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. 2012 Jan 10;20(5):972–983. doi: 10.1038/mt.2011.295

Figure 6.

Figure 6

Focal adhesion kinase (FAK) is required for the internalization of Mn++ induced adeno-associated virus (AAV) aggregates. (a–d) FAK+/+ and FAK−/− mouse embryonic fibroblasts (MEFs) were infected with Alexa568-recombinant adeno-associated virus 2 (rAAV2) in the (a, b) absence and (c, d) presence of Mn++ for 2 hours in the presence of Mn++, followed by 2 hours in virus- and Mn++-free media. Membranes were stained with AlexaFluor-488 Wheat Germ Agglutinin (Alexa488-WGA) to demark the plasma membrane and endosomes. A series of XY confocal images were taken through each cell (square images at the bottom of each panel) and a reconstructed YZ-vertical image was created using metamorph software (top rectangular panels) to help demonstrate the location of AAV objects relative to the plasma membrane. The location of the XY slice (square panels) is marked on the YZ panels by a yellow line. Similarly the location of the YZ slice (rectangular panels) is marked on the XY panels by a yellow line. AAV single channel images are depicted at both high and low threshold to demonstrate increased cluster size and intensity in the presence of Mn++ (c, d). In the absence of Mn++, rAAV2 is internalized regardless of FAK expression (a, b). In the presence of Mn++, FAK competent (c) cells internalize the large and bright AAV clusters, however, in FAK−/− MEFs these rAAV2 clusters are either broken apart or remain associated with the cell surface (d). The boxed regions in square panels are enlarged as an inset.