Fig. 4. The effect of heat shock on GFP8 and control cells. (A) A heat shock of 40°C was applied for 60 minutes. Cells were immobilized on slides and fixed by rinsing in -20°C methanol for 20 seconds. T0, cells before heat shock; T60, cells after heat shock. SFA-GFP fibers were largely depolymerized with the exception of a dot-like region near the basal bodies. T120, thin fibers extend from the brighter dot; T240, globular aggregates have formed, in addition to the cross-like structure. These aggregates were also visible in phase contrast (arrows) and were often located at the end of the developing SFA-GFP fibers (insert). T360, SFA-GFP fibers were reassembled almost completely. Bar, 10 µm. (B) In vivo observation of cells embedded in agarose during heat shock and recovery. Each picture represents an average of several images taken at six to seven different focus levels over a period of 5-6 seconds. For heat treatment the slide with the embedded cells was incubated on a temperature-controlled metal block. T0, embedded cells were still able to rotate, causing the SMAFs to appear several times on the averaged image. T70, cells have resorbed the flagella and the fibers were depolymerized. T130, T210, T290, repolymerization of SMAFs first visible as thread-like extensions from the stable core region. Bar, 10 µm. (C) Immunofluorescence analysis of heat-shocked control (1) and GFP8 cells (2, 3) using anti-SFA. (1) In control cells, SMAFs seem to be unaffected by heat shock. (2) GFP fluorescence and corresponding antibody staining (3) of a GFP8 cell after heat shock. The resulting signals show differences in shape and size, indicating an unequal distribution of wild-type and GFP-tagged protein. It should be noted that anti-SFA binds both SFA and SFA-GFP (compare also Fig. 5, bottom). Bar, 1 µm. (D) Northern blot of control and GFP8 cells using a partial cDNA of SFA as a probe. In GFP8 cells, several transcripts at and below 3.2 kb were detected 45 minutes after heat shock (T105), which were not present before (T0) or three hours after heat shock (T240), or in control cells. 24hD, RNA from cells cultivated in the dark for 24 hours. 30 µg of total RNA were loaded in each lane. The positions of RNA standard molecules are indicated. (E) Western blot analysis of GFP8 and control cells during heat shock using anti-SFA. Time points and sample type (whole cell, pellet or supernatant) are indicated above the bars and from each sample type similar amounts of protein were loaded. A heat shock of 60 minutes duration was applied from T0 to T60. Upper rows: in whole cells only minor variations in the amount of SFA and SFA-GFP were observed during heat shock. Middle rows: analysis of pellets and supernatants of GFP8 cells during heat shock. SFA-GFP was largely removed from the insoluble fraction after the heat shock. Note the difference in apparent molecular weight between soluble and insoluble SFA-GFP. Anti-centrin staining (BAS6.8) of the same samples is shown as a loading control. Lower rows: supernatant and pellet of control cells. Note the shift in molecular weight between soluble and insoluble forms of SFA. wc-c, whole cell control.