Recruitment timing and dynamics of transcription factors at the Hsp70 loci in living cells.

Spinning Disk Microscopy

Drosophila salivary glands, from the crosses described in the Supplemental Experimental Procedures , were dissected from third instar larva as previously described ( Yao et al., 2008 ) and were transferred, immediately with medium to a MatTek glass-bottomed culture dish (P35G-1.0-14-C) and a glass cover-slip was placed on top to reduce evaporation and movement of the glands. A Carl Zeiss Cell Observer SD system with the Yokogawa CSU-X1 spinning disk unit and a Hamamatsu C9100-13 EMCCD was used to obtain confocal 3D stacks with 2-channels and time intervals of 10–30 sec. Two identical Plan-Apochromat 40x/1.3 Oil Iris objectives were used; one was maintained at room temperature and one was heated to 36°C using a Bioptechs Objective Heater. A 40 μm pre-activation z-stack, with 1 μm sections, was taken using the room temperature objective in both channels alternating channels every slice. Then the 36°C objective was moved into position; HS times were started at the moment the objective contacted the slide. A xyzt series with 40 z-sections was obtained after readjusting the focal position. Time intervals were between 10–30 sec and the time series lasted 20 min ( Figure 2A ). Images were taken at a resolution of 512×512 pixels, using 16-bit color depth. Details regarding data analysis can be found in the Supplemental Experimental Procedures .

Laser Scanning Confocal Microscopy and Multiphoton Microscopy

Dissected glands were transferred with medium to a Bioptechs FCS3 Closed Chamber System with a 0.2 mm spacer. We used an upright confocal/multi-photon microscope system (Carl Zeiss LSM510 META). NHS images were obtained with a C-Apochromat 63x, 1.2 NA, water immersion objective. For HS, an identical objective was swapped in for the room temperature objective the same manner as described above.

FRAP

Dissected glands were imaged using MPM as described above. Perfusion of 3μM PJ34 or Media alone into the FCS3 chamber occurred as depicted in Figure 6A . We used a circular ROI limited to the dimensions of the Hsp70 loci. eGFP samples were photobleached with a Mai Tai laser (Spectrum Physics) at 910 nm with a power of 15–20 mW (measured after the objective). mRFP photobleaching used the same laser at 800 nm using 40–50 mW. These settings photobleached the samples to 40–60% initial intensity. Images were corrected for acquisition photobleaching by monitoring a small nuclear region. FRAP curves were normalized for pre-bleach images to equal one, and first image after the bleach equal to 0. Recovery times were obtained by fitting the FRAP data to f ( t )= A ×( 1 - C eq × e − koff ×t ) ( Sprague et al., 2004 ).

Supplementary Material 01