Clustering induces a lateral redistribution of alpha 2 beta 1 integrin from membrane rafts to caveolae and subsequent protein kinase C-dependent internalization

Abstract

Integrin alpha 2 beta 1 mediates the binding of several epithelial and mesenchymal cell types to collagen. The composition of the surrounding plasma membrane, especially caveolin-1- and cholesterol-containing membrane structures called caveolae, may be important to integrin signaling. On cell surface alpha 2 beta 1 integrin was located in the raft like membrane domain, rich in GPI-anchored proteins, rather than in caveolae. However, when antibodies were used to generate clusters of alpha 2 beta 1 integrin, they started to move laterally on cell surface along actin filaments. During the lateral movement small clusters fused together. Finally alpha 2 beta 1 integrin was found inside caveolae and subsequently internalized into caveosome-like perinuclear structures. The internalization process, unlike cluster formation or lateral redistribution, was dependent on protein kinase C alpha activity. Caveolae are known to be highly immobile structures and alpha 2 beta 1 integrin clusters represent a previously unknown mechanism to activate endocytic trafficking via caveolae. The process was specific to alpha 2 beta 1 integrin, because the antibody-mediated formation of alpha V integrin clusters activated their internalization in coated vesicles and early endosomes. In addition to natural ligands human echovirus-1 (EV1) gains entry into the cell by binding to alpha 2 beta 1 and taking advantage of alpha 2 beta 1 internalization via caveolae.

Figure 5.

Binding of α2β1 integrin to EV1 induces the phosphorylation of ERK MAP kinase in a PKCα activity-dependent manner. (A) ERK is transiently phosphorylated <15 min after α2β1 integrin binding to EV1 and the phosphorylation lasts ∼1 h (Western blot). (B) Inhibitors of ERK activation (PD) and p38 kinase (SB) do not inhibit infection in contrast to methyl β-cyclodextrin (CYCLO), an inhibitor of cholesterol metabolism and caveolae. (C) Transient transfections with cDNAs encoding the dominant negative MEK (DN MEK) or dominant negative Ras (DN Ras) do not inhibit α2β1 integrin internalization or infection. Constitutively active MEK (CA MEK) does not increase the internalization of α2β1 integrin either. (D) ERK is translocated to the nucleus 4 h p.i. The general PKC inhibitor bisindolylmaleimide (5 μM, BIS) inhibits the nuclear translocation of ERK, unlike a Ras inhibitor, perillyl alcohol (500 μM, POH). Quantification of ERK positive nuclei after treatments with bisindolylmaleimide (BIS), another PKC inhibitor safingol (10 μM, SAF) or Ras inhibitor (POH). Error bars show SE values (B and C: the graph on the left, D) or 95% confidence limits (C: the graph on the right).

Figure 6.

PKCα activity is crucial for EV1 replication and the clustering of α2β1 increases PKCα phosphorylation. (A) EV1 infection is prevented after inhibition of PKC activity with either bisindolylmaleimide (BIS) or safingol (SAF). (B) Dominant negative PKCα inhibits infection (p < 0.001, binomial t test), whereas over-expression of wild-type PKCα increases infection to some extent (p < 0.05, binomial t test). Dominant negative PKCε does not have any effect on infection. (C) PKCα is downregulated after a chronic treatment (6 h) with phorbol ester (PMA), which is shown by Western blotting. This treatment also inhibits EV1 infection. (D) Phosphorylation of PKCα detected by Western blotting during EV1 entry (EV1), a short treatment (30 min) with PMA and clustering of α2β1 integrin by anti-α2 mAb (a-α2 + Sec) and Fab of anti-α2 mAb as its control. Negative control is marked as C. In C and D antibodies against phosphorylated PKCα, total PKCα, tubulin and caveolin-1 were used. Error bars show SE values (A and C) or 95% confidence limits (B).

Figure 7.

Integrin α2β1-mediated internalization of EV1 via caveolae is dependent on PKCα activity. (A) Safingol can be added to cells 30 min before EV1 (-30′), at the same time as EV1 (0), or 5 min after EV1 (+5′) to prevent the infection. (B) Safingol does not affect the ability of α2β1 integrin to bind radioactive EV1. (C) Live cell imaging showing that safingol does not prevent the clustering-related redistribution leading to colocalization of α2β1 integrin (red) and caveolin-1 (green) on the plasma membrane. To show that safingol inhibits integrin (red) internalization a rectangular slice was taken from the center area of one safingol treated and one nontreated cell (both in the presence of clustering antibodies) and projections were viewed from the side. Caveolin-1 has been excluded from these images. Note that safingol is causing some rounding of cells. (D) Safingol prevents the internalization of EV1 (100 MOI). Internalization was quantified by labeling EV1 before and after permeabilization with different colors and then taking Z-scans through the cells with a confocal microscope and measuring the internalized vesicles using 3D for LSM software. (E) Safingol has no effect on the internalization of antibody induced αV clusters. Error bars show SE values.

Figure 1.

Integrin α2β1 is located in lipid rafts. (A) In flotation gradient centrifugation of 1% Triton X-100 lysed cell homogenate α2β1 integrin, caveolin-1, and GPI-APs (ASSP) localize at the 5-35% sucrose interphase. The localization of detergent-resistant membranes (DRM) have been indicated. Western blot of gradient fractions 1-9 (from top to bottom). (B) Lipid rafts containing GPI-APs (ASSP) are enriched in α2β1 integrin in SAOS-α2β1 cells. SAOS-pAW cells are α2β1 negative control cells. Indirect immunofluorescence labeling was used to reveal α2β1 integrin and caveolin-1, whereas GPI-APs were labeled directly with an ASSP-Alexa 546 conjugate. Confocal images are 3D projections of scanned cells. Scale bar, 10 μm. In merge images yellow color indicates colocalization of GPI-APs and α2β1 integrin.

Figure 2.

Formation of α2β1 clusters initiates redistribution of α2β1 out of lipid rafts. (A) In SAOS-α2β1 cells α2β1 integrin (green) is colocalized with GPI-APs (ASSP, red; control), but after incubation with anti-α2 mAb and secondary antibody (+a-α2) α2β1 integrin is located outside the lipid rafts. Scale bar, 10 μm. Blow-ups show lipid raft areas on cell edges (merge images). (B) A close-up about the formation of α2β1 integrin clusters (red) and integrin redistribution (see Video 1). Scale bar, 0.5 μM. (C) A whole-cell view shows that integrin clusters (red) move along actin filaments (GFP-actin; see Video 2). Scale bar, 10 μM. (D) A close-up of integrin clusters fusing together and moving along actin. Scale bar, 2 μM. B, C, and D are all live images of the same cell, B and D being digitally isolated close ups of C (actin is not shown in B). The live imaging (B-D) was performed at low temperature (27°C) in order to increase temporal resolution. (E) A rectangular slice of the cell from the 70-min time point was taken to verify that the actin filaments were indeed cortical, i.e., close to the plasma membrane. The white rectangle shows the location of this slice.

Figure 3.

Clusters of α2β1 integrin are internalized in caveolae. (A) Live cell imaging of cellular internalization of clustered α2β1 integrin. Integrin α2β1 is green and GPI-APs are red (ASSP). A rectangular slice was taken from the center area of one cell, and projections were viewed from above and the side (see Video 3). (B) Live cell imaging with fluorophore (green)-labeled anti-α2 integrin mAb without secondary antibody. A rectangular slice was taken from the center area of one cell, and the projection was viewed from the side. (C) In SAOS-α2β1 cells incubated with anti-α2 mAb and secondary antibody or EV1, α2β1 integrin (green) is colocalized with caveolin-1 (red; see Video 4). Scale bar, 10 μM. (D) Caveolin-1 colocalizes with EV1 2 h p.i. Scale bar, 10 μM. (E) Electron microscopy of gold particles linked to secondary antibodies against anti-α2 mAb indicates that clustered α2β1 integrin is internalized in caveolae (left) and later is found in caveosomes (right). Protein A gold particles (5 nm) label the secondary antibodies that were used to cluster the primary anti-α2 antibodies. Scale bar, 100 nm. (F) A caveosome containing internalized EV1 that is labeled with gold particles (5 nm) is shown for comparison. Scale bar, 100 nm. For EV1 infections MOI 100 was used.

Figure 4.

Clusters of αV and α2β1 integrins are internalized through distinct pathways. (A) Clusters of αV integrins (green) are not targeted to caveolin-1-containing vesicles (red). Scale bar, 10 μM. (B) Clusters of αV integrins, unlike clusters of α2β1 integrins, are found in vesicles positive for EEA1, a molecular marker for early endosomes. Scale bar, 10 μM. (C) Clusters of αV integrins, unlike clusters of α2β1 integrins, are internalized via the same pathway as transferrin. Scale bar, 10 μM. In A-C, white rectangles show the locations of the blow-ups. (D) αV integrin clusters labeled with gold particles (10 nm) 1 min after internalization can be seen inside coated vesicles by electron microscopy. Clathrin coat is indicated by arrows. Scale bar, 100 nm.

Figure 8.

The proposed model of α2β1 integrin-mediated activation of caveolae. Antibody or virus-mediated formation of α2β1 integrin clusters leads to lateral movement on cell surface in which integrin clusters seem to follow microfilaments. Often smaller integrin clusters seem to fuse to each other. Clustering and lateral movement are followed by PKCα dependent internalization in caveolae.