Exploring the role of macromolecular crowding and TNFR1 in cell volume control
- Indian Association for the Cultivation of Science, India
- Indian Institute of Science Education and Research Kolkata, India
Abstract
The excessive cosolute densities in the intracellular fluid create a physicochemical condition called macromolecular crowding (MMC). Intracellular MMC entropically maintains the biochemical thermodynamic equilibria by favouring associative reactions while hindering transport processes. Rapid cell volume shrinkage during extracellular hypertonicity elevates the MMC and disrupts the equilibria, potentially ushering cell death. Consequently, cells actively counter the hypertonic stress through regulatory volume increase (RVI) and restore the MMC homeostasis. Here, we establish fluorescence anisotropy of EGFP as a reliable tool for studying cellular MMC and explore the spatiotemporal dynamics of MMC during cell volume instabilities under multiple conditions. Our studies reveal that the actin cytoskeleton enforces spatially varying MMC levels inside adhered cells. Within cell populations, MMC is uncorrelated with nuclear DNA content but anti-correlated with the cell spread area. Although different cell lines have statistically similar MMC distributions, their responses to extracellular hypertonicity vary. The intensity of the extracellular hypertonicity determines a cell's ability for RVI, which correlates with Nuclear Factor Kappa Beta (NFkB) activation. Pharmacological inhibition and knockdown experiments reveal that Tumour Necrosis Factor Receptor 1 (TNFR1) initiates the hypertonicity induced NFkB signalling and RVI. At severe hypertonicities, the elevated MMC amplifies cytoplasmic microviscosity and hinders Receptor Interacting Protein Kinase 1 (RIPK1) recruitment at the TNFR1 complex, incapacitating the TNFR1-NFkB signalling and consequently, RVI. Together, our studies unveil the involvement of TNFR1-NFkB signalling in modulating RVI and demonstrate the pivotal role of MMC in determining cellular osmoadaptability.
Data availability
No new datasets were generated by this manuscript. The codes used in the manuscript for analyzing images, FRAP data, and single particle tracking are freely available online in GitHub, under the URL: https://github.com/bparijat/ImageJ-Macros__MatLab-codes/tree/main/MMC-TNFR1_in_CellVolumeControl. Descriptions of the codes are provided in a README file along with the codes. Any queries regarding operational details of the codes can be forwarded to the owner of the GitHub repository via direct messaging. Source data for Western blotting, immunofluorescence images, and histograms are provided with figures, and further queries can be forwarded to the authors.
Article and author information
Author details
Deepak Kumar Sinha
Funding
Department of Science and Technology, Ministry of Science and Technology, India (CRG/2022/005356)
- Deepak Kumar Sinha
Department of Biotechnology, Ministry of Science and Technology, India (BT/PR6995/BRB/10/1140/2012)
- Deepak Kumar Sinha
Wellcome Trust DBT India Alliance (IA/I/13/1/500885)
- Bidisha Sinha
Indian Association for the Cultivation of Science
- Parijat Biswas
- Subhamoy Jana
- Ridita Ray Basunia
Council of Scientific and Industrial Research, India
- Priyanka Roy
- Dipanjan Ray
University Grants Commission
- Jibitesh Das
- Bipasa Chaudhuri
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2024, Biswas et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Exploring the role of macromolecular crowding and TNFR1 in cell volume control
eLife 13:e92719.
https://doi.org/10.7554/eLife.92719
