Volumetric Compression Shifts Rho GTPase Balance and Induces Mechanobiological Cell State Transition

Summary.

In the context of the extracellular matrix (ECM) microenvironment, the complex interplay between cell volume, cytoskeletal dynamics, and mechanosensing governs cellular function, behavior, and survival. Understanding exactly how cell volume alterations impact intracellular organization and mechanosensing still requires comprehensive investigation. Collagen-1 interconnected fibers, a fundamental component of many tissue microenvironments, influence the elastic properties of the ECM and undergo rearrangements under applied external forces. These rearrangements ultimately lead to stress relaxation, time-dependent mechanical properties of the ECM, and changes in its dynamic interaction with the cytoskeleton. The cytoskeleton, and in particular actin filaments via their upstream regulators (the Rho GTPases Rac1, cdc42, RhoA), are involved in force generation, maintaining cell shape, and migration. In this preprint, the authors have addressed the lack of in-depth understanding of the effect of volumetric compression on cytoskeletal reprogramming, mechanosensing, and cell-matrix interactions.

The authors managed to achieve cell compression by volumetrically altering osmotic pressure using low molecular weight polyethylene glycol (PEG300). Cellular compression resulted in a shift in Rho GTPase signaling, leading to changes in cytoskeletal organization and cell shape dynamics. Interestingly, an increase in peripheral actin, along with an increase in cell adhesion was observed, while stress fibers were diminished.

These alterations ultimately impacted the mechanosensing ability of collagen 1, which relies on dynamic cell boundary fluctuations. These findings highlight the sensitivity and dynamic nature of Rho GTPase signaling in cells under compression, which is relevant in a wide range of fundamental (cellular and tissue) developmental, homeostatic, and pathological events.

Key findings.

(a) Cytoskeletal re-organization: Volumetric compression considerably modified actin arrangement, resulting in a decrease in interior stress fibers, but an increase in peripheral actin despite the inhibition of lamellipodia. The compression-induced cells appeared compact, and polygonal in shape, and were designated as being in a “frozen state” (Figure 1).

 

Figure 1: Adapted from Figure 8B. Controlling cell volume programs cell-ECM non-elastic interactions in 3D via protrusion dynamics.

(b) Altered cell mechanical functions and cell-matrix dynamics: Compressed and non-compressed cells did not differ in cell contractility, although their capacity for mechanics-dependent ECM interactions was altered. Compressed cells displayed decreased compaction of non-elastic ECM in its vicinity, which in turn affected local and global interactions.

(c) Mechanosensing: Although on stiff substrates compressed cells behaved similarly to non-compressed cells, which suggests that the general actomyosin-regulated stiffness-sensing pathways and adhesion are preserved, they were less reactive to mechanical remodeling of non-elastic substrates, showing a significant reduction in cell spreading and YAP nuclear translocation.

(d) Rho GTPase activities: Cell compression reduced Rac1 activity, and activated RhoA, resulting in decreased cell spreading and protrusion, while retaining contractility. Restoration of biochemical activities (Rac1 & RhoA) could override mechanical regulation, partially restoring cellular functions.

 Significance and Implications:

During physiological developmental processes, pathological conditions like cancer progression, and medical procedures such as injections or implantations, cells and tissues are subjected to mechanical compressions in their microenvironment, altering cell-cell, and cell-matrix interactions. This study sheds light on the complex regulations and interactions between volumetric compression and cellular mechanics, underlining the importance of Rho GTPase signaling as a key regulator. Unraveling the molecular players and mechanisms is a step forward toward potential therapeutic solutions that could alter cellular responses to mechanical stress under pathophysiological conditions.

Questions for the authors.

(1) Were any alternative methods considered to induce compression in cells?

(2) What could be the long-term effects of volumetric compression? Could there be adaptations in cells that are under compression over extended periods of time?

Author’s response:

(1) Were any alternative methods considered to induce compression in cells?

Yes. Alongside other cell compression studies, we also demonstrated mechanical compression induced by physical weight applied on top of the cells (Supplementary Figure 12 in this preprint). We showed that this mechanical compression could lead to similar cytoskeletal states we observed from applying the hyperosmolarity-induced volumetric compression. This led us to think this universal response is a self-protection mechanism to mechanical stresses.

(2) What could be the long-term effects of volumetric compression? Could there be adaptations in cells that are under compression over extended periods of time?

Yes. Other studies from our group have shown the adaptation to longer-term (5 days) compressive stress can occur, which involves dramatic changes in cell morphology, changes in transcriptional profile, anti-apoptosis phenotype, and even drug resistance. These changes could further lead to unfavorable prognosis in diseases such as cancer. We would like to direct our readers to our recent publications for this topic:

https://www.pnas.org/doi/abs/10.1073/pnas.2220062120

https://www.biorxiv.org/content/10.1101/2023.10.08.561453v1

Tags: cell mechanics, cell volume, mechanosensing, rho gtpase

doi: https://doi.org/10.1242/prelights.36341

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