Transient anchorage of cross-linked glycosyl-phosphatidylinositol-anchored proteins depends on cholesterol, Src family kinases, caveolin, and phosphoinositides

Abstract

How outer leaflet plasma membrane components, including glycosyl-phosphatidylinositol-anchored proteins (GPIAPs), transmit signals to the cell interior is an open question in membrane biology. By deliberately cross-linking several GPIAPs under antibody-conjugated 40-nm gold particles, transient anchorage of the gold particle-induced clusters of both Thy-1 and CD73, a 5' exonucleotidase, occurred for periods ranging from 300 ms to 10 s in fibroblasts. Transient anchorage was abolished by cholesterol depletion, addition of the Src family kinase (SFK) inhibitor PP2, or in Src-Yes-Fyn knockout cells. Caveolin-1 knockout cells exhibited a reduced transient anchorage time, suggesting the partial participation of caveolin-1. In contrast, a transmembrane protein, the cystic fibrosis transmembrane conductance regulator, exhibited transient anchorage that occurred without deliberately enhanced cross-linking; moreover, it was only slightly inhibited by cholesterol depletion or SFK inhibition and depended completely on the interaction of its PDZ-binding domain with the cytoskeletal adaptor EBP50. We propose that cross-linked GPIAPs become transiently anchored via a cholesterol-dependent SFK-regulatable linkage between a transmembrane cluster sensor and the cytoskeleton.

Figure 1.

Maximal cross-linking scheme that produces transient anchorage. (a) After incubating cells with biotinylated mouse primary antibodies recognizing specific GPIAPs (top), antibiotin gold particles are added on the cell membrane to form bonds with the primary antibodies (middle). Finally, tertiary polyclonal antibodies that bind to mouse IgG are added to further cross-link the GPIAPs (bottom). (b) During SPT, short periods with zero displacement were observed (arrows indicate representative transient anchorage events). (c) The periods of transient anchorage for maximally cross-linked CD73 on IMR 90 cells varied from several hundred milliseconds to >10 s and are bimodally distributed. (d) Preassembled complexes also showed a similar bimodal distribution of transient anchorage durations.

Figure 2.

Both SFK inhibition and cholesterol depletion suppressed transient anchorage. (a) C3H expressing Thy-1 (top) and IMR 90 cells expressing CD73 (bottom) were treated with PP2 (n = 79 for Thy-1 and n = 79 for CD73 where n is the number of trajectories analyzed) or cholesterol-removing agents, mβCD (n = 100 for Thy-1 and n = 70 for CD73), and/or cholesterol oxidase (n = 67 for CD73) before maximal cross-linking. SPT with maximal cross-linking was the positive control (n = 90 for Thy-1 and n = 88 for CD73). SPT without maximal cross-linking (without tertiary antibody addition) was the negative control (n = 69 for Thy-1 and n = 85 for CD73). Error bars indicate the SEM. (b) Western blotting was performed to check the expression of CD73 and whether the anti–human CD73 antibody can detect CD73 on the human and mouse cell lines used in this study. MEF, mouse embryonic fibroblast. (c) SFKs are required for the transient anchorage of CD73. SFK-deficient cells (n = 65) did not demonstrate transient anchorage upon maximal cross-linking, but transient anchorage can be rescued by the transfection of c-Src (n = 80) into the deficient cell line. (a and c) The results were collected over three to five independent experiments.

Figure 3.

Effects of PI3 inhibition on transient anchorage. (a) PI3 kinase inhibition induced more transient anchorage upon maximal cross-linking (n = 117) compared with the control cells (n = 120). The results were collected over three to four independent experiments. Error bars indicate the SEM. (b) Compared with Fig. 1 c, longer anchorage times were largely abolished in PI3 kinase–inhibited cells. (c) A portion of single particles after PI3 inhibition exhibited bidirectional movements, as shown by this representative trajectory.

Figure 4.

Caveolae participated in transient anchorage. (a and b) Partial colocalization between CD73 (a) and caveolin-1 (b) was seen on IMR 90 cells after maximal cross-linking. (c and d) Partial colocalization between Thy-1 (c) and caveolin-1 (d) was seen on C3H cells after maximal cross-linking. The specificity of primary and secondary antibodies used here has been examined by the manufacturers and they do not cross react. (a–d) Arrows show representative regions of colocalization. (e) In caveolin-1–deficient mouse embryonic cells (n = 92), transient anchorage of Thy-1was reduced to about one third compared with wild-type parental cells (n = 100). Transient anchorage could be suppressed by SFK inhibition and cholesterol depletion in both cell lines (n = 70 for caveolin^−/− + MβCD; n = 72 for caveolin^+/+ + MβCD; n = 88 for caveolin^−/− + PP2; and n = 75 for caveolin^+/+ + PP2). The results were collected over three to five independent experiments. Error bars indicate the SEM. MEF, mouse embryonic fibroblast.

Figure 5.

CFTR demonstrated transient anchorage independent of maximal cross-linking, SFK activities, and cholesterol presence. (a) CFTR tagged with an extracellular HA epitope was expressed in C3H cells and labeled with biotinylated anti-HA antibodies and antibiotin antibody–conjugated gold for SPT. CFTR displayed transient anchorage in the absence of tertiary cross-linking antibody (n = 74). SFK inhibition (by PP2; n = 60) or cholesterol depletion (by MβCD; n = 60) only slightly reduced the transient anchorage, whereas deletion of the PDZ-binding domain in CFTR (Δ4 mutant; n = 65) caused transient anchorage to be almost completely eliminated. Error bars indicate the SEM. (b) CFTR demonstrated a similar bimodal distribution of transient anchorage durations compared with the maximal linked CD73 in Fig. 2 c.

Figure 6.

Transient anchorage hypothesis. Cross-linking causes nanodomains to form on both the inner and outer leaflet, with a transmembrane protein bridging the two leaflets to transduce the signal. When an activated SFK randomly partitions into such a nanodomain, phosphorylation by the SFK causes a resident molecule, possibly the transmembrane protein, to attach to the actin cytoskeleton indirectly through other linker proteins. The attachment results in a transient anchorage until the SFK becomes deactivated and a recruited phosphatase dephosphorylates the linking resident molecule in the nanodomain (see Discussion for details). ERM, ezrin/radixin/moesin proteins; PIP₂, phosphatidyl-inositol bisphosphate; SFK, Src family kinase.