Confinement Sensing and Signal Optimization via Piezo1/PKA and Myosin II Pathways.

Cells adopt distinct signaling pathways to optimize cell locomotion in different physical microenvironments. However, the underlying mechanism that enables cells to sense and respond to physical confinement is unknown. Using microfabricated devices and substrate-printing methods along with FRET-based biosensors, we report that, as cells transition from unconfined to confined spaces, intracellular Ca(2+) level is increased, leading to phosphodiesterase 1 (PDE1)-dependent suppression of PKA activity. This Ca(2+) elevation requires Piezo1, a stretch-activated cation channel. Moreover, differential regulation of PKA and cell stiffness in unconfined versus confined cells is abrogated by dual, but not individual, inhibition of Piezo1 and myosin II, indicating that these proteins can independently mediate confinement sensing. Signals activated by Piezo1 and myosin II in response to confinement both feed into a signaling circuit that optimizes cell motility. This study provides a mechanism by which confinement-induced signaling enables cells to sense and adapt to different physical microenvironments.

Distinct signaling mechanisms regulate migration in unconfined versus confined spaces.

Using a microchannel assay, we demonstrate that cells adopt distinct signaling strategies to modulate cell migration in different physical microenvironments. We studied α4ß1 integrin-mediated signaling, which regulates cell migration pertinent to embryonic development, leukocyte trafficking, and melanoma invasion. We show that α4ß1 integrin promotes cell migration through both unconfined and confined spaces. However, unlike unconfined (2D) migration, which depends on enhanced Rac1 activity achieved by preventing α4/paxillin binding, confined migration requires myosin II-driven contractility, which is increased when Rac1 is inhibited by α4/paxillin binding. This Rac1-myosin II cross talk mechanism also controls migration of fibroblast-like cells lacking α4ß1 integrin, in which Rac1 and myosin II modulate unconfined and confined migration, respectively. We further demonstrate the distinct roles of myosin II isoforms, MIIA and MIIB, which are primarily required for confined and unconfined migration, respectively. This work provides a paradigm for the plasticity of cells migrating through different physical microenvironments.

Dual functions of [alpha]4[beta]1 integrin in epicardial development: initial migration and long-term attachment.

The epicardium of the mammalian heart arises from progenitor cells outside the developing heart. The epicardial progenitor (EPP) cells migrate onto the heart through a cyst-mediated mechanism in which the progenitors are released from the tissue of origin as cysts; the cysts float in the fluid of the pericardial cavity and attach to the naked myocardial surface of the heart, and cells in the cysts then migrate out to form an epithelial sheet. In this paper, we show that the gene encoding the alpha4 subunit of alpha4beta1 integrin (alpha4beta1) is essential for this migratory process. We have generated a knockin mutation in mice replacing the alpha4 integrin gene with the lacZ reporter gene, placing lacZ under the control of the alpha4 integrin promoter. We show that in homozygous mutant embryos, the migration of EPP progenitor cells is impaired due to inefficient budding of the cysts and a failure of the cells in the cysts to migrate on the heart. This study provides direct genetic evidence for essential roles for alpha4beta1 integrin-mediated cell adhesion in the migration of progenitor cells to form the epicardium, in addition to a previous finding that alpha4beta1 is essential for maintaining the epicardium (Yang, J.T., H. Rayburn, and R.O. Hynes. 1995. Development. 121:549-560).