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First published online 14 September 2004
doi: 10.1242/jcs.01373

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Hsp70 dynamics in vivo: effect of heat shock and protein aggregation

Xian-Chun Zeng^1,*, Samir Bhasin^1,*, Xufeng Wu¹, Joeng-Goo Lee¹, Shivani Maffi¹, Christopher J. Nichols¹, Kyung Jin Lee¹, J. Paul Taylor², Lois E. Greene¹ and Evan Eisenberg^1,

¹ Laboratory of Cell Biology, NHLBI, NIH, 50 South Drive MSC 8017, Bethesda, MD 20892-0301, USA
² Neurogenetics Branch, NINDS, NIH, 10 Center Drive, MSC 1250, Bethesda, MD 20893-1250, USA

View larger version (59K): [in a new window]   Fig. 1. Properties of GFP-Hsp70. (A) Protection of GFP-Hsp70 against apoptosis from the expression of Htt(Q72). The extent of apoptosis was measured 72 hours after transfection in control cells and cells expressing GFP-Htt(Q25), GFP-Htt(Q72), GFP-Htt(Q72) and flag-tagged Hsp70, myc-tagged Htt(Q72) and GFP vector, and myc-tagged Htt(Q72) and GFP-Hsp70. Apoptosis was measured by immunostaining for cleaved caspase 3 using rhodamine-conjugated secondary antibody. The data represent an average of three experiments in which 100 GFP cells were analyzed for positive caspase staining. (B) Distribution of GFP-Hsp70 in control (left) and heat-shocked cells (right).

View larger version (95K): [in a new window]   Fig. 2. The transport of (A-F) GFP-Hsp70 and (GL) GFP-Hsp70(K71E) into and out of the nucleus in control (A-C,G-I) and heat-shocked cells (D-F,J-L). Cells before (A.D,G,J) photobleaching, immediately after (B,E,H,K) photobleaching and 15 minutes following (C,F,I,L) photobleaching. Outlined areas indicate photobleached regions. Fluorescence of pre-bleached cells was intensified during imaging to insure that the fluorescence signal of the post-photobleached cells was sufficiently high to accurately measure transport.

View larger version (13K): [in a new window]   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.

View larger version (16K): [in a new window]   Fig. 4. Cellular distribution of GFP-Hsp70 and GFP-Hsp70(K71E) in control and heat-shocked cells. HeLa cells transfected with GFP-Hsp70 (Fig. 4A) or GFP-Hsp70(K71E) (Fig. 4B) were imaged before (black bars) and immediately following (white bars) heat shock. The average fluorescence intensities of the cytosol and the nucleus were measured and the ratio of the fluorescence of the nucleus relative to the cytosol was calculated. A minimum of 250 cells per experimental condition were imaged and the percent of cells with each Hsp70 distribution range was determined. Hsp70 distribution was calculated as percentage of the total cells imaged.

View larger version (31K): [in a new window]   Fig. 5. Mobility of GFP-Hsp70 and GFP-Hsp70 (K71E) relative to GFP in (A) cytosol and (B) nucleus. GFP or GFP-Hsp70 were bleached and the fluorescence recovery after photobleaching was measured. The fluorescence recovery after photobleaching is shown for GFP (), GFP-Hsp70 () and GFP-Hsp70(K71E) ().

View larger version (39K): [in a new window]   Fig. 6. Heat shock affects mobility of GFP-Hsp70 in the nucleus and GFP-Hsp70(K71E) in the nucleus and cytoplasm. HeLa cells were heat shocked and the fluorescence recovery after photobleaching was measured. (A) fluorescence recovery of GFP vector in the nucleus(), GFP-Hsp70 in the cytoplasm (), GFP-Hsp70 in the nucleoplasm () and GFP-Hsp70 in the nucleolus (solid circles). (B) fluorescence recovery of GFP-Hsp70(K71E) in the cytoplasm () and nucleoplasm ().

View larger version (95K): [in a new window]   Fig. 7. GFP-Hsp70 or GFP-CBP shells around RFP-Htt(Q72) inclusions. HeLa cells cotransfected with RFP-Htt(Q72) and either GFP-Hsp70 or GFP-CBP were imaged 48 hours after transfection. (A,D,G) RFP-Htt(Q72); (B) GFP-Hsp70; (H) E:GFP-CBP. Merged images are C, F and I.

View larger version (18K): [in a new window]   Fig. 8. Interaction of GFP-Hsp70 and GFP-Hsp70(K71E) with Htt inclusions. Cells were transfected with RFP-Htt(Q72) and either GFP-Hsp70 or GFP-Hsp70(K71E). GFP-Hsp70 () or GFP-Hsp70(K71E) () shells around Htt inclusions were photobleached.

View larger version (33K): [in a new window]   Fig. 9. Effect of Htt(Q72) on the mobility of GFP-Hsp70 and GFP-Hsp70(K71E) in the nucleoplasm. HeLa cells were cotransfected with myc-tagged Htt(Q72) and either GFP-Hsp70 () or GFP-Hsp70(K71E) () and then imaged 48 hours after transfection. The dotted line, superimposed on the solid circles, was obtained for the GFP-Hsp70 data in Fig. 5B while the dashed line was obtained for the GFP-Hsp70(K71E) data in Fig. 5B. The data sets in Fig. 5B were obtained under the identical conditions as the data obtained in the presence of Htt.