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Fig. 3. The relative rate of transport into and out of the nucleus before, immediately following, and six-hours post heat shock of GFP-Hsp70 and GFP-Hsp70(K71E). Transport out of the nucleus was determined by bleaching the cytosol and then measuring the percent of the nuclear fluorescence that entered the bleached cytosol in 15 minutes. Transport into the nucleus was determined by bleaching the nucleus and then measuring the percent of the cytosolic fluorescence that entered the bleached nucleus in 15 minutes. Either the nucleus or the cytoplasm was bleached multiple times until the GFP fluorescence was essentially unmeasurable. The cells were imaged with the pinhole set to 12 µm to visualize the entire depth of the cell. To increase the resolution when the in and out-rates of GFP-Hsp70 were measured, images were scanned every 15 seconds using a pixel depth of 12x12 bits. To directly compare in and out-rates, data were corrected by assuming that one-sixth of the cell volume is occupied by the nucleus. Note that this correction is not necessary when comparing either in-rates with each other or out-rates with each other under varied conditions. Data from at least 10 cells were collected for each condition and their average and standard deviation were determined. GFP-Hsp70, black bars, and GFP-Hsp70(K71E), white bars.
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