CD317/tetherin is an organiser of membrane microdomains

Abstract

The integral membrane protein tetherin has been associated with an eclectic mix of cellular processes, including restricting the release of a range of enveloped viruses from infected cells. The unusual topology of tetherin (it possesses both a conventional transmembrane domain and a glycosylphosphatidylinositol anchor), its localisation to membrane microdomains (lipid rafts) and the fact that its cytosolic domain can be linked (indirectly) to the actin cytoskeleton, led us to speculate that tetherin might form a 'tethered picket fence' and thereby play a role in the organisation of lipid rafts. We now show that knocking down expression of tetherin leads to changes in the distribution of lipid raft-localised proteins and changes in the organisation of lipids in the plasma membrane. These changes can be reversed by re-expression of wild-type tetherin, but not by any of a range of tetherin-based constructs, indicating that no individual feature of the tetherin sequence is dispensable in the context of its lipid raft organising function.

Keywords: Laurdan; Lipid raft; Membrane; Tetherin.

Fig. 1.

Tetherin knockdown disrupts raft localisation of membrane proteins. (A) Left: Immunoblot of HeLa cell lysates for CD317/tetherin or lamin A/C after treatment with the indicated siRNA for 48 hours. Right: Immunoblot of distribution of endogenous tetherin in sucrose density gradient fractions from ice-cold TX100 extracts of HeLa cells. (B) Representative immunoblots showing distribution of H-Ras-GFP or transferrin receptor (TfR) (as indicated) in sucrose density gradient fractions following Na₂CO₃ extraction from HeLa cells in which expression of lamin A/C has been knocked down (control KD) or in HeLa cells in which expression of tetherin has been knocked down (CD317 KD) using siRNA. The histogram shows the results of densitometric analysis of the band corresponding to H-Ras-GFP in each of the fractions from the sucrose gradient (n = 3). (C) Representative immunoblots showing distribution of flotillin 2 in sucrose density gradient fractions from ice-cold TX100 extracts of HeLa cells in which expression of lamin A/C has been knocked down (lamin KD), HeLa cells in which expression of tetherin has been knocked down (CD317 KD) and tetherin knockdown HeLa cells expressing siRNA-resistant wild-type tetherin (WT-HuCD317-HA SR; WT rescue). Numbers indicate fraction numbers; P, pellet. The histogram shows the results of densitometric analysis of the band corresponding to flotillin 2 in fractions 1–4 (raft) and fractions 5–12 (non-raft) from the sucrose gradient (n = 4). The black bar above immunoblots indicates those fractions considered as raft fractions.

Fig. 2.

Tetherin knockdown disrupts membrane microdomain order. (A) Increase in membrane fluidity follows CD317 knockdown. HeLa cells were transfected with siRNA targeting CD317 (tetherin KD) or control siRNA (control) and, concomitantly, plasmids encoding either GPI-YFP or CD99-GFP proteins. At 48 hours post-transfection, regions of the plasma membrane were photobleached and fluorescence recovery of the photobleached regions measured; n = 20 (CD317 KD, GPI-YFP), 21 (control, GPI-YFP), 13 (CD317 KD, CD99 GFP) and 12 (control, CD99-GFP). (B) Laurdan microscopy reveals decrease in membrane order following tetherin knockdown. HeLa cells transiently transfected with TfR siRNA (TfR KD), CD317 siRNA (CD317 KD), CD317 siRNA and WT-HuCD317-HA SR constructs (CD317 KD+WT), or mock treated only, were labelled with Laurdan dye, fixed in formaldehyde, and subsequently treated with wheat germ agglutinin (WGA)-TexasXRed to label the cell surface. Additionally, cells were either labelled with transferrin-Alexa Fluor 647 (Tf-647) concomitant with Laurdan treatment (mock, TfR KD cells), or, post-fixation, immunolabelled with CD317 or HA antibodies, as indicated. Right column: pseudocoloured Laurdan GP images. (C–F) GP values (GP is a measure of relative membrane order; see Materials and Methods for details) were determined for fixed and labelled cells and the average GP distributions (C,E) or mean GP values (± s.e.m.) (D,F) plotted. Each dot represents one image field; n = 11 for each treatment. (C,D) GP values considering the whole cell; (E,F) GP values considering only surface (WGA-positive) regions of the cell. ***P<0.0001 using Student's t-test, for all pair-wise comparisons indicated.

Fig. 3.

Tetherin-based constructs. (A) Schematic of tetherin constructs (see text for details). Rounded rectangles represent TM domains (dark blue, tetherin; yellow, CD8; cyan, CD44); green triangles, GPI anchor; orange bars, HA tag. (B,C) Localisation of tetherin constructs in tetherin knockdown HeLa cells. Cells were either permeabilised by methanol fixation followed by immunofluorescence detection of the HA epitope to visualise whole cell localisation of CD317 (B), or incubated with a HA antibody at the surface at 4°C before formaldehyde fixation to assess cell-surface delivery of the protein (C). (D) Lipid raft/non-lipid raft localisation of tetherin constructs. HeLa cells transfected with the indicated construct were lysed and DRMs prepared as described in Materials and Methods. Raft localisation (protein in flotillin-2 positive fractions) was quantified by densitometry, with values given as protein localised to rafts as a percentage of total protein.

Fig. 4.

Rescue of flotillin 2 raft localisation by tetherin-based constructs. Lipid raft/non-lipid raft localisation of flotillin 2 in HeLa cells, in which expression of endogenous tetherin has been knocked down using siRNA, expressing the indicated tetherin-based construct. HeLa cells transfected with the indicated construct were lysed and DRMs prepared as described in the methods. Raft localisation of flotillin 2 (i.e. to the flotillin-2-positive fractions from wild-type cells) was quantified by densitometry, with rescue of flotillin 2 raft localisation by wild-type tetherin set to 100%. Data represent mean ± s.e.m. from three independent experiments.

Fig. 5.

CD317 activates the NF-κB pathway. (A) HEK293T cells were transfected with reporter plasmids (50 ng of pNF-κB-Luc, 12.5 ng of pRL-SV40) and between 0.1 and 50 ng of an experimental plasmid encoding HuCD317-HA-SR. 24 hours later, cells were lysed and firefly and Renilla luciferase activities measured. Data are presented as firefly:Renilla activity ratios versus cell surface expression of tetherin as determined by FACS analysis. (B) HEK293T cells were transfected with reporter plasmids, as described in A, and 50 ng of experimental plasmids encoding HuCD317-HA-SR or one of its mutant variants. For a negative control, HA-tagged placental alkaline phosphatase (HuPLAP-HA) was used. 24 hours later, cells were lysed and firefly and Renilla luciferase activities measured. Firefly:Renilla activity ratios were standardised for differences in protein expression by FACS analysis. FACS of each construct was performed in duplicate 12-well plates contemporaneously with the luciferase experiments, each treatment of which was performed in octuplicate. Data are presented as firefly:Renilla activity ratios normalised to cell surface expression levels of each of the tetherin constructs (as determined by FACS). Data represent the mean ± s.e.m. from two independent experiments.

Fig. 6.

Cartoon representation of tetherin at cell surface. The dimeric extracellular parallel coiled coil domain of tetherin is shown in red, with the C-terminal end associated with a lipid raft (yellow) by virtue of GPI anchors. N-linked glycosylation is indicated by green hexagons. (A) Single lipid raft. (B,C) Head to head dimerisation of the N-termini of tetherin dimers leads to the linking of adjacent lipid rafts. A recent publication (Lehmann et al., 2011) has suggested, based on super-resolution microscopy techniques, that tetherin is present at the cell surface in clusters of 5–11 dimers.