Clustering Induces a Lateral Redistribution of 21 Integrin from Membrane Rafts to Caveolae and Subsequent Protein Kinase C-dependent Internalization

MBC Videos

This article contains the following 4 movies:

• Movie 1 Integrin clustering in a close up (3.65 MB) Formation of alpha2beta1 clusters was induced with anti alpha2ƒnintegrin antibody, using anti-mouse Alexa 546 as the secondary antibody. To visualize clustering in more detail, temporal resolution was improved in two ways: the time interval between acquiring confocal Z-stacks was decreased to 5 min and the temperature was lowered to approx. 27°C. The data, acquired over 115 min, were processed into series of 3D projections.

  Red: alpha2beta1 integrin.

  In this movie the formation of integrin clusters is first seen in one representative cell, with the time running on screen from 0 to 115 min. The clusters seem to be forming lines along which they move. Next a few integrin clusters from the same cell are shown more closely (the zooming in and the isolation of the clusters have been done digitally). The clustering is shown with the time running on screen from 0 to 55 min, and the formation of one large integrin cluster from several smaller ones can clearly be seen. The clusters can also be seen to move in a certain direction.

• Movie 2 Integrin clusters appear to be moving along actin filaments (5 MB) The data used here is the same as in Movie 1. However, the cell was actually transfected with actin-GFP, which was left out from the projections when creating Movie 1. Now the actin filaments are shown.

  Red: alpha2beta1 integrin, green: actin-GFP.

  The formation of integrin clusters is first seen as in Movie 3, with the time running on screen from 0 to 115 min. The lines along which the clusters are moving can be seen to correspond to the green cortical actin filaments. Next one actin filament and a few integrin clusters from the same cell are shown more closely (the zooming in and the isolation have again been done digitally). The clustering is shown with the time running on screen from 0 to 60 min. The formation of one large integrin cluster from several smaller ones can again be seen, but, perhaps more importantly, the clusters can clearly be seen to move along the actin filament.

  Notes:

  1. The actin filaments visualized are mostly cortical, i.e. close to the cell surface (see also Fig. 2 E).

  2. The few clusters visualized in close up are not the same as in Movie 3, although they are, of course, from the same cell.

• Movie 3 Formation and internalization of integrin clusters in the presence of anti alpha2 mAb (6.36 MB) Cluster formation of alpha2 integrins was induced with anti alpha2 mAb and anti mouse Alexa 488 (secondary antibody) in living cells. Cells were observed with a confocal microscope for 90 min. Z-stacks were taken every 10 min, and the data were processed into series of 3D projections. Sample temperature was approx. 37°C.

  Green: alpha2 integrin, red: GPI-APs.

  In this movie three representative cells are first rotated to give an impression of their three-dimensional structure. A rectangular slice is then cut from the centre of one cell, as shown. The slice is rotated to give an impression of its three dimensional structure before integrin clustering. The time series from 0 to 90 min is then shown with the time running on screen. The slice is viewed simultaneously from above and from the side. The green dots (alpha2beta1 integrin) can be seen to cluster into larger aggregates and move from the cell surface to inside the cell. Finally the slice is rotated again to illustrate its structure after clustering.

  Notes:

  1. GPI anchored proteins are also internalized to some extent, but this is not evident in these images due to the ¡§shadow¡¨ rendering method used. (This method is designed to produce clear and ¡§realistic¡¨ 3D projections, and therefore it, unlike the often used maximum intensity rendering, does not show colocalization as mixing of colors. Thus, because GPI anchored proteins are internalized less than the integrin, their red color signal is weaker and is overshadowed by green in these projections.)

  2. Time point 0 means the starting point of imaging, which was about 5 min after the secondary antibody was added. Therefore some clustering can already be seen.

• Movie 4 Integrin internalization in caveolin-1 containing vesicles (8.96 MB) Formation of alpha2beta1 clusters was induced and cells imaged as in Movie 3. Cells expressing caveolin-1-GFP were used, and the secondary antibody was anti mouse Alexa 546.

  Green: caveolin-1, red: alpha2beta1 integrin.

  In the movie two representative cells (one containing green caveolin-1 and one not) are first rotated to give an impression of their structure. A thin section through the cells is then taken and viewed from the side together with the cells. The time series from 0 to 90 min is shown with the time running on screen. Both the red integrin and the green caveolin can be seen to cluster into larger aggregates and also to move from the cell surface to inside the cell. Due to the maximum intensity rendering method used colocalization of caveolin and integrin can be seen as yellow colour. Importantly, the colocalization is confirmed by the aggregates appearing yellow also in the side view of the thin section. Finally the cells are rotated again to illustrate their structure after clustering.

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