Heat Stress Triggers Nuclear Invagination and Spatial Compartmentalization of Protein Metabolism

Abstract

Heat stress is a common challenge for cells, causing multiple types of cellular damage while triggering complex stress responses, including the highly conserved mechanism known as the heat shock response (HSR). However, the subcellular coordination of these stress responses remains poorly understood. In this study, we identify reversible nuclear morphological changes under heat stress, characterised by kidney-shaped invaginations. These nuclear invaginations are associated with intermediate filament collapse and the regional clustering of organelles. Through immunofluorescence imaging and proteomic analysis, we further reveal that nuclear invagination regions function as specialised compartments where newly synthesised proteins are concentrated and protein degradation demand is heightened. Moreover, this compartmentalization is not only essential for cellular adaptation and recovery from heat stress but also correlates with the differential heat tolerance across cell lines. Our findings highlight a previously unappreciated mechanism by which cells spatially reorganise protein metabolism to optimise stress responses, providing new insights into cellular stress adaptation.

References

[1] K. L. O'Neill, D. W. Fairbairn, M. J. Smith, and B. S. Poe, “Critical Parameters Influencing Hyperthermia-Induced Apoptosis in Human Lymphoid Cell Lines,” Apoptosis 3, no. 5 (1998): 369–375.

[2] E. Falcieri, F. Luchetti, S. Burattini, B. Canonico, S. Santi, and S. Papa, “Lineage-Related Sensitivity to Apoptosis in Human Tumor Cells Undergoing Hyperthermia,” Histochemistry and Cell Biology 113, no. 2 (2000): 135–144.

[3] T.-T. Wang, A. S. Chiang, J. J. Chu, T. J. Cheng, T. M. Chen, and Y. K. Lai, “Concomitant Alterations in Distribution of 70kDa Heat Shock Proteins, Cytoskeleton and Organelles in Heat Shocked 9L Cells,” International Journal of Biochemistry & Cell Biology 30, no. 6 (1998): 745–759.

[4] F. Luchetti, F. Mannello, B. Canonico, et al., “Integrin and Cytoskeleton Behaviour in Human Neuroblastoma Cells During Hyperthermia-Related Apoptosis,” Apoptosis 9, no. 5 (2004): 635–648.

[5] R. A. Coss and W. A. M. Linnemans, “The Effects of Hyperthermia on the Cytoskeleton: A Review,” International Journal of Hyperthermia 12, no. 2 (2009): 173–196.

[6] A. Pawlik, J. M. Nowak, D. Grzanka, L. Gackowska, J. Michalkiewicz, and A. Grzanka, “Hyperthermia Induces Cytoskeletal Alterations and Mitotic Catastrophe in p53-Deficient H1299 Lung Cancer Cells,” Acta Histochemica 115, no. 1 (2013): 8–15.

[7] R. A. Kammerer, D. Kostrewa, J. Zurdo, et al., “Exploring Amyloid Formation by a De Novo Design,” Proceedings of the National Academy of Sciences of the United States of America 101, no. 13 (2004): 4435–4440.

[8] J. R. Buchan and R. Parker, “Eukaryotic Stress Granules: The Ins and Outs of Translation,” Molecular Cell 36, no. 6 (2009): 932–941.

[9] E. W. Wallace, J. L. Kear-Scott, E. V. Pilipenko, et al., “Reversible, Specific, Active Aggregates of Endogenous Proteins Assemble Upon Heat Stress,” Cell 162, no. 6 (2015): 1286–1298.

[10] T. M. Franzmann and S. Alberti, “Protein Phase Separation as a Stress Survival Strategy,” Cold Spring Harbor Perspectives in Biology 11, no. 6 (2019): a034058.

[11] P. Gallardo, S. Salas-Pino, and R. R. Daga, “Reversible Protein Aggregation as Cytoprotective Mechanism Against Heat Stress,” Current Genetics 67, no. 6 (2021): 849–855.

[12] W. J. Welch and J. P. Suhan, “Morphological Study of the Mammalian Stress Response: Characterization of Changes in Cytoplasmic Organelles, Cytoskeleton, and Nucleoli, and Appearance of Intranuclear Actin Filaments in Rat Fibroblasts After Heat-Shock Treatment,” Journal of Cell Biology 101, no. 4 (1985): 1198–1211.

[13] R. M. Rivera, K. L. Kelley, G. W. Erdos, and P. J. Hansen, “Alterations in Ultrastructural Morphology of Two-Cell Bovine Embryos Produced In Vitro and In Vivo Following a Physiologically Relevant Heat Shock,” Biology of Reproduction 69, no. 6 (2003): 2068–2077.

[14] Z. Török, I. Horváth, P. Goloubinoff, et al., “Evidence for a Lipochaperonin: Association of Active Proteinfolding GroESL Oligomers With Lipids Can Stabilize Membranes Under Heat Shock Conditions,” Proceedings of the National Academy of Sciences 94, no. 6 (1997): 2192–2197.

[15] L. Vigh, B. Maresca, and J. L. Harwood, “Does the Membrane's Physical State Control the Expression of Heat Shock and Other Genes?,” Trends in Biochemical Sciences 23, no. 10 (1998): 369–374.

[16] G. Balogh, I. Horváth, E. Nagy, et al., “The Hyperfluidization of Mammalian Cell Membranes Acts as a Signal to Initiate the Heat Shock Protein Response,” FEBS Journal 272, no. 23 (2005): 6077–6086.

[17] K. Richter, M. Haslbeck, and J. Buchner, “The Heat Shock Response: Life on the Verge of Death,” Molecular Cell 40, no. 2 (2010): 253–266.

[18] K. Ahmed, Y. Tabuchi, and T. Kondo, “Hyperthermia: An Effective Strategy to Induce Apoptosis in Cancer Cells,” Apoptosis 20, no. 11 (2015): 1411–1419.

[19] N. R. Datta, S. G. Ordóñez, U. S. Gaipl, et al., “Local Hyperthermia Combined With Radiotherapy and−/or Chemotherapy: Recent Advances and Promises for the Future,” Cancer Treatment Reviews 41, no. 9 (2015): 742–753.

[20] M. A. Nashiry, G. R. A. Froemming, Y. S. Keong, A. B. M. Ismail, A. M. Din, and A. M. al-Khateeb, “Severe Hyperthermia Induces Apoptosis Mediated by Caspases Activation and Suppression of Hsp90-Alpha Expression in Osteosarcoma Cells,” Indonesian Biomedical Journal 11, no. 2 (2019): 167–174.

[21] E. M. Scutigliani, Y. Liang, H. Crezee, R. Kanaar, and P. M. Krawczyk, “Modulating the Heat Stress Response to Improve Hyperthermia-Based Anticancer Treatments,” Cancers 13, no. 6 (2021): 1243.

[22] F. Ritossa, “A New Puffing Pattern Induced by Temperature Shock and DNP in Drosophila,” Experientia 18, no. 12 (1962): 571–573.

[23] M. Ashburner and J. J. Bonner, “The Induction of Gene Activity in Drosophila by Heat Shock,” Cell 17, no. 2 (1979): 241–254.

[24] N. S. Peterson, G. Moller, and H. K. Mitchell, “Genetic Mapping of the Coding Regions for Three Heat-Shock Proteins in Drosophila Melanogaster,” Genetics 92, no. 3 (1979): 891–902.

[25] K. D. Sarge, V. Zimarino, K. Holm, C. Wu, and R. I. Morimoto, “Cloning and Characterization of Two Mouse Heat Shock Factors With Distinct Inducible and Constitutive DNA-Binding Ability,” Genes & Development 5, no. 10 (1991): 1902–1911.

[26] J. T. Westwood and C. Wu, “Activation of Drosophila Heat Shock Factor: Conformational Change Associated With a Monomer-To-Trimer Transition,” Molecular and Cellular Biology 13, no. 6 (1993): 3481–3486.

[27] K. D. Sarge, S. P. Murphy, and R. I. Morimoto, “Activation of Heat Shock Gene Transcription by Heat Shock Factor 1 Involves Oligomerization, Acquisition of DNA-Binding Activity, and Nuclear Localization and Can Occur in the Absence of Stress,” Molecular and Cellular Biology 13, no. 3 (2023): 1392–1407.

[28] S. Lindquist, “The Heat-Shock Response,” Annual Review of Biochemistry 55, no. 1 (1986): 1151–1191.

[29] E. T. Powers, R. I. Morimoto, A. Dillin, J. W. Kelly, and W. E. Balch, “Biological and Chemical Approaches to Diseases of Proteostasis Deficiency,” Annual Review of Biochemistry 78, no. 1 (2009): 959–991.

[30] T. A. Allen, S. von Kaenel, J. A. Goodrich, and J. F. Kugel, “The SINE-Encoded Mouse B2 RNA Represses mRNA Transcription in Response to Heat Shock,” Nature Structural & Molecular Biology 11, no. 9 (2004): 816–821.

[31] P. D. Mariner, R. D. Walters, C. A. Espinoza, et al., “Human Alu RNA Is a Modular Transacting Repressor of mRNA Transcription During Heat Shock,” Molecular Cell 29, no. 4 (2008): 499–509.

[32] P. Yakovchuk, J. A. Goodrich, and J. F. Kugel, “B2 RNA and Alu RNA Repress Transcription by Disrupting Contacts Between RNA Polymerase II and Promoter DNA Within Assembled Complexes,” Proceedings of the National Academy of Sciences 106, no. 14 (2009): 5569–5574.

[33] A. De Benedetti and C. Baglioni, “Activation of Hemin-Regulated Initiation Factor-2 Kinase in Heat-Shocked HeLa Cells,” Journal of Biological Chemistry 261, no. 1 (1986): 338–342.

[34] R. L. Matts, Z. Xu, J. K. Pal, and J. J. Chen, “Interactions of the Heme-Regulated eIF-2 Alpha Kinase With Heat Shock Proteins in Rabbit Reticulocyte Lysates,” Journal of Biological Chemistry 267, no. 25 (1992): 18160–18167.

[35] R. L. Matts, R. Hurst, and Z. Xu, “Denatured Proteins Inhibit Translation in Hemin-Supplemented Rabbit Reticulocyte Lysate by Inducing the Activation of the Heme-Regulated eIF-2.Alpha. Kinase,” Biochemistry 32, no. 29 (2002): 7323–7328.

[36] M. Holcik and N. Sonenberg, “Translational Control in Stress and Apoptosis,” Nature Reviews. Molecular Cell Biology 6, no. 4 (2005): 318–327.

[37] P. Anderson and N. Kedersha, “Stressful Initiations,” Journal of Cell Science 115, no. 16 (2002): 3227–3234.

[38] N. Kedersha, G. Stoecklin, M. Ayodele, et al., “Stress Granules and Processing Bodies Are Dynamically Linked Sites of mRNP Remodeling,” Journal of Cell Biology 169, no. 6 (2005): 871–884.

[39] P. Anderson and N. Kedersha, “Stress Granules: The Tao of RNA Triage,” Trends in Biochemical Sciences 33, no. 3 (2008): 141–150.

[40] D. Mateju, T. M. Franzmann, A. Patel, et al., “An Aberrant Phase Transition of Stress Granules Triggered by Misfolded Protein and Prevented by Chaperone Function,” EMBO Journal 36, no. 12 (2017): 1669–1687.

[41] H. A. Parag, B. Raboy, and R. G. Kulka, “Effect of Heat Shock on Protein Degradation in Mammalian Cells: Involvement of the Ubiquitin System,” EMBO Journal 6, no. 1 (1987): 55–61.

[42] J. R. Buchan, R. M. Kolaitis, J. P. Taylor, and R. Parker, “Eukaryotic Stress Granules Are Cleared by Autophagy and Cdc48/VCP Function,” Cell 153, no. 7 (2013): 1461–1474.

[43] K. Dokladny, M. N. Zuhl, M. Mandell, et al., “Regulatory Coordination Between Two Major Intracellular Homeostatic Systems: Heat Shock Response and Autophagy,” Journal of Biological Chemistry 288, no. 21 (2013): 14959–14972.

[44] K. Dokladny, O. B. Myers, and P. L. Moseley, “Heat Shock Response and Autophagy—Cooperation and Control,” Autophagy 11, no. 2 (2015): 200–213.

[45] R. W. Walters, D. Muhlrad, J. Garcia, and R. Parker, “Differential Effects of Ydj1 and Sis1 on Hsp70-Mediated Clearance of Stress Granules in Saccharomyces cerevisiae,” RNA 21, no. 9 (2015): 1660–1671.

[46] N. Jain, A. Rai, R. Mishra, and S. Ganesh, “Loss of Malin, but Not Laforin, Results in Compromised Autophagic Flux and Proteasomal Dysfunction in Cells Exposed to Heat Shock,” Cell Stress & Chaperones 22, no. 2 (2017): 307–315.

[47] C. Vigouroux, M. Auclair, E. Dubosclard, et al., “Nuclear Envelope Disorganization in Fibroblasts From Lipodystrophic Patients With Heterozygous R482Q/W Mutations in the Lamin A/C Gene,” Journal of Cell Science 114, no. Pt 24 (2001): 4459.

[48] A. Muchir, B. G. van Engelen, M. Lammens, et al., “Nuclear Envelope Alterations in Fibroblasts From LGMD1B Patients Carrying Nonsense Y259X Heterozygous or Homozygous Mutation in Lamin A/C Gene,” Experimental Cell Research 291, no. 2 (2003): 352–362.

[49] H. J. Worman, L. G. Fong, A. Muchir, and S. G. Young, “Laminopathies and the Long Strange Trip From Basic Cell Biology to Therapy,” Journal of Clinical Investigation 119, no. 7 (2009): 1825–1836.

[50] M. Stiekema, M. A. M. J. van Zandvoort, F. C. S. Ramaekers, and J. L. V. Broers, “Structural and Mechanical Aberrations of the Nuclear Lamina in Disease,” Cells 9, no. 8 (2020): 1884.

[51] A. K. Velichko, E. N. Markova, N. V. Petrova, S. V. Razin, and O. L. Kantidze, “Mechanisms of Heat Shock Response in Mammals,” Cellular and Molecular Life Sciences 70, no. 22 (2013): 4229–4241.

[52] D. A. Fletcher and R. D. Mullins, “Cell Mechanics and the Cytoskeleton,” Nature 463, no. 7280 (2010): 485–492.

[53] M. Li, L. Peng, Z. Wang, et al., “Roles of the Cytoskeleton in Human Diseases,” Molecular Biology Reports 50, no. 3 (2023): 2847–2856.

[54] M. Crisp, Q. Liu, K. Roux, et al., “Coupling of the Nucleus and Cytoplasm: Role of the LINC Complex,” Journal of Cell Biology 172, no. 1 (2006): 41–53.

[55] M. L. Lombardi, D. E. Jaalouk, C. M. Shanahan, B. Burke, K. J. Roux, and J. Lammerding, “The Interaction Between Nesprins and Sun Proteins at the Nuclear Envelope Is Critical for Force Transmission Between the Nucleus and Cytoskeleton,” Journal of Biological Chemistry 286, no. 30 (2011): 26743–26753.

[56] M. B. Omary, N. O. Ku, G. Z. Tao, D. M. Toivola, and J. Liao, ““Heads and Tails” of Intermediate Filament Phosphorylation: Multiple Sites and Functional Insights,” Trends in Biochemical Sciences 31, no. 7 (2006): 383–394.

[57] B. MacTaggart and A. Kashina, “Posttranslational Modifications of the Cytoskeleton,” Cytoskeleton 78, no. 4 (2021): 142–173.

[58] M. L. Styers, G. Salazar, R. Love, A. A. Peden, A. P. Kowalczyk, and V. Faundez, “The Endo-Lysosomal Sorting Machinery Interacts With the Intermediate Filament Cytoskeleton,” Molecular Biology of the Cell 15, no. 12 (2004): 5369–5382.

[59] D. M. Toivola, G. Z. Tao, A. Habtezion, J. Liao, and M. B. Omary, “Cellular Integrity Plus: Organelle-Related and Protein-Targeting Functions of Intermediate Filaments,” Trends in Cell Biology 15, no. 11 (2005): 608–617.

[60] N. Schwarz and R. E. Leube, “Intermediate Filaments as Organizers of Cellular Space: How They Affect Mitochondrial Structure and Function,” Cells 5, no. 3 (2016): 30.

[61] S. Etienne-Manneville, “Cytoplasmic Intermediate Filaments in Cell Biology,” Annual Review of Cell and Developmental Biology 34 (2018): 1–28.

[62] W. H. Goldmann, “Intermediate Filaments and Cellular Mechanics,” Cell Biology International 42, no. 2 (2018): 132–138.

[63] K. A. Spriggs, M. Bushell, and A. E. Willis, “Translational Regulation of Gene Expression During Conditions of Cell Stress,” Molecular Cell 40, no. 2 (2010): 228–237.

[64] O. L. Kantidze, A. K. Velichko, and S. V. Razin, “Heat Stress-Induced Transcriptional Repression,” Biochemistry 80, no. 8 (2015): 990–993.

[65] M. E. Tanenbaum, L. A. Gilbert, L. S. Qi, J. S. Weissman, and R. D. Vale, “A Protein-Tagging System for Signal Amplification in Gene Expression and Fluorescence Imaging,” Cell 159, no. 3 (2014): 635–646.

[66] D. C. Dieterich, A. J. Link, J. Graumann, D. A. Tirrell, and E. M. Schuman, “Selective Identification of Newly Synthesized Proteins in Mammalian Cells Using Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT),” Proceedings of the National Academy of Sciences 103, no. 25 (2006): 9482–9487.

[67] R. M. Vabulas, S. Raychaudhuri, M. Hayer-Hartl, and F. U. Hartl, “Protein Folding in the Cytoplasm and the Heat Shock Response,” Cold Spring Harbor Perspectives in Biology 2, no. 12 (2010): a004390.

[68] T. Gidalevitz, V. Prahlad, and R. I. Morimoto, “The Stress of Protein Misfolding: From Single Cells to Multicellular Organisms,” Cold Spring Harbor Perspectives in Biology 3, no. 6 (2011): a009704.

[69] S. S. Lam, J. D. Martell, K. J. Kamer, et al., “Directed Evolution of APEX2 for Electron Microscopy and Proximity Labeling,” Nature Methods 12, no. 1 (2014): 51–54.

[70] B. J. DiDomenico, G. E. Bugaisky, and S. Lindquist, “The Heat Shock Response Is Self-Regulated at Both the Transcriptional and Posttranscriptional Levels,” Cell 31 (1982): 593–603.

[71] P. Workman, F. Burrows, L. E. Neckers, et al., “Drugging the Cancer Chaperone HSP90: Combinatorial Therapeutic Exploitation of Oncogene Addiction and Tumor Stress,” Annals of the New York Academy of Sciences 1113 (2007): 202–216.

[72] S. K. Calderwood and A. Murshid, “Molecular Chaperone Accumulation in Cancer and Decrease in Alzheimer's Disease: The Potential Roles of HSF1,” Frontiers in Neuroscience 11 (2017): 192.

[73] P. Thomas and T. G. Smart, “HEK293 Cell Line: A Vehicle for the Expression of Recombinant Proteins,” Journal of Pharmacological and Toxicological Methods 51, no. 3 (2005): 187–200.

[74] M. Uhlen, L. Fagerberg, B. M. Hallström, et al., “Proteomics. Tissue-Based Map of the Human Proteome,” Science 347, no. 6220 (2015): 1260419.

[75] M. Uhlen, C. Zhang, S. Lee, et al., “A Pathology Atlas of the Human Cancer Transcriptome,” Science 357, no. 6352 (2017): eaan2507.

[76] J. Lammerding, L. G. Fong, J. Y. Ji, et al., “Lamins A and C but Not Lamin B1 Regulate Nuclear Mechanics,” Journal of Biological Chemistry 281, no. 35 (2006): 25768–25780.

[77] M. Guo, I. Y. Wong, A. S. Moore, et al., “Vimentin Intermediate Filaments as Structural and Mechanical Coordinators of Mesenchymal Cells,” Nature Cell Biology 27, no. 8 (2025): 1210–1218.

[78] Y. Tang, J. Ren, and C. C. Li, “Establishment of a GFP::LMNB1 Knockin Cell Line (CSUi002-A-1) From a Dystonia Patient-Specific iPSC by CRISPR/Cas9 Editing,” Stem Cell Research 55 (2021): 102505.

Rights & permissions

2026 The Author(s). Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.