Dynamics of the association of heat shock protein HSPA6 (Hsp70B’) and HSPA1A (Hsp70–1) with stress-sensitive cytoplasmic and nuclear structures in differentiated human neuronal cells
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Heat shock proteins (Hsps) are cellular repair agents that counter the effects of protein misfolding that is a characteristic feature of neurodegenerative diseases. HSPA1A (Hsp70–1) is a widely studied member of the HSPA (Hsp70) family. The little-studied HSPA6 (Hsp70B’) is present in the human genome and absent in mouse and rat; hence, it is missing in current animal models of neurodegenerative diseases. Differentiated human neuronal SH-SY5Y cells were employed to compare the dynamics of the association of YFP-tagged HSPA6 and HSPA1A with stress-sensitive cytoplasmic and nuclear structures. Following thermal stress, live-imaging confocal microscopy and Fluorescence Recovery After Photobleaching (FRAP) demonstrated that HSPA6 displayed a prolonged and more dynamic association, compared to HSPA1A, with centrioles that play critical roles in neuronal polarity and migration. HSPA6 and HSPA1A also targeted nuclear speckles, rich in RNA splicing factors, and the granular component of the nucleolus that is involved in rRNA processing and ribosomal subunit assembly. HSPA6 and HSPA1A displayed similar FRAP kinetics in their interaction with nuclear speckles and the nucleolus. Subsequently, during the recovery from neuronal stress, HSPA6, but not HSPA1A, localized with the periphery of nuclear speckles (perispeckles) that have been characterized as transcription sites. The stress-induced association of HSPA6 with perispeckles displayed the greatest dynamism compared to the interaction of HSPA6 or HSPA1A with other stress-sensitive cytoplasmic and nuclear structures. This suggests involvement of HSPA6 in transcriptional recovery of human neurons from cellular stress that is not apparent for HSPA1A.
KeywordsHSPA6 (Hsp70B’) HSPA1A (Hsp70–1) FRAP Live imaging SH-SY5Y
This study is supported by grants from NSERC to I.R.B.
Supplementary Movie 1: YFP-HSPA6. Live imaging time sequence of the intracellular localization of YFP-tagged HSPA6 in differentiated human neuronal cells following a 20 min heat shock at 43 °C. The same conventions used in Figs. 1, 2, 3, 4 were employed in the movie: boxed areas = centrioles; dashed arrows = nuclear speckles; solid arrows = granular component of nucleolus; arrowheads = perispeckles. (AVI 88007 kb)
Supplementary Movie 2: YFP-HSPA1A. Live imaging time sequence of YFP-tagged HSPA1A intracellular localization following heat shock of differentiated human neuronal cells. The same conventions used in Figs. 1, 2, 3, 4 were employed in the movie: boxed areas = centrioles; dashed arrows = nuclear speckles; solid arrows = granular component of nucleolus. (AVI 92712 kb)
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