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Fig. 4. Syndecan-4 is internalized in the clathrin- and dynamin-independent manner. (a-c) Syndecan-4 endocytosis was studied in FcR-S4 expressing RFPEC transiently transfected with various constructs. Antibody clustering of FcR-S4 chimeras carried out as described in Fig. 1 and 2 leads to internalization of syndecan-4 (a). Note the absence of co-localization of internalized syndecan-4 with clathrin in cells expressing clathrin-eGFP 5 minutes after clustering (b,c). The effect of clathrin dominant negative was studied in cells transiently expressing a c-myc tagged AP180C construct (d). Cell expressing AP180C demonstrates the same FcR-S4 internalization as non-transfected cells (e,f,g,h). The FcR-S4 remaining on the cell surface is shown in green and internalized FcR-S4 is in red. (i-m) The role of dynamin was studied in cells transiently expressing a dominant-negative HA-tagged dyn2^K44A construct (i). Note that the cell expressing Dyn2^K44A demonstrates high level of FcR-S4 internalization (r,i,l,m). h and m show the color images in g and l, merged with their respective DIC images. In g, h and i internalized FcR-S4 is in red, and in f,g,h,k,i FcR-S4 constructs remaining on the cell surface are in yellow and green. Scale bars, 10 µm.

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