Differential Targeting of Hsp70 Heat Shock Proteins HSPA6 and HSPA1A with Components of a Protein Disaggregation/Refolding Machine in Differentiated Human Neuronal Cells following Thermal Stress

Abstract

Heat shock proteins (Hsps) co-operate in multi-protein machines that counter protein misfolding and aggregation and involve DNAJ (Hsp40), HSPA (Hsp70), and HSPH (Hsp105α). The HSPA family is a multigene family composed of inducible and constitutively expressed members. Inducible HSPA6 (Hsp70B') is found in the human genome but not in the genomes of mouse and rat. To advance knowledge of this little studied HSPA member, the targeting of HSPA6 to stress-sensitive neuronal sites with components of a disaggregation/refolding machine was investigated following thermal stress. HSPA6 targeted the periphery of nuclear speckles (perispeckles) that have been characterized as sites of transcription. However, HSPA6 did not co-localize at perispeckles with DNAJB1 (Hsp40-1) or HSPH1 (Hsp105α). At 3 h after heat shock, HSPA6 co-localized with these members of the disaggregation/refolding machine at the granular component (GC) of the nucleolus. Inducible HSPA1A (Hsp70-1) and constitutively expressed HSPA8 (Hsc70) co-localized at nuclear speckles with components of the machine immediately after heat shock, and at the GC layer of the nucleolus at 1 h with DNAJA1 and BAG-1. These results suggest that HSPA6 exhibits targeting features that are not apparent for HSPA1A and HSPA8.

Keywords: DNAJ (Hsp40); HSPA1A (Hsp70-1); HSPA6 (Hsp70B'); HSPA8 (Hsc70); HSPH1 (Hsp105α); human neuronal SH-SY5Y cells.

Figure 1

HSPA6 was targeted to the periphery of nuclear speckles (perispeckles) following heat shock in differentiated human neuronal cells. (A) Prior to heat shock, HSPA6 (green) was distributed in the neuronal cytoplasm. After heat shock, HSPA6 localized to foci at the periphery of nuclear speckles (closed arrowheads) identified by the marker protein SON (red, open arrowheads). DAPI (blue) was used to identify neuronal nuclei. ImageJ line scans demonstrated that HSPA6 fluorescent peaks were offset from SON peaks. ImageJ line scans confirm that (B) DNAJB1, (C) HSPH1, (D) HSPB1, and (E) HSPA8 did not co-localize with HSPA6. (F) HSPA6-positive foci co-localized with the perispeckle marker RNA polymerase II (closed arrowheads). Scale bar represents 5 μm (A–F). Inset scale bar in (A) represents 0.5 μm.

Figure 2

HSPA1A co-localized at nuclear speckles with components of a mammalian protein disaggregation/refolding machine after thermal stress. (A) Prior to heat shock, HSPA1A (green) was distributed in the neuronal cytoplasm. After heat shock, HSPA1A co-localized with the nuclear speckle marker protein SON (red, open arrowheads) at 20 min and 1 h, confirmed by ImageJ line scans. DAPI (blue) was used to identify neuronal nuclei. Components of a mammalian protein disaggregation/refolding machine including (B) DNAJB1, (C) HSPH1, and (D) HSPB1, also targeted nuclear speckles (open arrowheads) after heat shock at 20 min and 1 h, as determined by co-localization with nuclear speckle markers SON (HSPA1A) and SC35 (HSPH1, DNAJB1, and HSPB1). Scale bar represents 5 μm.

Figure 3

HSPA1A, but not HSPA6, was targeted to the GC layer of the nucleolus at 1 h following heat shock. (A) At 1 h, HSPA1A (green, upper panel), but not HSPA6 (green, lower panel) co-localized with nucleophosmin (NPM) (red, arrow), a marker of the GC layer of the nucleolus. This localization was not observed at 20 min, confirmed by ImageJ line scans shown on the right. (B) DNAJA1 also co-localized with nucleophosmin at 1 h (arrows). (C) BAG-1 co-localized at 1 h with HSPA1A and DNAJA1 (arrows) that were shown to localize to the nucleolus in (A,B), however the “disaggregase” HSPH1 did not (D, arrow). The open arrowheads represent HSPA1A and HSPH1 targeting to nuclear speckles, previously shown in Figures 2A,C. Scale bar represents 5 μm.

Figure 4

At 3 h after thermal stress, HSPA6 co-localized with nucleophosmin, a marker of the granular component (GC) of the nucleolus. (A) HSPA6 co-localized at the 3 h time point with a marker of the GC layer of the nucleolus, nucleophosmin (arrow, upper panel), but not with the nuclear speckle marker SON (open arrowheads, lower panel). (B) HSPA1A did not co-localize with nucleophosmin (upper panel) or SON (lower panel) at 3 h. (C) Components of a mammalian disaggregation/refolding machine, including DNAJB1 and HSPH1, were also targeted to the GC layer of the nucleolus at 3 h (arrows), however (D) BAG-1 was not. DAPI (blue in merged panels and ImageJ line scans) was used to identify neuronal nuclei. Scale bar represents 5 μm.

Figure 5

Constitutively expressed HSPA8 exhibited similar heat shock-induced targeting as HSPA1A, however HSPA6 did not. (A) HSPA8 targeted SON-positive nuclear speckles at 20 min and 1 h after heat shock. (B) HSPA8 also targeted the GC layer of the nucleolus (identified by the marker protein nucleophosmin) at 1 h and co-localized with HSPA1A. (C) HSPA8 did not co-localize with HSPA6 at 20 min and 1 h at perispeckles or (D) at the GC layer of the nucleolus at 3 h. DAPI (blue) was used to identify neuronal nuclei. Scale bar represents 5 μm.