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First published online 11 December 2002
doi: 10.1242/jcs.00253

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Heat shock and Cd2+ exposure regulate PML and Daxx release from ND10 by independent mechanisms that modify the induction of heat-shock proteins 70 and 25 differently

Isabelle Nefkens1,*, Dmitri G. Negorev1,*, Alexander M. Ishov1, Jennifer S. Michaelson2, Edward T. H. Yeh3, Robert M. Tanguay4, Werner E. G. Müller5 and Gerd G. Maul1,{ddagger}

1 The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA
2 Department of Explorative Science, Biogen, Cambridge, MA 02142, USA
3 The University of Texas-Houston Health Science Center, Houston, TX 77030, USA
4 Laboratory of Cell and Developmental Genetics, Department of Medicine, Pav. Marchand, Université Laval, Ste-Foy, QC, G1K 7P4, Canada
5 Institut für Physiologische Chemie, Universität Mainz, D-55099, Mainz, Germany



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  		Fig. 1. Immunolocalization of ND10-associated proteins after heat shock (HS) of HEp-2 cells. The proteins labeled in each panel are indicated in the upper left and right of the panel in the relevant color. Duration and extent of thermal stress and times of recovery are given at the bottom of each panel. (A) Control cells triple-labeled for Daxx, SC35 and coilin. (B) Control cells triple-labeled for Daxx heat-shock factor (HSF1) and nucleoli/nuclear envelope (NO/NE, fibrillarin and lamin B by a mixture of two human autoantibodies). (C) Control cells labeled for PML and Daxx. Both proteins colocalize to ND10 but some staining is found throughout the nucleus, excluding the nucleolus. (D) Control cells labeled for Sp100 and SUMO-1. Most Sp100-positive sites are also positive for SUMO-1. (E) Same as A but after 12 minutes of heat shock. Daxx is dispersed but not SC35 or coilin. (F) Same as B but after 12 minutes of heat shock. Daxx is dispersed but not the nucleolus and nuclear envelope. There is a slight repositioning of HSF1. (G) Same as C but exposed to 42°C for 12 minutes. More PML- and Sp100-containing sites are detected. Not all the new small sites contain both proteins. (H) Same as D but exposed to 42°C for 12 minutes. SUMO-1 is absent from all PML aggregates. (I) Same as A but exposed to 42°C for 1 hour. Little or no Daxx is seen in the PML aggregates but SC35 and coilin are maintained in their normal distribution. (J) Same as B but exposed to 42°C for 30 hours. Daxx is totally dispersed and fibrillarin has left the nucleolus. HSF1 has aggregated in specific domains. (K) Same as C but exposed to 42°C for 1 hour. Daxx is absent from PML aggregates. (L) Same as D but exposed to 42°C for 1 hour. Neither SUMO-1 nor Sp100 is present in concentrated aggregates, although Sp100 is still present in a `sandy' distribution in some cells (top). (M) Cells exposed to 42°C for 1 hour were allowed to recover for 2 hours at 37°C. SUMO-1 starts to reappear in some PML aggregates. (N) Different cells within the same experiment as in M but showing the variation between no recovery of Daxx at ND10 and localized return of Daxx to a subset of ND10, demonstrating the high diversity of the recovery process. (O) Apparent total segregation of Daxx and PML to a few sites after 5 hours of recovery from HS.

 


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  		Fig. 2. Immunolocalization of ND10-associated proteins after Cd2+ exposure of HEp-2 cells. Proteins shown are indicated at the upper left and right of each panel in the color used. For control images, see Fig. 1A-C. (A) Daxx, SC35 and coilin. Daxx is totally dispersed, whereas SC35 and coilin are apparently distributed as in control cells. (B) The nuclear envelope and HSF1. Daxx is dispersed and HSF1 is aggregated, whereas the nuclear envelope and nucleoli appear intact. (C) Most but not all cells exposed to 50 µM Cd2+ for 4 hours show Daxx and PML dispersed. (D) Same as A but stained for SUMO and Sp100. SUMO is dispersed but most cells retain a few Sp100 aggregates. (E) Cells treated with SB203580 for 3 hours and exposed to 50 µM Cd2+ for 5 hours and labeled for Sp100 and SUMO-1. ND10 are not dispersed. (F) HEp-2 cells exposed to PD98059 for 5 hours and labeled for Sp100 and SUMO-1. ND10 are not dispersed.

 


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  		Fig. 3. Quantitative evaluation of ND10 reformation after dispersion by 1 hour of heat shock (42°C). (A) Decrease in frequency of PML aggregates after increasing recovery periods. (B) Increase of Daxx aggregates colocalizing with the PML dots after increasing recovery periods.

 


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  		Fig. 4. Disappearance of slower mobility isoforms of PML and Sp100 upon heat shock (HS). (A) Western blot of HEp-2 cells exposed to 42°C for various times and probed for PML. (B) Same blot as A after stripping and subsequent probing with anti-Daxx. (C) Same blot as A after stripping and subsequent probing with anti-Sp100. Asterisks indicate the major non-conjugated form of PML and Sp100, respectively; arrows indicate the SUMO-1-conjugated forms of PML and Sp100.

 


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  		Fig. 5. Immunolocalization of ND10-associated proteins after transient expression of SENP-1. Proteins are indicated at the top of each panel in the color used. (A-D) HEp-2 cells triple-labeled for His-SENP-1, SUMO-1 and PML. (E-H) HEp-2 cells triple-labeled for His—SENP-1, Daxx and PML. (I-L) HEp-2 cells labeled for His-SENP-1, Sp100 and PML.

 


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  		Fig. 6. Production of Daxx in MEF-T and ES Daxx{Delta}260-Tcells. Untreated cells were probed by western blotting with anti-Daxx antibodies. Arrowheads indicate the normal Daxx and the truncated Daxx{Delta}260 mutant.

 


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  		Fig. 7. Induction of Hsp70 and Hsp25 in different cell lines lacking PML or Daxx. Heat-shock (HS) factor 1 (HSF1) and tubulin as a loading control were also probed by western blotting on the same transfer membrane.

 


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  		Fig. 8. Hsp70 and Hsp25 mRNA accumulation after 1 hour of hyperthermal stress. PML-/-, Daxx-/- cells and their respective control cells were exposed to 1 hour of 42°C (HS) and increasing hours of recovery times. Levels of accumulated mRNA were analyzed by reverse-transcription PCR. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as a non-inducible control.

 


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  		Fig. 9. Effect of CdCl2 exposure on the production of Hsp70 and Hsp25. PML+/+ and PML-/- cells were exposed to different concentrations of CdCl2 and probed for various proteins by western blotting using the same transfer membrane after stripping.

 

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