Figure 4: Acute formation of vacuoles and vacuole resistance to SOCE.

An xyt series of confocal images of t-system rhod-5N shows the t-system responding to caffeine-induced SOCE and reuptake of Ca²⁺ (images 110, 176 and 221 s). The spatially averaged profile at bottom right of the figure indicates the internal bathing solution the fibre was immersed in. The pale blue vertical bars on the graph indicate the timing of the solution changes and the horizontal bars at top indicate the internal solution composition. The time-stamp on the images correspond to that on the graph. The initial exposure of the fibre to caffeine caused SR Ca²⁺ depletion and SOCE that caused a uniform depletion of [Ca²⁺]_t-sys. The following substitution of caffeine for 1.3 μM [Ca²⁺]_cyto caused the t-system to take up Ca²⁺ and for vacuoles to form (longitudinal structures, seen as bright yellow in images 254 to 729 s). The substitution of 1.3 μM [Ca²⁺]_cyto for caffeine caused the transverse tubules to deplete of Ca²⁺ without affecting the Ca²⁺ held in the vacuoles (bright longitudinal structures remain in the presence of caffeine and the spatially averaged fluorescence signal indicated in the graph does not drop as low as in the initial exposure to caffeine). The secondary exposure to 200 nM Ca²⁺ allows the transverse tubules to again take up Ca²⁺ and the final exposure to caffeine again causes only a partial reduction in the spatially averaged [Ca²⁺]_t-sys. Scale bar: 25 μm.