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1 DOI: /ncb2215 Figure S1 Number of egfp-vps4a bursts versus cellular expression levels. The total number of egfp-vps4a bursts, counted at the end of each movie (frame 2000, after 1h 28 min) are plotted as a function of mean number of detected photons(corrected for background and given in photons per pixel) coming from cellular egfp-vps4a molecules. No correlation between the number of bursts and the expression level is observed. Photon counts were normalized from each cell to standard settings of 1 mw laserpower, 295 EM-Gain, 4.9 Pre-Amp, 10 MHz ADC, and are used as a measure of the cellular egfp-vps4a expression level. 1
2 Figure S2 The time dependence of colocalizing and non-colocalizing egfp-vps4a bursts. The number of bursts colocalizing (filled black squares) or non-colocalizing (open grey squares) with HIV mcherry assembly sites are plotted as a function of time. The number of bursts occurring within a 528 s time window (200 frames) were calculated and then evaluated for colocalization with HIV assembly sites (see Methods for details). The mean and s.e.m. from five different cells are shown. 2
3 FigureS3 mcherry-vps4a(e228q) bursts. a Time projection of TIRF microscopy image stacks (3873 s) showing arrested, static bursts of the ATP hydrolysis defective egfp-vps4a(e228q) mutant (green) not colocalizing with HIV mcherry VLPs (magenta) at the cell membrane (overlay). b Arrested bursts detected for mcherry-vps4a(e228q) mutant (magenta) colocalized with egfp-vps4a(wt) protein (green) in cells not expressing HIV mcherrry. The images show overlays of time-projected movies (5285 s). In the wide-field images of each cell, the accumulation of the VPS4A(E228Q) mutant in enlarged endosomal compartments can be seen. In contrast to egfp-vps4a(wt), almost no mcherry-vps4a(e228q) was observed in the cytosol. Scale bars: 1 µm.c Background corrected intensity trace of a Gag. mcherry assembly site together with the corresponding intensity trace of egfp-vps4a(e228q), indicating that there was no burst. The vertical line indicates the start of phase II. 3
4 1: monomeric egfp (2 cells, 11 ROIs) 2: egfp-vps4a(wt) + HIVmCherry (background, 1 cell, 8 ROIs) 3: egfp-vps4a(wt) + HIVmCherry (dark grey, 1 cell, 4 ROIs; background (light grey) FigureS4 egfp-vps4a molecules per burst determined using a SDCM. To verify the average number of egfp-vps4a subunits per burst, we performed a moment analysis on data collected using a spinning disk confocal microscope. The average brightness from cells expressing monomeric egfp was used as a control and the results were normalized to 1 GFP equivalent unit (bar 1). In fixed cells, eight regions of interest were taken where no bursts were visible and the average brightness was calculated (bar 2). The molecular brightness corresponds to monomeric GFP as was the case in Fig. 3b. Four regions of interest were selected that contained a number of bursts, showing an average burst brightness of ~40 GEUs. This corresponds very well with the distribution of burst intensities observed in TIRF (Fig. 3c). 4
5 Figure S5 Analysis of photobleaching on Image Correlation Spectroscopy analysis (ICS). For the ICS analysis, movies of fixed cells transfected with egfp-vsp4a and HIV mcherry were collected. To analyze the affect of photobleaching on the experiment, the same regions of interest for several frames of a single cell movie (3 ROIs) were analysed using ICS. All error bars are given as s.e.m. a The average number of particles within the point-spread function (PSF) and b the quantal brightness of the bright and dim species are shown during the course of the movie. The quantal brightness of the monomeric species remains constant while the brightness of the bursts decreases by only a few percent over 200 frames. Due to the limited photobleaching, there is little change in the number density of the monomeric species. 5
6 Figure S6 Expression level of egfp-vps4a relative to endogenous VPS4A analyzed by immunostaining of VPS4A. The distribution of the VPS4A overexpression levels was determined from 3 different sets of measurements on 58 cells. The histogram relates the ratio of average intensities, obtained from transfected cells labelled by immunostaining, to the average intensities of non-transfected cells. The ratio has mean value of 28 ± 3 (s.e.m.). The average intensity of autofluorescence and non-specific staining was subtracted prior to calculation of this ratio. Transfection conditions were chosen to correspond with the conditions used in the live-cell imaging experiments. The cells were stained with E-8 anti-vps4 monoclonal mouse primary antibody from Santa Cruz Biotechnology (sc ) and goat antimouse secondary antibody from Invitrogen labelled with Alexa 568 dye. 6
7 Supplementary movie legends Movie S1 egfp-vps4a(wt) bursts during HIV mcherry assembly. This movie illustrates the dynamics of egfp-vpsawt bursts (green), which mainly colocalize with assembling HIV mcherry VLPs (magenta) at the membrane of a co-transfected HeLa wt cell (crop from Fig. 1a) measured with a rate of 1 frame per 2.64 s (200 ms acquisition time, ~1.7 mw at 488 nm) over 1 h 30 min and 22 hpt. In the field of view 12 HIV mcherry (46 % of all particles) showed a clear release event after an egfp-vps4a(wt) burst. Thus the quasi efficiency of a burst leading to a release event is 40 %, which is consistent with the number derived from the analysis of all manually tracked particles. Approximately 70 % of the bursts colocalized with HIV particles and around 65 % of these bursts, that colocalized with HIV, showed release during the course of the movie. The movie was binned over 2 frames giving a frame rate of 5.28 s and is displayed at a rate of 30 frames per second (Quicktime, 7.1 MB). Movie S2 Example of a release event after egfp-vps4a(wt) bursts. An assembling HIV mcherry particle (upper crop, trajectory as greenline) as shown in time series of Fig. 1b as well as in the example trace of Fig. 4b that disappears after VPS4A activity indicated by two individual egfp-vps4a(wt) bursts (lower crop, white boxes). The co-transfected HeLa cells were imaged 19 hpt at a rate of 1 frame per 2.64 s (200 ms acquisition time, ~1.0 mw at 488 nm) over 1 h 18 min. The movie was binned over 3 frames giving a frame rate of 7.92 s and is displayed at a rate of 30 frames per second (Quicktime, 3.6 MB). Movie S3 Cell expressing egfp-vps4(wt) alone. This movie shows a HeLa cell expressing egfp-vps4a(wt) in the absence of HIV expression. It was acquired 17 hpt at a rate of 1 frame per 2.64 s (200 ms acquisition time, ~1.1 mw at 488 nm) over a total duration of 58 min. A binning of 4 frames, corresponding to a frame rate of s was applied and runs at 30 frames per second (Quicktime, 7.7 MB). The intensity scaling is different than in Movie 1 to show details of the cytosolic VPS4A in the absence of bursts. Movie S4 egfp-vps4a(wt) with HIV mcherry (late-). HIV mcherry (late-) assembles to VLPs (magenta) without showing significant recruitment of egfp-vps4a(wt) bursts (green). The movie, referring to Fig. 1d, was acquired 22 hpt at a rate of 1 frame per 2.64 s (200 ms acquisition time, ~1.5 mw at 488 nm) over 1 h 32 min. For display purposes, only frames 400 to 2000 (18 min to 1 h 28 min) of the movie were used and it was further shortened by a frame binning of 2, corresponding to a frame rate of 5.28 s. It runs at a rate of 30 frames per second (Quicktime, 7.8 MB). Movie S5 HIV mcherry in cells expressing egfp-vps4a(wt) versus cells expressing egfp-vps4(e228q) Regions of interest showing the assembly of nascent HIV mcherry particles when co-transfected with egfp-vps4a(wt) (upper half) or egfp-vsp4a(e228q) (lower half). Towards the end of the movie, the more dynamic nature of the HIV complexes in the presence of egfp-vsp4wt is clearly observable. Data were collected at 1 frame per 2.64 s (200 ms acquisition time, ~1.7 mw at 488 nm) over 1 h 30 min and 22 hpt for egpf-vsp4awt and at 1 frame per 2.64 s (200 ms acquisition time, ~1.6 mw at 488 nm) over 1 h 05 min and 22 hpt for egfp-vsp4a(e228q). The movie was binned over 2 frames giving a frame rate of 5.28 s and is displayed at a rate of 30 frames per second (Quicktime, 7.2 MB). 7
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