Figure 8

Cell recovery temporally correlates with AdcA dephosphorylation. A. KAx-3 cells were let to adhere on a glass coverslip in culture medium for 1 h. Cells were then treated with 200 mM sorbitol in culture medium and observed by time-lapse videomicroscopy using DIC optics. The images illustrate the cells at different stages post sorbitol addition. Scale bar: 5 μm. B. KAx-3 in shaking culture received simultaneously 2 mg/ml FITC-dextran and various concentrations of sorbitol (0, 100, 200 and 400 mM). Aliquots were collected as a function of time and treated as described in Methods. The volume of internalized fluid was expressed in fl/cell. Data correspond to a representative experiment. KAx-3 overexpressing AdcA_GFP were used in parallel to follow the localisation of AdcA during the stress response. Cells from a shaking culture were removed prior (t = 0, a, a’) and after sorbitol addition (t = 30 min (b,b’) and t = 210 min (c,c’)) and observed directly by fluorescence microscopy. Two representative cells are shown. C. Shaking cultures of KAx-3 and adcA null cells were treated with 200 mM sorbitol or 10 μM cytochalasin A (only for KAx-3). Adaptation to treatments was monitored by the resumption of the endocytic activity as described in B using FITC-dextran as a fluid-phase marker. Mean ± s.e.m. (n = 3). D. Ax2 and dstC^- cells were subjected to 200 mM sorbitol and their endocytic activity was evaluated as described above. Mean ± s.e.m. (n = 3).