Collective heterogeneity of mitochondrial potential in contact inhibition of proliferation

Abstract

In the epithelium, cell density and cell proliferation are closely connected to each other through contact inhibition of proliferation (CIP). Depending on cell density, CIP proceeds through three distinct stages, namely the free-growing stage at low density, the pre-epithelial transition stage at medium density, and the post-epithelial transition stage at high density. Previous studies have elucidated how cell morphology, motion, and mechanics vary in these stages. However, it remains unknown whether cellular metabolism also has a density-dependent behavior. By measuring the mitochondrial membrane potential at different cell densities, here we reveal a heterogeneous landscape of metabolism in the epithelium, which appears qualitatively distinct in three stages of CIP. Moreover, it did not follow the trend of other CIP-associated parameters, which increase or decrease monotonically with increasing cell density. Importantly, epithelial cells established a collective heterogeneity in mitochondrial potential exclusively in the pre-epithelial transition stage, where the multicellular clusters of high and low-potential cells emerged. However, in the post-epithelial transition stage, the potential field became relatively homogeneous. The collective metabolic heterogeneity in the pre-epithelial transition stage was independent of the mitochondrial content and spatially correlated with the local cell density. Next, to study the underlying dynamics, we constructed a system-biological model, which predicted the role of cell proliferation in metabolic potential towards establishing collective heterogeneity. Further experiments revealed that the metabolic pattern indeed spatially correlated with the proliferative capacity of cells, as measured by the nuclear localization of a pro-proliferation protein, YAP. Finally, experiments perturbing the actomyosin contractility revealed that while metabolic heterogeneity was maintained in absence of actomyosin contractility, its ab initio emergence depended on the latter. Taken together, our results revealed a density-dependent collective heterogeneity in the metabolic field of a pre-epitheli al transition stage epithelial monolayer, which may have significant implications for epithelial form and function. STATEMENT OF SIGNIFICANCEEpithelial contact inhibition of proliferation (CIP) plays a key role in tissue homeostasis, morphogenesis, and development. The biochemical changes in cells during different stages of CIP are not as well-documented as the biophysical changes. We unveil a heterogeneous landscape of metabolism which appears distinct in different stages of CIP. Importantly, in the pre-epithelial transition stage, the epithelial cells establish a collective metabolic heterogeneity wherein multicellular clusters of high and low-potential cells emerge, despite the uniform genetic and nutrient conditions for the cells. The collective heterogeneity is correlated to the local fluctuations in geometrical parameters and the proliferative capacity of cells. Finally, we demonstrate the role of cell mechanics in the establishment of collective heterogeneity.