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

Abstract

The molecular chaperone Hsp70 interacts with misfolded proteins and also accumulates in the nucleus during heat shock. Using GFP-Hsp70 and fluorescence recovery after photobleaching, we show that Hsp70 accumulates in the nucleus during heat shock not only because its inflow rate increases but also because of a marked decrease in its outflow rate. Dynamic imaging also shows that GFP-Hsp70 has greatly reduced mobility when it interacts with organelles such as nucleoli in heat-shocked cells or the large inclusions formed from fragments of mutant huntingtin protein. In heat-shocked cells, nucleoplasmic Hsp70 has reduced mobility relative to the cytoplasm, whereas the ATPase-deficient mutant of Hsp70, Hsp70(K71E), is almost completely immobilized both in the nucleoplasm and the cytoplasm. Moreover, the Hsp70 mutant shows reduced mobility in the presence of diffusive huntingtin fragments with expanded polyglutamine repeats. This provides strong evidence that Hsp70 interacts not only with organelles but also with diffusive proteins in the nucleoplasm and cytoplasm during heat shock as well as with diffusive huntingtin fragments.

ATP Binding Regulates Oligomerization and Endosome Association of RME-1 Family Proteins

“…When data sets were compared, identical conditions were used in photobleaching the cells including the number of bleaches, the area of the photobleach region, and the time course of imaging at low laser power. Measuring diffusive GFP constructs on a fast time scale, we used the method as described in Zeng et al (21).…”

Depletion of GAK/auxilin 2 inhibits receptor-mediated endocytosis and recruitment of both clathrin and clathrin adaptors

“…The following constructs were used to transfect HeLa cells: GFPclathrin light chain a, DsRed-clathrin light chain a, GFP-AP2 (␣-chain labeled), GFP-GGA3 (a gift from R. Puertollano, NIH, Bethesda, MD, USA), GFP-epsin (a gift from P. De Camilli, Yale University, New Haven, CT, USA), flag-tagged Hsp70, and flag-tagged Hsp70(K71E) Wu et al, 2003;Zeng et al, 2004). To make mRFP-GAK, we used mRFP expression vector (gift from R. Tsien, Stanford University, Stanford, CA, USA) and then subcloned GAK into EcoRI and KpnI restriction sites.…”

Cellular prion protein (PrPC) protects neuronal cells from the effect of huntingtin aggregation

“…The Htt polyglutamine constructs (HttQ25 and HttQ103) containing glutamine repeat expansion had an EGFP-tag on its C-terminal (Zeng et al, 2004). A mouse prion gene with the hamster epitope tag (a gift from S. Priola, Rocky Mountain Lab, MT) and a hamster prion gene (a gift from D. Ramanujan Hegde, NIH, Bethesda, MD) was subcloned using HindIII and XhoI restriction sites into pCDNA4 vector (Invitrogen).…”

“…A mouse prion gene with the hamster epitope tag (a gift from S. Priola, Rocky Mountain Lab, MT) and a hamster prion gene (a gift from D. Ramanujan Hegde, NIH, Bethesda, MD) was subcloned using HindIII and XhoI restriction sites into pCDNA4 vector (Invitrogen). Cells were seeded and transfected with Htt plasmids as described previously (Zeng et al, 2004). At 48 hours after transfection, cells on coverslips were fixed with 4% paraformaldehyde and 3 g/ml 4Ј,6-diamidino-2-phenylindole (Invitrogen) in 1ϫ PBS at room temperature for 20 minutes and mounted.…”