Cell mechanobiology in confinement using an integration of bioengineering, materials systems and in vivo models

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Summary- Cells in vivo travel through confining three-dimensional (3D) pores between fibrillar extracellular matrix (ECM) networks or channel-like tracks bordered by ECM bundles, vessels, myofibers or nerves. The mechanisms enabling cell locomotion in diverse microenvironments are adaptive in response to the physical and biochemical cues, such as confinement, stiffness, viscoelastic properties and composition of ECM. Adaptive systems/modules include cell-ECM interactions, the actomyosin cytoskeleton and cell volume regulation. Recently, we and others have also identified the key role of the nucleus in confined migration. However, numerous fundamental and translational questions remain unanswered on the crosstalk between nuclear mechanosensing, cytoskeleton and cell volume regulation, and their contributions to confined migration in health and disease. The overarching goal of this project is to employ state-of-the-art bioengineering, materials and imaging tools as well as in vivo models to provide a novel unified framework for efficient migration in confinement by deciphering the interplay between nuclear mechanics, cytoskeleton and ion channels. This R01 application will test the hypothesis that the nucleus senses and responds to physical confinement by exquisitely regulating the spatial activation of RhoA along the longitudinal cell axis in confined spaces via the synergistic roles of confinement-induced nuclear stiffening and anillin/Ect2 nuclear exit to the cytoplasm. This hypothesis is supported by intriguing preliminary data showing that cell entry into confining µ-channels induces nuclear stiffening which activates RhoA and supports ion channel-dependent nuclear blebbing and rupture. Nuclear rupture induces the exit of anillin and the RhoGEF Ect2 from the nucleus to the cytoplasm. Anillin accumulates specifically at the cell poles, where it locally bridges Ect2, RhoA and actomyosin, thereby exacerbating RhoA- myosin II contractility. In Aim 1, we will decipher the mechanisms of anillin exit to the cytoplasm, and demonstrate its critical role as a scaffolding protein, which bridges Ect2, RhoA and actomyosin at the cell poles, thereby regulating the spatial activation of RhoA and bleb-based migration in confinement. We will also elucidate the novel crosstalk between cell volume regulation and anillin/Ect2/RhoA in nuclear blebbing and rupture in confinement. Lastly, we will decipher the contributions of nuclear pushing from the cell rear versus nuclear pulling from the cell front (i.e., nuclear piston model) to migration as a function of the degree of confinement. In Aim 2, we will extend the applicability of our findings to 3D gels and confining µ-channels of prescribed physiologically relevant mechanical properties in vitro. We will also visualize the distinct localization patterns of anillin, Ect2 and key ion channels in natural tissue tracks of different dimensions in vivo, and test how perturbations of these molecules impact local and distant tissue invasion in vivo. This work will also develop and establish novel bioengineering tools (e.g., optogenetic probes, µ-fluidic chamber) for better understanding cell motility in health and disease.