino)-1,2,4,5-tetrazine-Cy5 (H-Tet-Cy5) and H-Tet-KK114 (Sednev MV et al., Bioconjug Chem 24: , 2013)

10 Figure S6: Click-labeling of Env407 ncaa variants analyzed by in-gel fluorescence. Complete images corresponding to the cropped images shown as main Figure 5A analyzed by in-gel fluorescence (A) and immunoblotting (B). Background observed by in-gel fluorescence was low, with exception of a band with an apparent molecular mass of ~48 kda. Immunoblot analysis (B) using polyclonal rat antiserum raised against recombinant PylRS and secondary antibody IRDye 680 rat IgG (red), together with polyclonal gp120 antibody and secondary antibody IRDye 800CW donkey rabbit IgG (green) suggested that the additional band observed by in-gel fluorescence corresponded to the overexpressed orthogonal PylRS, presumably released from dead cells, bound to Cy5-coupled ncaa.

11 Figure S7: Individual recovery curves of FRAP analysis of click-labeled Env at the plasma membrane. HEK293T cells were co-transfected with penv407 TAG, ptrna pyl /pylrs AF and perf1 (E55D) and grown in the presence of 250 M BCN-Lys. At 40 h.p.t. cells were labeled with 2 M H-Tet-Cy5 as described in STAR methods and live-cell SDCM imaging was performed. Initial movie sequences were recorded for 3 s, followed by photobleaching. Subsequently, fluorescence recovery was recorded for 10 s. The recovery curves show the fluorescence intensities recorded over time after photobleaching from five different cells. Fluorescence recovery data were fitted to a single exponential association equation (magenta curves). The halftimes and mobile fractions were calculated from the exponential fits. Main Figure 6B shows averaged data from these individual measurements.

12 Supplementary movie S1: Time lapse microscopy of the fluorescence recovery of click labeled HIV-1 Env. The full movie is for the same experiment described in main Figure 6A. The video is played with a rate of 20 frames/s.