Mapping of tetraspanin-enriched microdomains that can function as gateways for HIV-1 - PubMed (original) (raw)

Mapping of tetraspanin-enriched microdomains that can function as gateways for HIV-1

Sascha Nydegger et al. J Cell Biol. 2006.

Abstract

Specific spatial arrangements of proteins and lipids are central to the coordination of many biological processes. Tetraspanins have been proposed to laterally organize cellular membranes via specific associations with each other and with distinct integrins. Here, we reveal the presence of tetraspanin-enriched microdomains (TEMs) containing the tetraspanins CD9, CD63, CD81, and CD82 at the plasma membrane. Fluorescence and immunoelectron microscopic analyses document that the surface of HeLa cells is covered by several hundred TEMs, each extending over a few hundred nanometers and containing predominantly two or more tetraspanins. Further, we reveal that the human immunodeficiency virus type 1 (HIV-1) Gag protein, which directs viral assembly and release, accumulates at surface TEMs together with the HIV-1 envelope glycoprotein. TSG101 and VPS28, components of the mammalian ESCRT1 (endosomal sorting complex required for transport), which is part of the cellular extravesiculation machinery critical for HIV-1 budding, are also recruited to cell surface TEMs upon virus expression, suggesting that HIV-1 egress can be gated through these newly mapped microdomains.

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Figures

Figure 1.

Figure 1.

Identification of CD63-enriched microdomains at the surface of HeLa cells. HeLa cells were stained with an anti-CD63 antibody after fixation and permeabilization (A; a middle section is shown) or after fixation without permeabilization (B [middle and bottom sections are shown] and C [a bottom section is shown]), followed by incubation with a fluorophore-conjugated secondary antibody. The selective staining of surface CD63 (B and C) allows visualization of discrete microdomains where this antigen clusters at the plasma membrane. Bar, 10 μm. (C, right) Sevenfold-magnified view of the boxed region in the cell shown in the left panel. Bar, 1 μm.

Figure 2.

Figure 2.

CD9, CD63, CD81, and CD82 cocluster in surface TEMs. To identify surface TEMs containing three of the tetraspanins analyzed here, HeLa cells were surface stained as described in Materials and methods. Insets show sixfold-magnified views of the boxed region in the cell. The percentages of TEMs containing one (30 ± 9), two (24 ± 7), or three (46 ± 14) of the tetraspanins visualized in these triple stainings were assessed as described in Materials and methods (n > 300). Bar, 10 μm.

Figure 3.

Figure 3.

Quantitative analysis of surface TEM composition. Cells were triple stained with anti-tetraspanin antibodies as described in the legend for Fig. 2, and the relative presence in surface TEMs of CD9, CD63, CD81, and CD82 was analyzed as described in Materials and methods. (A) A representative micrograph reveals that the relative contribution of the tetraspanins to the build-up of individual TEMs varies. (B) Statistical analyses confirming the stochastic nature of the tetraspanin contributions to TEM formation. Each bar represents a single TEM that showed staining for three of the above tetraspanins. The relative contribution of each tetraspanin was determined as described in Materials and methods. The bars were sorted in ascending order according to CD63 signal. Bar, 0.5 μm.

Figure 4.

Figure 4.

Visualization of CD63- and CD9-enriched microdomains by immuno-EM. HeLa cells were incubated with either an anti-CD63 or an anti-CD9 antibody or a combination of both antibodies and processed as described in Materials and methods. Representative electron micrographs of tetraspanin immunogold labeling associated with discrete membrane microdomains on HeLa cell plasma membrane are shown for cells stained for CD63 (A), CD9 (B), or both tetraspanins (C and D). CD9- and CD63-associated gold particles (20 and 10 nm, respectively) are seen by transparency. CD63 clusters are encircled with dashed lines and are marked with arrows in D; CD9 clusters are encircled with dotted lines, and microdomains containing both antigens are encircled by dashed/dotted lines. Bars, 0.5 μm.

Figure 5.

Figure 5.

Distribution of surface TEMs relative to influenza virus HA-enriched microdomains. HeLa cells expressing HA were surface stained for HA and CD63 (A) or CD9 (B). Insets show sixfold-magnified views of the boxed region in the cell. Colocalization analysis (see Materials and methods) showed that 7.7 ± 5.3% of CD63 and 9.7 ± 5.8% of CD9 colocalized with HA. Bar, 10 μm.

Figure 6.

Figure 6.

HIV-1 Env and Gag colocalize with surface CD63. (A) HeLa cells expressing full-length HIV-1 were surface stained for CD63 and HIV-1 Env. (B) To visualize Gag, cells were subsequently permeabilized and treated with an anti-Gag antibody followed by incubation with a fluorophore-conjugated secondary antibody. Bottom sections and middle sections are shown for each cell. Bar, 10 μm.

Figure 7.

Figure 7.

Immuno-EM analysis of HIV-1 Gag accumulation at surface TEMs containing CD9 or CD63. HeLa cells were incubated with either an anti-CD63 or an anti-CD9 antibody, and cells were treated as described for Fig. 4. CD9- and CD63-associated gold particles (10 nm) and Gag-associated gold particles (15 nm) are seen by transparency. Representative electron micrographs of HIV-1 Gag immunogold labeling associated with discrete plasma membrane microdomains are shown for cells stained for either CD63 or CD9. Bar, 0.2 mm.

Figure 8.

Figure 8.

TSG101 and VPS28 are recruited to CD63-containing surface TEMs where Gag clusters. (A) HeLa cells expressing HIV-1 Gag, TSG101-YFP, and VPS28-CFP were fixed, permeabilized, and stained for Gag. (B) HeLa cells expressing HIV-1 Gag, TSG101-GFP, and FLAG-VPS28 were fixed, surface stained for CD63, permeabilized, and stained for Gag. Bottom sections are shown for each cell. Blow-ups show 11-fold–magnified views of the boxed region in the cell. Bar, 10 μm.

Figure 9.

Figure 9.

Quantitative analysis of viral Env and Gag colocalization with tetraspanins. HeLa cells expressing HIV Gag and Env individually, together but expressed from different plasmids, or coexpressed from a proviral vector were incubated with anti-Env and anti-CD63 (A) or anti-CD9 (B) antibodies without permeabilization. After fixation, the cells were permeabilized and incubated with an anti-Gag antibody and with fluorophore-conjugated secondary antibodies. Fluorescent images were captured and analyzed for colocalization (see Materials and methods). At least 10 cells were analyzed for each condition. The bar diagrams summarize the data from that analysis. The percentage of Gag or Env that colocalizedwith either CD63 or CD9 is shown. White bars show Gag or Env colocalization with CD63 or CD9 if the viral antigens were expressed separately, gray bars show Gag or Env colocalization with CD63 or CD9 if the viral antigens were coexpressed from separate plasmids, and black bars show Gag or Env colocalization with CD63 or CD9 if the viral antigens were coexpressed from a proviral plasmid. Error bars indicate SD.

Figure 10.

Figure 10.

HIV-1 at surface TEMs in Jurkat T lymphocytes. Jurkat T lymphocytes were surface stained for CD9 and CD63 (A). Surface TEMs containing CD9 and CD63 are comparable in size, and their distribution is similar to the one in HeLa cells (Fig. 2). (B and C) Jurkat T lymphocytes transfected with HIV-1 provirus–carrying plasmids and thus producing infectious virus were surface stained for Env and CD63 or CD9 to visualize virus and surface TEMs, respectively. Bar, 5 μm.

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