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Hsp70 architecture: the formation of novel polymeric structures of Hsp70.1 and Hsc70 after proteotoxic stress.

Steel R, Cross RS, Ellis SL, Anderson RL - PLoS ONE (2012)

Bottom Line: In heated fibroblasts expressing Hsp70.1, Hsp70.1 and Hsc70 complexes are efficiently disaggregated before the cells undergo-heat induced apoptosis.In the absence of Hsp70.1, fibroblasts have increased rates of heat-induced apoptosis and maintain stable insoluble Hsc70 structures.The differences in the intracellular distribution of Hsp70.1 and Hsc70, combined with the ability of Hsp70.1, but not Hsc70, to promote the disaggregation of insoluble Hsp70.1/Hsc70 complexes, indicate that these two closely related proteins perform distinctly different cellular functions in heated cells.

View Article: PubMed Central - PubMed

Affiliation: Peter MacCallum Cancer Centre, St Andrew's Place, East Melbourne, Victoria, Australia.

ABSTRACT
Heat induces Hsp70.1 (HSPA1) and Hsc70 (HSPA8) to form complex detergent insoluble cytoplasmic and nuclear structures that are distinct from the cytoskeleton and internal cell membranes. These novel structures have not been observed by earlier immunofluorescence studies as they are obscured by the abundance of soluble Hsp70.1/Hsc70 present in cells. While resistant to detergents, these Hsp70 structures display complex intracellular dynamics and are efficiently disaggregated by ATP, indicating that this pool of Hsp70.1/Hsc70 retains native function and regulation. Hsp70.1 promotes the repair of proteotoxic damage and cell survival after stress. In heated fibroblasts expressing Hsp70.1, Hsp70.1 and Hsc70 complexes are efficiently disaggregated before the cells undergo-heat induced apoptosis. In the absence of Hsp70.1, fibroblasts have increased rates of heat-induced apoptosis and maintain stable insoluble Hsc70 structures. The differences in the intracellular distribution of Hsp70.1 and Hsc70, combined with the ability of Hsp70.1, but not Hsc70, to promote the disaggregation of insoluble Hsp70.1/Hsc70 complexes, indicate that these two closely related proteins perform distinctly different cellular functions in heated cells.

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Proteotoxic stress induces Hsp70.1/Hsc70 binding to insoluble cell structures.MEF-H2 were maintained at 37°C (unheated) or heated at 44°C for 20 min and fixed immediately with 4% paraformaldehyde. Hsp70.1 and Hsc70 were detected by immunofluorescence (green) and the nuclei stained with PI (red): Hsp70.1, unheated (A); Hsp70.1, heated (B); Hsc70, unheated (E); Hsc70, heated (F). MEF-H2 were treated as described above but permeabilized with 100 µg/ml digitonin prior to fixation: Hsp70.1, unheated (C); Hsp70.1, heated (D); Hsc70, unheated (G); Hsc70, heated (H). MEF-H2 heated at 44°C for 2 min, permeabilized, Hsp70.1 staining (I); MEF-H2 cells heated at 44°C for 20 min, digitonin permeabilized and stained for Hsp70.1 (J) and Hsc70 (K); MEF-H2 heated at 44°C for 20 min, double stained for Hps70.1 (green) and Hsc70 (red) (L). Scale bars represent 20 µm.
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pone-0052351-g001: Proteotoxic stress induces Hsp70.1/Hsc70 binding to insoluble cell structures.MEF-H2 were maintained at 37°C (unheated) or heated at 44°C for 20 min and fixed immediately with 4% paraformaldehyde. Hsp70.1 and Hsc70 were detected by immunofluorescence (green) and the nuclei stained with PI (red): Hsp70.1, unheated (A); Hsp70.1, heated (B); Hsc70, unheated (E); Hsc70, heated (F). MEF-H2 were treated as described above but permeabilized with 100 µg/ml digitonin prior to fixation: Hsp70.1, unheated (C); Hsp70.1, heated (D); Hsc70, unheated (G); Hsc70, heated (H). MEF-H2 heated at 44°C for 2 min, permeabilized, Hsp70.1 staining (I); MEF-H2 cells heated at 44°C for 20 min, digitonin permeabilized and stained for Hsp70.1 (J) and Hsc70 (K); MEF-H2 heated at 44°C for 20 min, double stained for Hps70.1 (green) and Hsc70 (red) (L). Scale bars represent 20 µm.

Mentions: Immunofluorescence has been used extensively to observe Hsp70.1 and Hsc70 in mammalian cells, but aside from the heat induced localization of these two proteins to the nucleus and nucleolus, immunofluorescence has revealed little detail of Hsp70 function [22], [23], [24]. In formaldehyde fixed MEF-H2 that constitutively express exogenous human Hsp70.1 [10], Hsp70.1 displayed a diffuse nuclear and cytoplasmic distribution, indicative of a soluble protein (Fig. 1A). Little change in Hsp70.1 distribution was observed immediately after heating, although there was a small increase in protein present in the nucleus (Fig. 1B).


Hsp70 architecture: the formation of novel polymeric structures of Hsp70.1 and Hsc70 after proteotoxic stress.

Steel R, Cross RS, Ellis SL, Anderson RL - PLoS ONE (2012)

Proteotoxic stress induces Hsp70.1/Hsc70 binding to insoluble cell structures.MEF-H2 were maintained at 37°C (unheated) or heated at 44°C for 20 min and fixed immediately with 4% paraformaldehyde. Hsp70.1 and Hsc70 were detected by immunofluorescence (green) and the nuclei stained with PI (red): Hsp70.1, unheated (A); Hsp70.1, heated (B); Hsc70, unheated (E); Hsc70, heated (F). MEF-H2 were treated as described above but permeabilized with 100 µg/ml digitonin prior to fixation: Hsp70.1, unheated (C); Hsp70.1, heated (D); Hsc70, unheated (G); Hsc70, heated (H). MEF-H2 heated at 44°C for 2 min, permeabilized, Hsp70.1 staining (I); MEF-H2 cells heated at 44°C for 20 min, digitonin permeabilized and stained for Hsp70.1 (J) and Hsc70 (K); MEF-H2 heated at 44°C for 20 min, double stained for Hps70.1 (green) and Hsc70 (red) (L). Scale bars represent 20 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3526589&req=5

pone-0052351-g001: Proteotoxic stress induces Hsp70.1/Hsc70 binding to insoluble cell structures.MEF-H2 were maintained at 37°C (unheated) or heated at 44°C for 20 min and fixed immediately with 4% paraformaldehyde. Hsp70.1 and Hsc70 were detected by immunofluorescence (green) and the nuclei stained with PI (red): Hsp70.1, unheated (A); Hsp70.1, heated (B); Hsc70, unheated (E); Hsc70, heated (F). MEF-H2 were treated as described above but permeabilized with 100 µg/ml digitonin prior to fixation: Hsp70.1, unheated (C); Hsp70.1, heated (D); Hsc70, unheated (G); Hsc70, heated (H). MEF-H2 heated at 44°C for 2 min, permeabilized, Hsp70.1 staining (I); MEF-H2 cells heated at 44°C for 20 min, digitonin permeabilized and stained for Hsp70.1 (J) and Hsc70 (K); MEF-H2 heated at 44°C for 20 min, double stained for Hps70.1 (green) and Hsc70 (red) (L). Scale bars represent 20 µm.
Mentions: Immunofluorescence has been used extensively to observe Hsp70.1 and Hsc70 in mammalian cells, but aside from the heat induced localization of these two proteins to the nucleus and nucleolus, immunofluorescence has revealed little detail of Hsp70 function [22], [23], [24]. In formaldehyde fixed MEF-H2 that constitutively express exogenous human Hsp70.1 [10], Hsp70.1 displayed a diffuse nuclear and cytoplasmic distribution, indicative of a soluble protein (Fig. 1A). Little change in Hsp70.1 distribution was observed immediately after heating, although there was a small increase in protein present in the nucleus (Fig. 1B).

Bottom Line: In heated fibroblasts expressing Hsp70.1, Hsp70.1 and Hsc70 complexes are efficiently disaggregated before the cells undergo-heat induced apoptosis.In the absence of Hsp70.1, fibroblasts have increased rates of heat-induced apoptosis and maintain stable insoluble Hsc70 structures.The differences in the intracellular distribution of Hsp70.1 and Hsc70, combined with the ability of Hsp70.1, but not Hsc70, to promote the disaggregation of insoluble Hsp70.1/Hsc70 complexes, indicate that these two closely related proteins perform distinctly different cellular functions in heated cells.

View Article: PubMed Central - PubMed

Affiliation: Peter MacCallum Cancer Centre, St Andrew's Place, East Melbourne, Victoria, Australia.

ABSTRACT
Heat induces Hsp70.1 (HSPA1) and Hsc70 (HSPA8) to form complex detergent insoluble cytoplasmic and nuclear structures that are distinct from the cytoskeleton and internal cell membranes. These novel structures have not been observed by earlier immunofluorescence studies as they are obscured by the abundance of soluble Hsp70.1/Hsc70 present in cells. While resistant to detergents, these Hsp70 structures display complex intracellular dynamics and are efficiently disaggregated by ATP, indicating that this pool of Hsp70.1/Hsc70 retains native function and regulation. Hsp70.1 promotes the repair of proteotoxic damage and cell survival after stress. In heated fibroblasts expressing Hsp70.1, Hsp70.1 and Hsc70 complexes are efficiently disaggregated before the cells undergo-heat induced apoptosis. In the absence of Hsp70.1, fibroblasts have increased rates of heat-induced apoptosis and maintain stable insoluble Hsc70 structures. The differences in the intracellular distribution of Hsp70.1 and Hsc70, combined with the ability of Hsp70.1, but not Hsc70, to promote the disaggregation of insoluble Hsp70.1/Hsc70 complexes, indicate that these two closely related proteins perform distinctly different cellular functions in heated cells.

Show MeSH
Related in: MedlinePlus