Figure 1.
Depiction of WT α2/δ-1 (A), the PIN-G reporter (Genbank AY841887.1) [51] (B) and chimeric (C) constructs.
Throughout, numbering is based on the full-length polypeptide prior to signal peptide cleavage and cartoons for all constructs are approximately to scale. A. Wild type α2/δ showing positions of the α2 and δ polypeptides and Von Willebrand factor A (VWA) domain. In α2/δ-1, residues 1–25 encode the signal peptide (SP). The δ subunit is further subdivided into exofacial (δe), putative transmembrane (δTM) and intracellular (δi) regions. The putative minimal GPI anchoring motif, located within a cysteine-rich region (δc) proximal to the external face of the lipid bilayer, contains, in turn, the ω residue (Gly1060) to which GPI is attached, a short spacer (dashed line) and a largely hydrophobic region. Indents between residues 1060 and 1061 indicate chimera fusion site where all downstream δ sequences in constructs PIN-δc or PIN-α2/δ were replaced by the transmembrane and intracellular carboxy terminal residues of PIN-G (constructs PIN-δc-PINTMI and PIN-α2/δ-PINTMI). The parent construct PIN-G (B) contains a signal peptide derived from the Igκ chain, an exofacial haemagglutin (HA) epitope tag, green fluorescent protein (GFP) a carboxy terminal sequence (PINTMI) containing the transmembrane spanning domain from the platelet-derived growth factor receptor and a 17 residue intracellular inert region, whose modification with endocytic or other cytoplasmically exposed targeting motifs can be used to re-direct the reporter to specific intracellular organelles [51]. Chimera (C) include PIN-δc, where the entire transmembrane and intracellular region (Residues 327–370) of PIN-G was replaced by WT α2/δ residues 1027–1091 (i.e. δc, δTM, δi); PIN-δc-PINTMI corresponding to a PIN-G construct containing δc residues (1027–1060) inserted prior to the PINTMI region. Additional chimera include PIN-α2/δ, corresponding to exofacial PIN-G residues 1–326 fused to the amino terminus of WT α2/δ-1, and PIN-α2/δ-PINTMI, where the C-terminal residues (WT α2/δ-1: 1061–1091 (see A)) were replaced by the entire transmembrane and intracellular region (Residues 327–370) of PIN-G. While the putative GPI anchoring motif (lines in C) is present in PIN-δc and PIN-α2/δ, it is absent in PIN-G and disrupted in PIN-δc-PINTMI and PIN-α2/δ-PINTMI chimera. Vertical solid and dashed lines denote α2/δ and transmembrane domain boundaries, respectively.
Table 1.
Comparison of predicted GPI-anchoring potential for WT α2δ-1, PIN-α2δ chimera, mutant α2δ-2 GAS:WKW and Thy-1.
Figure 2.
Effect of WT α2δ-1, PIN-α2δ and PIN-α2δ-PINTMI on Cav2.2/β1b currents.
(A)Average current density-voltage (I-V) plots for Cav2.2/β1b currents in the absence of α2/δ-1 (open circle) and in the presence of WT α2/δ-1 (closed circle), PIN-α2δ (open square) and PIN-α2δ-PINTMI (closed square). Continuous lines indicate the Boltzmann fits to I-V plots using the function described in the Methods. (B) Representative peak current traces from cells expressing Cav2.2/β1b in the absence of α2δ-1 and Cav2.2/β1b co-expressed with WT α2δ-1, PIN-α2δ and PIN-α2δ-PINTMI. Currents were evoked using 150 ms depolarising steps in 5 mV intervals (−30 to +65 mV), from a holding potential, Vh, −80 mV. Data are shown as the mean ± S.E.M.
Figure 3.
Surface and total cellular distribution of PIN-α2/δ chimera expressed in COS-7 cells.
A. PIN-α2/δ. B. PIN-α2/δ-PINTMI. C. PIN-δc. D. PIN-δc-PINTMI. Cells were labelled with anti-HA and Cy5 secondary antibodies using a surface-labelling specific protocol (Methods) and the distribution of surface (red) and total (green, GFP) PIN construct expression determined by fluorescence imaging. Note strong labelling at cell margins for PIN-δc and PIN-δc-PINTMI and highly punctate labelling for PIN-α2/δ and PIN-α2/δ-PINTMI. Scale bar 15 µm.
Figure 4.
Distribution profile of PIN-α2/δ chimera in detergent-resistant membranes is not affected by disruption of the putative GPI anchoring motif.
COS-7 cells were transfected with the corresponding PIN chimera and the membranes analysed via immunoblotting of fractions from sucrose density gradients containing 1% Triton-X-100, using antibodies to caveolin (endogenous) (Panel A) or anti-HA (Panel B)(for PIN chimera). Representative blots in panels A and B, correspond to cells transfected with PIN-α2/δ, PIN-α2/δ-PINTMI, PIN-δc and PIN-δc-PINTMI. Note the absence of PIN-δc or PIN-δc-PINTMI in raft fractions (3–6) and the presence in raft fractions of both PIN-α2/δ and PIN-α2/δ-PINTMI (asterisk in B). Immunodetection loading controls are denoted by ‘T’.
Figure 5.
Effect of PI-PLC cell pre-treatment on the cell surface distribution of GFP-GPI (control) and PIN-α2/δ expressed in COS-7 cells.
Panels A–I correspond to GFP-GPI fluorescence in the absence (A–C) and presence (G–I) of PI-PLC cell treatment. For clarity, panels A and G depict just the surface (red channel, anti-GFP) labelling corresponding to the merged (red (surface) and green (GFP, surface + intracellular) images shown in B and H. Panels C and I correspond to high magnification views of the boxed areas shown in A and G, respectively. Note strong surface labelling and evidence of clustering of GFP-GPI, in the absence of PI-PLC and diminution of surface cluster and interstitial fluorescence after PI-PLC treatment. Since contiguity between GFP-GPI clusters precluded standard particle analysis, the effect of PI-PLC on GFP-GPI clustering was analysed further by generating contour maps (panels D and J) (level scale (0–255) shown to right) of the labelling seen in panels C and I, respectively. Line scans based on the contour maps were then constructed to show differences in fluorescence intensity in the absence (white and yellow in D and F) or presence (red and orange in D and F) of PI-PLC cell treatment. Panel K shows the effect of PI-PLC cell pre-treatment on the signal to background fluorescence for raw images (n>8) collected using identical imaging conditions. *** denotes statistically significant difference (P<0.001); Student's t-test. Panels L–T correspond to images from cells transfected with PIN-α2/δ in the absence (L–N) and presence (R–T) of PI-PLC. Panels L and M (−PI-PLC) and R and S (+PI-PLC) show merged images for total (surface + intracellular)(green, GFP) and surface (red, anti-GFP)) for separate cells. Panels N and T correspond to high magnification views of the boxed areas shown in L and R (red, (surface) channel only). Note the presence of extensive PIN-α2/δ clustering irrespective of whether or not the cells had been treated with PI-PLC. Panels O and U correspond to contour maps (above) of the labelling seen in panels N and T, respectively (level scale (0–255) shown to right). Line scans corresponding to the contour maps were then constructed to show differences in fluorescence intensity in the absence (white and yellow in P and Q) or presence (red and orange in P and Q) of PI-PLC cell treatment. Panel V shows the effect of PI-PLC cell pre-treatment on the signal to background fluorescence for raw images (n>8) collected using identical imaging conditions. Note lack of effect of PI-PLC on PIN-α2/δ distribution (O and U) or intensity (V). All images are representative examples from data sets comprised of >8 images (>2 experiments). Scale bars are as follows: panels A, B, G, H, L, M, R and S, 20 µm; panels C, I, N and T, 4 µm.
Figure 6.
Effect of GPI-anchor removal through cell pre-treatment with PI-PLC.
COS-7 cells were transfected with either GFP-GPI or HA-α2/δ and the membranes analysed via immunoblotting of fractions from sucrose density gradients containing 1% Triton-X-100, using antibodies to GFP (GFP-GPI), the HA-epitope tag (HA-α2/δ), caveolin (endogenous) or flotillin (endogenous). Representative blots at left and right correspond to cells before and after pre-treatment with PI-PLC, respectively. Note the presence of all proteins in the buoyant (raft) fraction prior to PI-PLC exposure and restriction of GFP-GPI, HA-α2/δ and caveolin, but not flotillin (B., asterisk) in denser non-raft fractions following PI-PLC exposure. Immunodetection loading controls are denoted by ‘T’.
Figure 7.
Effect of PI-PLC cell pre-treatment on the distribution of endogenous caveolin and flotillin in COS-7 cells.
Panels A and B correspond to caveolin labelling in the absence (A) and presence (B) of PI-PLC cell pre-treatment. Panel C depicts intensity profiles (averaged in y axis) corresponding to boxes shown in A and B (red and black lines corresponding to profiles with and without PI-PLC, respectively). By averaging the fluorescence intensity, such ‘box scans’ reduce the noisiness seen in individual line scans. Note aggregation of caveolin fluorescence proximal to the nucleus (B) and increase in intensity (C) in images from cells pre-treated with PI-PLC. Panels D and E depict flotillin labelling in the absence (D) and presence (E) of PI-PLC cell pre-treatment. The corresponding box scans are shown in F (red line: +PI-PLC; black line: −PI-PLC). Note similarity in flotillin distribution irrespective of cell pre-treatment with PI-PLC. Scale bars: 15 µm.
Table 2.
Comparison of experimental approaches and conclusions in the present study and that of Davies et al., [20].