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. 2010 Nov;11(11):1401-14.
doi: 10.1111/j.1600-0854.2010.01111.x.

HIV-1 assembly differentially alters dynamics and partitioning of tetraspanins and raft components

Dimitry N Krementsov  1 , Patrice Rassam, Emmanuel Margeat, Nathan H Roy, Jürgen Schneider-Schaulies, Pierre-Emmanuel Milhiet, Markus Thali
Affiliations

HIV-1 assembly differentially alters dynamics and partitioning of tetraspanins and raft components

Dimitry N Krementsov et al. Traffic. 2010 Nov.

Abstract

Partitioning of membrane proteins into various types of microdomains is crucial for many cellular functions. Tetraspanin-enriched microdomains (TEMs) are a unique type of protein-based microdomain, clearly distinct from membrane rafts, and important for several cellular processes such as fusion, migration and signaling. Paradoxically, HIV-1 assembly/egress occurs at TEMs, yet the viral particles also incorporate raft lipids. Using different quantitative microscopy approaches, we investigated the dynamic relationship between TEMs, membrane rafts and HIV-1 exit sites, focusing mainly on the tetraspanin CD9. Our results show that clustering of CD9 correlates with multimerization of the major viral structural component, Gag, at the plasma membrane. CD9 exhibited confined behavior and reduced lateral mobility at viral assembly sites, suggesting that Gag locally traps tetraspanins. In contrast, the raft lipid GM1 and the raft-associated protein CD55, while also recruited to assembly/budding sites, were only transiently trapped in these membrane areas. CD9 recruitment and confinement were found to be partially dependent on cholesterol, while those of CD55 were completely dependent on cholesterol. Importantly, our findings support the emerging concept that cellular and viral components, instead of clustering at preexisting microdomain platforms, direct the formation of distinct domains for the execution of specific functions.

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Figures

Figure 1
Figure 1. Clustering of PM CD9 by Gag in live cells
Live HeLa cells were labeled on ice using full-length anti-CD9 antibody and full-length secondary (A) or Fab anti-CD9 and Fab secondary (B) then imaged at 37°C. C) HeLa cells expressing NL4-3(MA-mRFP) were labeled with Fab anti-CD9 and Fab secondary on ice, then imaged at 37°C. D) Human CD4+ T cells infected with NL4-3(MA-GFP) were labeled as in (C). E) HeLa cells expressing Venus-fused monomeric Gag were labeled and imaged as in (C). Bottom optical Z-sections (i.e. cell–coverslip contact) are shown, except in (D), which shows middle sections. Images in (A and B) second column represent a 10× magnification of the boxed region. Scale bar represents 10 μm.
Figure 2
Figure 2. Gag-CD9 colocalization is increased at later time-points and at higher Gag expression levels
HeLa cells transfected with GagGFP were fixed at 4 or 8 h posttransfection, then surface labeled for CD9 and processed for colocalization analysis. A) Average colocalization of Gag and CD9 at 4 versus 8 h posttransfection (data represent the mean of 10 or more cells, error bars represent SEM) (see Materials and Methods). B) Colocalization data for individual cells from 4-, 8- and 16-h time-points were pooled and plotted as a function of mean Gag intensity. Each point represents a single cell – the cells displayed in (D) and (E) are indicated with arrows. A best fit line obtained using linear regression is displayed. C) HeLa cells expressing GagGFP were imaged continuously for 5 h at 37°C, one frame/10 min. The mean GFP intensity of a representative cell is displayed. Representative bottom sections of cells included for the analysis in (A), exhibiting low (26%, GFP intensity = 2755) and high (70%, GFP intensity = 4823) colocalization, are shown in (D) and (E), respectively. Gag intensity was scaled differently between (D) and (E), in order to enhance visualization of the weaker signals in (D). Images in right hand column represent 9.5× magnifications of the boxed area. Scale bar represents 10 μm. AU, arbitrary units.
Figure 3
Figure 3. Gag expression alters the mobility of CD9
Representative FRAP analyses of untransfected Vero cells (A) or Vero cells expressing GagGFP (B) and (C) and labeled for surface CD9 with Fab primary and secondary. The cell shown in (C) expresses a higher amount of Gag than the cell in (B). The bleach ROIs are indicated with a circle. Images in right column in (B) and (C) represent a 3× magnification of a region surrounding the bleach ROI. Note that the bleach areas in (B) and (C) are different sizes. Scale bar represents 10 μm. D) Average recovery curves for CD9 in the absence of Gag [at planar membranes, red, and at microvilli (MV), blue] or in the presence of Gag (green). Values indicate mean fractional fluorescence ± SEM.
Figure 4
Figure 4. Mobility of GM1 is not altered by Gag
Representative FRAP analyses of untransfected Vero cells (A) or Vero cells expressing GagGFP (B) and labeled for surface GM1 with CTxB-Alexa 647. The bleach ROIs are indicated with a circle. Images in right column in (B) represent a 3× magnification of a region surrounding the bleach ROI. Scale bar represents 10 μm. C) Average recovery curves for GM1 in the absence (red) or in the presence of Gag (green). Values indicate mean fractional fluorescence ± SEM.
Figure 5
Figure 5. Single-molecule analysis of CD9, CD55 and CD46 behavior in the presence or absence of Gag
Upper panel: Micrographs on the left show superimposition of GFP and GagGFP fluorescence signal with several representative single molecule trajectories (white lines) obtained after tracking Atto647N-conjugated Fab fragments of anti-CD9, anti-CD46 or anti CD55 antibodies in HeLa cells. Histograms on the right represent the percentage of each diffusion mode (Brownian, mixed and confined) relative to the total number of trajectories for CD9, CD46 or CD55 for cells transfected with GFP (white bars) or with GagGFP (black bars). Error bars correspond to SEM. * and *** respectively indicate p values below 0.01 and 0.0001 for comparison of the diffusion modes percentage in GagGFP-expressing cells versus GFP-expressing control cells for CD9, CD46 and CD55, as determined by the Mann–Whitney U-test. Lower panel: The micrograph on the left shows CD9 representative trajectories with Brownian (B), mixed (M) or confined (C) behavior (white lines) superimposed with the GagGFP fluorescence ensemble labeling. Histograms on the right represent the percentage of trajectories that colocalized with Gag-enriched areas (see Materials and Methods) as well as the proportion of the different diffusion modes displayed at these Gag-enriched areas: confined (crosshatched bars), mixed (hatched bars) and Brownian (dotted bars).
Figure 6
Figure 6. Influence of cholesterol on Gag-induced clustering of membrane components
Colocalization fluorescence experiments between CD9, CD46 or CD55 (in red) with Gag-enriched areas (in green) in Gag-expressing HeLa cells treated (D, E, F) or not (A, B, C) with COase. Histograms (G) represent the percentage of colocalization between CD9, CD46 or CD55 with Gag-enriched areas (defined as the number of yellow pixels over the total number of yellow and green pixels) in HeLa cells treated (gray bars) or not (black bars) with COase. *** indicates p values below 0.0001 for comparison of the percentage of colocalization in GagGFP-expressing cells treated with COase versus GagGFP-expressing control cells for CD9, CD46 and CD55, as determined by the Mann–Whitney U-test.
Figure 7
Figure 7. Influence of cholesterol on Gag-induced trapping of CD9, CD46 and CD55 at the single-molecule level
The left panels represent the distribution of all the apparent diffusion coefficients calculated from the MSD-τ plot for CD9, CD46 and CD55 in GFP-or Gag-GFP-expressing cells, treated or not with COase. Each point represents one trajectory. The black arrowheads highlight the increase of the confined trajectories. The histograms on the right represent the percentage of each diffusion mode (Brownian, mixed and confined) relative to the total number of trajectories for CD9, CD46 or CD55 for cells transfected with GFP (white bars) or with Gag-GFP (black bars), treated or not with COase. *, ** and *** respectively indicate a p value below 0.01, 0.001 and 0.0001 for comparison of the apparent diffusion coefficient (left panel) or percentage of diffusion modes (right panel) in GagGFP-expressing cells versus GFP-expressing control cells for CD9, CD46 and CD55, as determined by the Mann–Whitney U-test.
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