Fig. 4. Localization of TPC2 in the myofibers of primary cultures of SMCs.

(A) Schematic representations of two sarcomeres showing the localization of (Aa) myosin in green and (Ab) TPC2 (in red), RyR (in green) and IP₃R (diagonal shading). (B-E, panels a-f) Series of optical sections projected as single images to show the localization of TPC2 with regards to other proteins in the myofibrils or SR. Cells were dual-immunolabeled with: (B) myosin heavy chain and TPC2 antibodies; (C) myosin heavy chain and LAMP1 antibodies; (D) RyR and TPC2 antibodies; or (E) RyR and IP₃R type III antibodies. The pattern of localization is shown in cells cultured for either (B-E, panels a-c) ~24 h or (B-E, panels d-f) ~48 h, and for each series of antibodies used, the pattern of localization of each protein is shown both alone (panels a,d and b,e) and when superimposed on the image of the other protein labeled (panels c,f). Scale bars, 1 μm. (B-E, panel g) Line-scan analyses. See key to symbols in Fig. 3 for explanation of yellow dashed lines and the blue and pink arrowheads; white arrowheads indicate TPC2-exclusion zones around the sarcomeric z-line and yellow arrowheads indicate regions of TPC2 and myosin heavy chain overlap. (F,G) Side (panel a) and end (panel b) views of SMCs to show the localization of (F) TPC2 and (G) LAMP1 in relation to the myosin heavy chain. (F,G, panel c) Schematics to illustrate the different views observed.