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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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Hsp70.1/Hsc70 complexes are disassociated by ATP but not by detergent.MEF-H2 stained for Hsp70.1 (A, B, C, D, I, J, K) or Hsc70 (E, F, G, H) (green). Nuclei stained with PI (red). All heated cells were incubated at 44°C for 20 min: unheated/digitonin permeabilized (A, E); heated/digitonin permeabilized (B, F); heated/digitonin permeabilized/incubated with PBS at room temp for 10 min (C, G); heated/digitonin permeabilized/incubated with 2 mM ATP at RT for 10 min (D, H). Unheated MEF-H2 permeabilized with 0.2% TX100 (I); heated and permeabilized with 0.2% TX100 (J); heated and permeabilized with RIPA (K). Scale bars represent 20 µm.
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pone-0052351-g002: Hsp70.1/Hsc70 complexes are disassociated by ATP but not by detergent.MEF-H2 stained for Hsp70.1 (A, B, C, D, I, J, K) or Hsc70 (E, F, G, H) (green). Nuclei stained with PI (red). All heated cells were incubated at 44°C for 20 min: unheated/digitonin permeabilized (A, E); heated/digitonin permeabilized (B, F); heated/digitonin permeabilized/incubated with PBS at room temp for 10 min (C, G); heated/digitonin permeabilized/incubated with 2 mM ATP at RT for 10 min (D, H). Unheated MEF-H2 permeabilized with 0.2% TX100 (I); heated and permeabilized with 0.2% TX100 (J); heated and permeabilized with RIPA (K). Scale bars represent 20 µm.

Mentions: The binding of Hsp70.1 and Hsc70 to protein substrates via the peptide-binding domain is inhibited by ATP [26]. As shown in Fig. 2A–H, the incubation of digitonin permeabilized MEF-H2 with 2 mM ATP efficiently extracted the immobilized Hsp70.1 and Hsc70 (Fig. 2 D & H). Incubating cells in PBS under identical conditions had little impact on the binding of both proteins although some degradation of the internal cell structures was observed (Fig. 2 C & G). The efficient extraction of Hsp70.1 and Hsc70 by ATP confirms that the aggregation of Hsp70.1/Hsc70 is dependent on interactions involving the substrate binding domain, regulated by the ATP binding domain, and is not due merely to Hsp70.1/Hsc70 denaturation.


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)

Hsp70.1/Hsc70 complexes are disassociated by ATP but not by detergent.MEF-H2 stained for Hsp70.1 (A, B, C, D, I, J, K) or Hsc70 (E, F, G, H) (green). Nuclei stained with PI (red). All heated cells were incubated at 44°C for 20 min: unheated/digitonin permeabilized (A, E); heated/digitonin permeabilized (B, F); heated/digitonin permeabilized/incubated with PBS at room temp for 10 min (C, G); heated/digitonin permeabilized/incubated with 2 mM ATP at RT for 10 min (D, H). Unheated MEF-H2 permeabilized with 0.2% TX100 (I); heated and permeabilized with 0.2% TX100 (J); heated and permeabilized with RIPA (K). Scale bars represent 20 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3526589&req=5

pone-0052351-g002: Hsp70.1/Hsc70 complexes are disassociated by ATP but not by detergent.MEF-H2 stained for Hsp70.1 (A, B, C, D, I, J, K) or Hsc70 (E, F, G, H) (green). Nuclei stained with PI (red). All heated cells were incubated at 44°C for 20 min: unheated/digitonin permeabilized (A, E); heated/digitonin permeabilized (B, F); heated/digitonin permeabilized/incubated with PBS at room temp for 10 min (C, G); heated/digitonin permeabilized/incubated with 2 mM ATP at RT for 10 min (D, H). Unheated MEF-H2 permeabilized with 0.2% TX100 (I); heated and permeabilized with 0.2% TX100 (J); heated and permeabilized with RIPA (K). Scale bars represent 20 µm.
Mentions: The binding of Hsp70.1 and Hsc70 to protein substrates via the peptide-binding domain is inhibited by ATP [26]. As shown in Fig. 2A–H, the incubation of digitonin permeabilized MEF-H2 with 2 mM ATP efficiently extracted the immobilized Hsp70.1 and Hsc70 (Fig. 2 D & H). Incubating cells in PBS under identical conditions had little impact on the binding of both proteins although some degradation of the internal cell structures was observed (Fig. 2 C & G). The efficient extraction of Hsp70.1 and Hsc70 by ATP confirms that the aggregation of Hsp70.1/Hsc70 is dependent on interactions involving the substrate binding domain, regulated by the ATP binding domain, and is not due merely to Hsp70.1/Hsc70 denaturation.

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