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

Abstract

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.

Keywords: FRAP; HSPA1A (Hsp70–1); HSPA6 (Hsp70B’); Live imaging; SH-SY5Y.

Fig. 1

YFP-tagged HSPA6 and HSPA1A were recruited to and dynamically associated with centrioles after thermal stress in differentiated human neuronal cells. a Live imaging demonstrated that YFP-HSPA6 and YFP-HSPA1A localized to putative centrioles (indicated by boxed areas) in the cytoplasm of differentiated human neuronal SH-SY5Y cells following heat shock at 43 °C for 20 min. 0 min = prior to heat shock. 20 min = after 20 min heat shock. Scale bar = 10 μm. b Time course of the localization of YFP-HSPA6 and YFP-HSPA1A with putative centrioles by live imaging. FI fluorescence intensity. Scale bar = 1.5 μm. c The centriole-specific marker protein, γ-tubulin, confirmed that the YFP-tagged HSPA6- and HSPA1A-positive cytoplasmic structures were centrioles following immunocytochemistry on fixed-cell preparations. Scale bars = 10 and 1.5 μm. d Upper panel Live imaging of the time course of photobleaching recovery of YFP-HSPA6 and YFP-HSPA1A after FRAP of individual centrioles. Pre-B pre-bleached. Scale bar = 1.5 μm; Lower panel FRAP data of YFP-tagged HSPA6 and HSPA1A at centrioles (n = 25) from multiple photobleaching experiments

Fig. 2

Targeting and dynamic interaction of YFP-tagged HSPA6 and HSPA1A with nuclear speckles following heat shock. a YFP-HSPA6 and YFP-HSPA1A rapidly localized to nuclear components (indicated by dashed arrows) in human neuronal cells after thermal stress. 20 min = after 20 min heat shock. Scale bar = 10 μm. b Time course of the association of YFP-tagged HSPA6 and HSPA1A with nuclear components. Scale bar = 1.5 μm. c Nuclear YFP-HSPA6 and YFP-HSPA1A colocalized with the nuclear speckle marker, SON. Scale bar = 10 and 1.5 μm. d Upper panel FRAP recovery profiles of an individual nuclear speckle after photobleaching. Scale bar = 1.5 μm. Lower panel FRAP data of YFP-tagged HSPA6 and HSPA1A at nuclear speckles (n = 25)

Fig. 3

Localization of YFP-tagged HSPA6 and HSPA1A to the nucleolus in human neuronal cells after thermal stress. a YFP-HSPA6 and YFP-HSPA1A subsequently localized to donut-shaped components in neuronal nuclei (indicated by solid arrows). 100 min = 100 min after start of heat shock. Scale bar = 10 μm. b Association of YFP-tagged HSPA6 and HSPA1A with donut-shaped nuclear structures. Scale bar = 1.5 μm. c nucleophosmin (NP), a marker protein for the granular component of the nucleolus, colocalized with the donut-shaped nuclear structures that are YFP-tagged HSPA6 and HSPA1A positive. Scale bar = 10 μm and 1.5 μm. d Upper panel Photobleaching recovery profiles of individual YFP-HSPA6 and YFP-HSPA1A positive nucleoli. Scale bar = 1.5 μm. Lower panel FRAP data of YFP-tagged HSPA6 and HSPA1A at the nucleolus (n = 25)

Fig. 4

Following neuronal stress, YFP-HSPA6, but not YFP-HSPA1A, demonstrated a highly dynamic association with the periphery of nuclear speckles (perispeckles). a Following association with nucleolar components, YFP-HSPA6 localized to bright star-like foci (indicated by arrowheads) in the nucleus of neuronal cells, whereas YFP-HSPA1A did not. 180 min = 180 min after start of heat shock. Scale bar = 10 μm. b Localization of YFP-HSPA6 to bright star-like nuclear foci. Scale bar = 1.5 μm. c Bright star-like nuclear foci (termed “perispeckles”) that are YFP-HSPA6 positive (green signal indicated by arrowheads) are located at the periphery of SON-positive nuclear speckles (red signal indicated by dotted arrows). Scale bars = 10 and 1.5 μm. d Upper panel Recovery profile after photobleaching of an individual YFP-HSPA6 positive perispeckle. Scale bar = 1.5 μm. Lower panel FRAP data of YFP-tagged HSPA6 and HSPA1A at perispeckles (n = 15).