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Cell Stress and Chaperones

, Volume 21, Issue 6, pp 993–1003 | Cite as

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

  • Sadek Shorbagi
  • Ian R. BrownEmail author
Original Paper

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

HSPA6 (Hsp70B’) HSPA1A (Hsp70–1) FRAP Live imaging SH-SY5Y 

Notes

Acknowledgments

This study is supported by grants from NSERC to I.R.B.

Supplementary material

Fig. S1

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)

Fig. S2

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)

References

  1. Agholme L, Lindstrom T, Kagedal K, Marcusson J, Hallbeck M (2010) An in vitro model for neuroscience: differentiation of SH-SY5Y cells into cells with morphological and biochemical characteristics of mature neurons. J Alzheimers Dis 20:1069–1082. doi: 10.3233/JAD-2010-091363 PubMedGoogle Scholar
  2. Allen TA, Von Kaenel S, Goodrich JA, Kugel JF (2004) The SINE-encoded mouse B2 RNA represses mRNA transcription in response to heat shock. Nat Struct Mol Biol 11:816–821. doi: 10.1038/nsmb813 CrossRefPubMedGoogle Scholar
  3. Asea AA, Brown IR (eds) (2008) Heat shock proteins and the brain: implications for neurodegenerative diseases and neuroprotection. Springer Science + Business Media B.V.Google Scholar
  4. Boisvert FM, van Koningsbruggen S, Navascues J, Lamond AI (2007) The multifunctional nucleolus. Nat Rev Mol Cell Biol 8:574–585. doi: 10.1038/nrm2184 CrossRefPubMedGoogle Scholar
  5. Borrell V, Reillo I (2012) Emerging roles of neural stem cells in cerebral cortex development and evolution. Dev Neurobiol 72:955–971. doi: 10.1002/dneu.22013 CrossRefPubMedGoogle Scholar
  6. Brito DA, Gouveia SM, Bettencourt-Dias M (2012) Deconstructing the centriole: structure and number control. Curr Opin Cell Biol 24:4–13. doi: 10.1016/j.ceb.2012.01.003 CrossRefPubMedGoogle Scholar
  7. Brown JM et al. (2008) Association between active genes occurs at nuclear speckles and is modulated by chromatin environment. J Cell Biol 182:1083–1097. doi: 10.1083/jcb.200803174 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Cheung YT, Lau WK, Yu MS, Lai CS, Yeung SC, So KF, Chang RC (2009) Effects of all-trans-retinoic acid on human SH-SY5Y neuroblastoma as in vitro model in neurotoxicity research. Neurotoxicology 30:127–135. doi: 10.1016/j.neuro.2008.11.001 CrossRefPubMedGoogle Scholar
  9. Chow AM, Brown IR (2007) Induction of heat shock proteins in differentiated human and rodent neurons by celastrol. Cell Stress Chaperones 12:237–244CrossRefPubMedPubMedCentralGoogle Scholar
  10. Chow AM, Mok P, Xiao D, Khalouei S, Brown IR (2010) Heteromeric complexes of heat shock protein 70 (HSP70) family members, including Hsp70B’, in differentiated human neuronal cells. Cell Stress Chaperones 15:545–553. doi: 10.1007/s12192-009-0167-0 CrossRefPubMedPubMedCentralGoogle Scholar
  11. de Anda FC, Meletis K, Ge X, Rei D, Tsai LH (2010) Centrosome motility is essential for initial axon formation in the neocortex. J Neurosci 30:10391–10406. doi: 10.1523/JNEUROSCI.0381-10.2010 CrossRefPubMedGoogle Scholar
  12. de Anda FC, Pollarolo G, Da Silva JS, Camoletto PG, Feiguin F, Dotti CG (2005) Centrosome localization determines neuronal polarity. Nature 436:704–708. doi: 10.1038/nature03811 CrossRefPubMedGoogle Scholar
  13. Deane CA, Brown IR (2016) Induction of heat shock proteins in differentiated human neuronal cells following co-application of celastrol and arimoclomol. Cell Stress Chaperones. doi: 10.1007/s12192-016-0708-2 PubMedGoogle Scholar
  14. Dunkel P, Chai CL, Sperlagh B, Huleatt PB, Matyus P (2012) Clinical utility of neuroprotective agents in neurodegenerative diseases: current status of drug development for Alzheimer’s, Parkinson’s and Huntington’s diseases, and amyotrophic lateral sclerosis. Expert Opin Investig Drugs 21:1267–1308. doi: 10.1517/13543784.2012.703178 CrossRefPubMedGoogle Scholar
  15. El Andaloussi-Lilja J, Lundqvist J, Forsby A (2009) TRPV1 expression and activity during retinoic acid-induced neuronal differentiation. Neurochem Int 55:768–774. doi: 10.1016/j.neuint.2009.07.011 CrossRefPubMedGoogle Scholar
  16. Espinoza CA, Goodrich JA, Kugel JF (2007) Characterization of the structure, function, and mechanism of B2 RNA, an ncRNA repressor of RNA polymerase II transcription. RNA 13:583–596. doi: 10.1261/rna.310307 CrossRefPubMedPubMedCentralGoogle Scholar
  17. Florio M et al. (2015) Human-specific gene ARHGAP11B promotes basal progenitor amplification and neocortex expansion. Science 347:1465–1470. doi: 10.1126/science.aaa1975 CrossRefPubMedGoogle Scholar
  18. Ge X, Frank CL, Calderon de Anda F, Tsai LH (2010) Hook3 interacts with PCM1 to regulate pericentriolar material assembly and the timing of neurogenesis. Neuron 65:191–203. doi: 10.1016/j.neuron.2010.01.011 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Geschwind DH, Rakic P (2013) Cortical evolution: judge the brain by its cover. Neuron 80:633–647. doi: 10.1016/j.neuron.2013.10.045 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Goodman T, Crandall JE, Nanescu SE, Quadro L, Shearer K, Ross A, McCaffery P (2012) Patterning of retinoic acid signaling and cell proliferation in the hippocampus. Hippocampus 22:2171–2183. doi: 10.1002/hipo.22037 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Hall LL, Smith KP, Byron M, Lawrence JB (2006) Molecular anatomy of a speckle. Anat Rec A Discov Mol Cell Evol Biol 288:664–675. doi: 10.1002/ar.a.20336 CrossRefPubMedPubMedCentralGoogle Scholar
  22. Hernandez-Verdun D, Roussel P, Thiry M, Sirri V, Lafontaine DL (2010) The nucleolus: structure/function relationship in RNA metabolism. Wiley Interdiscip Rev RNA 1:415–431. doi: 10.1002/wrna.39 CrossRefPubMedGoogle Scholar
  23. Hieda M, Winstanley H, Maini P, Iborra FJ, Cook PR (2005) Different populations of RNA polymerase II in living mammalian cells. Chromosom Res 13:135–144. doi: 10.1007/s10577-005-7720-1 CrossRefGoogle Scholar
  24. Huang Y, Mucke L (2012) Alzheimer mechanisms and therapeutic strategies. Cell 148:1204–1222. doi: 10.1016/j.cell.2012.02.040 CrossRefPubMedPubMedCentralGoogle Scholar
  25. Jacobs S, Lie DC, DeCicco KL, Shi Y, DeLuca LM, Gage FH, Evans RM (2006) Retinoic acid is required early during adult neurogenesis in the dentate gyrus. Proc Natl Acad Sci U S A 103:3902–3907. doi: 10.1073/pnas.0511294103 CrossRefPubMedPubMedCentralGoogle Scholar
  26. Khalouei S, Chow AM, Brown IR (2014a) Stress-induced localization of HSPA6 (HSP70B’) and HSPA1A (HSP70-1) proteins to centrioles in human neuronal cells. Cell Stress Chaperones 19:321–327. doi: 10.1007/s12192-013-0459-2 CrossRefPubMedGoogle Scholar
  27. Khalouei S, Chow AM, Brown IR (2014b) Localization of heat shock protein HSPA6 (HSP70B’) to sites of transcription in cultured differentiated human neuronal cells following thermal stress. J Neurochem 131:743–754. doi: 10.1111/jnc.12970 CrossRefPubMedGoogle Scholar
  28. Kuijpers M, Hoogenraad CC (2011) Centrosomes, microtubules and neuronal development. Mol Cell Neurosci 48:349–358. doi: 10.1016/j.mcn.2011.05.004 CrossRefPubMedGoogle Scholar
  29. Lizarraga SB et al. (2010) Cdk5rap2 regulates centrosome function and chromosome segregation in neuronal progenitors. Development 137:1907–1917. doi: 10.1242/dev.040410 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Lopes FM et al. (2010) Comparison between proliferative and neuron-like SH-SY5Y cells as an in vitro model for Parkinson disease studies. Brain Res 1337:85–94. doi: 10.1016/j.brainres.2010.03.102 CrossRefPubMedGoogle Scholar
  31. Lopez-Carballo G, Moreno L, Masia S, Perez P, Barettino D (2002) Activation of the phosphatidylinositol 3-kinase/Akt signaling pathway by retinoic acid is required for neural differentiation of SH-SY5Y human neuroblastoma cells. J Biol Chem 277:25297–25304. doi: 10.1074/jbc.M201869200 CrossRefPubMedGoogle Scholar
  32. Lui JH, Hansen DV, Kriegstein AR (2011) Development and evolution of the human neocortex. Cell 146:18–36. doi: 10.1016/j.cell.2011.06.030 CrossRefPubMedPubMedCentralGoogle Scholar
  33. Maden M (2007) Retinoic acid in the development, regeneration and maintenance of the nervous system. Nat Rev Neurosci 8:755–765. doi: 10.1038/nrn2212 CrossRefPubMedGoogle Scholar
  34. Muchowski PJ, Wacker JL (2005) Modulation of neurodegeneration by molecular chaperones. Nat Rev Neurosci 6:11–22. doi: 10.1038/nrn1587 CrossRefPubMedGoogle Scholar
  35. Noonan E, Giardina C, Hightower L (2008a) Hsp70B’ and Hsp72 form a complex in stressed human colon cells and each contributes to cytoprotection. Exp Cell Res 314:2468–2476. doi: 10.1016/j.yexcr.2008.05.002 CrossRefPubMedGoogle Scholar
  36. Noonan EJ, Fournier G, Hightower LE (2008b) Surface expression of Hsp70B’ in response to proteasome inhibition in human colon cells. Cell Stress Chaperones 13:105–110. doi: 10.1007/s12192-007-0003-3 CrossRefPubMedPubMedCentralGoogle Scholar
  37. Noonan EJ, Place RF, Giardina C, Hightower LE (2007a) Hsp70B’ regulation and function. Cell Stress Chaperones 12:393–402CrossRefPubMedPubMedCentralGoogle Scholar
  38. Noonan EJ, Place RF, Rasoulpour RJ, Giardina C, Hightower LE (2007b) Cell number-dependent regulation of Hsp70B’ expression: evidence of an extracellular regulator. J Cell Physiol 210:201–211. doi: 10.1002/jcp.20875 CrossRefPubMedGoogle Scholar
  39. Pahlman S, Ruusala AI, Abrahamsson L, Mattsson ME, Esscher T (1984) Retinoic acid-induced differentiation of cultured human neuroblastoma cells: a comparison with phorbolester-induced differentiation. Cell Differ 14:135–144CrossRefPubMedGoogle Scholar
  40. Pratt WB, Gestwicki JE, Osawa Y, Lieberman AP (2015) Targeting Hsp90/Hsp70-based protein quality control for treatment of adult onset neurodegenerative diseases. Annu Rev Pharmacol Toxicol 55:353–371. doi: 10.1146/annurev-pharmtox-010814-124332 CrossRefPubMedGoogle Scholar
  41. Ramirez VP, Stamatis M, Shmukler A, Aneskievich BJ (2015) Basal and stress-inducible expression of HSPA6 in human keratinocytes is regulated by negative and positive promoter regions. Cell Stress Chaperones 20:95–107. doi: 10.1007/s12192-014-0529-0 CrossRefPubMedGoogle Scholar
  42. Richter K, Haslbeck M, Buchner J (2010) The heat shock response: life on the verge of death. Mol Cell 40:253–266. doi: 10.1016/j.molcel.2010.10.006 CrossRefPubMedGoogle Scholar
  43. Rieder D et al. (2014) Co-expressed genes prepositioned in spatial neighborhoods stochastically associate with SC35 speckles and RNA polymerase II factories. Cell Mol Life Sci 71:1741–1759. doi: 10.1007/s00018-013-1465-3 CrossRefPubMedGoogle Scholar
  44. Rieder D, Trajanoski Z, McNally JG (2012) Transcription factories. Front Genet 3:221. doi: 10.3389/fgene.2012.00221 CrossRefPubMedPubMedCentralGoogle Scholar
  45. Sharma A, Takata H, Shibahara K, Bubulya A, Bubulya PA (2010) Son is essential for nuclear speckle organization and cell cycle progression. Mol Biol Cell 21:650–663. doi: 10.1091/mbc.E09-02-0126 CrossRefPubMedPubMedCentralGoogle Scholar
  46. Sirri V, Urcuqui-Inchima S, Roussel P, Hernandez-Verdun D (2008) Nucleolus: the fascinating nuclear body. Histochem Cell Biol 129:13–31. doi: 10.1007/s00418-007-0359-6 CrossRefPubMedGoogle Scholar
  47. Snapp EL, Altan N, Lippincott-Schwartz J (2003) Measuring protein mobility by photobleaching GFP chimeras in living cells. Curr Protoc Cell Biol Chapter 21:Unit 21.1. doi: 10.1002/0471143030.cb2101s19 PubMedGoogle Scholar
  48. Spector DL, Lamond AI (2011) Nuclear speckles. Cold Spring Harb Perspect Biol 3. doi: 10.1101/cshperspect.a000646
  49. Sprague BL, McNally JG (2005) FRAP analysis of binding: proper and fitting. Trends Cell Biol 15:84–91. doi: 10.1016/j.tcb.2004.12.001 CrossRefPubMedGoogle Scholar
  50. Taverna E, Gotz M, Huttner WB (2014) The cell biology of neurogenesis: toward an understanding of the development and evolution of the neocortex. Annu Rev Cell Dev Biol 30:465–502. doi: 10.1146/annurev-cellbio-101011-155801 CrossRefPubMedGoogle Scholar
  51. Westerheide SD, Morimoto RI (2005) Heat shock response modulators as therapeutic tools for diseases of protein conformation. J Biol Chem 280:33097–33100. doi: 10.1074/jbc.R500010200 CrossRefPubMedGoogle Scholar
  52. Yakovchuk P, Goodrich JA, Kugel JF (2009) B2 RNA and Alu RNA repress transcription by disrupting contacts between RNA polymerase II and promoter DNA within assembled complexes. Proc Natl Acad Sci U S A 106:5569–5574. doi: 10.1073/pnas.0810738106 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Cell Stress Society International 2016

Authors and Affiliations

  1. 1.Centre for the Neurobiology of Stress, Department of Biological SciencesUniversity of Toronto ScarboroughTorontoCanada

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