Illuminating the Role of Rho GTPase Oscillations in Mechanosensation and Stem Cell Fate
Summary by Leanne Iannucci: Sampayo, R. G., Sakamoto, M., Wang, M., Kumar, S. & Schaffer, D. V. Mechanosensitive stem cell fate choice is instructed by dynamic fluctuations in activation of Rho GTPases. Proceedings of the National Academy of Sciences 120, doi:10.1073/pnas.2219854120 (2023)
Image credit: Midjourney
Stem cells can sense and decode the mechanical properties (e.g., softness vs. stiffness) of their surrounding extracellular matrix (ECM) to inform their cell fate decisions [1]. A family of proteins called Rho GTPases help cells alter their cytoskeletons to create protrusions to probe their local environments and drive signal transduction [2]. However, it is unknown how this family of proteins signals temporally (e.g., statically or dynamically) and how the signaling mechanistically conveys cell fate specification instructions to the cell. Using neural stem cells (NSCs) as a model cell type, Sampayo et al. [3] showed that Rho GTPase signals in a oscillating manner: in stiff environments Rho GTPase activity occurs at a high frequency, but in softer environments signaling fluctuates at a much slower rate. Additionally, substrate stiffness drove a fate decision within NSCs. When NSCs were seeded on a stiff substrate, they became more astrocyte-like, but soft substrates led to more neuronal-like fates.
To investigate whether this temporal fluctuation of Rho GTPase activity drives cell specification, the authors used an optogenetic Rho GTPase activator to control Rho GTPase family members (RhoA and Cdc42) with high spatiotemporal resolution. The Rho GTPases are fused to the protein Cry2 which clusters when exposed to blue light and dissociates in the dark. Therefore, Rho GTPase activity can be switched on and off at the same frequencies observed naturally in response to different ECM stiffnesses. By exogenously re-creating the Rho GTPase cycling frequencies generated by the stiff and soft substrates in NSCs using optogenetics, they were able to phenocopy the astrogenesis and neurogenesis, respectively, even though cells were grown on matrices with the same mechanical properties. This shows that Rho GTPase dynamics influence stem cell lineage commitment.
How exactly are these cyclic fluctuations of Rho GTPases driving cell fate? The authors observed that in response to high, but not low, frequency activation, NSCs exhibit phosphorylation of SMAD1/5, which is a downstream effector of the Transforming Growth Factor Beta (TGFbeta) and Bone Morphogenetic Protein (BMP) signaling pathways. The authors then blocked TGFbeta/BMP receptor activity using small molecule inhibitors and still observed the same effector phosphorylation, indicating that the Rho GTPases are signaling along these pathways in a receptor-independent and non-canonical manner. Additionally, as it is known that Rho GTPases are involved in cytoskeletal regulation, the authors disrupted the ability of the actin cytoskeleton to assemble using a drug, cytochalasin D. This treatment blocked SMAD 1/5 activation. They concluded that the actin assembly is needed to trigger SMAD1/5 phosphorylation via Rho GTPase activity and initiate downstream astrogenesis pathways.
Together, these results show that when NSCs are on stiff substrates, high frequency Rho GTPase activation drives cytoskeletal reorganization, activation of SMAD 1/5 (in a TGFbeta/BMP receptor-independent manner), and cell fate progression towards astrogenesis. On soft substrates via low Rho GTPase frequency activity, pSMAD1/5 expresion levels are not persistently activated, leading to NSC neurogenesis. In conclusion, this study from Sampayo et al. helped elucidate how stem cells interpret mechanical signals to make fate decisions and can potentially inform regenerative therapies for disease.
1. Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E. Matrix Elasticity Directs Stem Cell Lineage Specification. Cell 126, 677-689, doi:10.1016/j.cell.2006.06.044 (2006).
2. Etienne-Manneville, S. & Hall, A. Rho GTPases in cell biology. Nature 420, 629-635, doi:10.1038/nature01148 (2002).
3. Sampayo, R. G., Sakamoto, M., Wang, M., Kumar, S. & Schaffer, D. V. Mechanosensitive stem cell fate choice is instructed by dynamic fluctuations in activation of Rho GTPases. Proceedings of the National Academy of Sciences 120, doi:10.1073/pnas.2219854120 (2023).