Collective and single cell behavior in epithelial contact inhibition

📝 Abstract

Control of cell proliferation is a fundamental aspect of tissue physiology central to morphogenesis, wound healing and cancer. Although many of the molecular genetic factors are now known, the system level regulation of growth is still poorly understood. A simple form of inhibition of cell proliferation is encountered in vitro in normally differentiating epithelial cell cultures and is known as “contact inhibition”. The study presented here provides a quantitative characterization of contact inhibition dynamics on tissue-wide and single cell levels. Using long-term tracking of cultured MDCK cells we demonstrate that inhibition of cell division in a confluent monolayer follows inhibition of cell motility and sets in when mechanical constraint on local expansion causes divisions to reduce cell area. We quantify cell motility and cell cycle statistics in the low density confluent regime and their change across the transition to epithelial morphology which occurs with increasing cell density. We then study the dynamics of cell area distribution arising through reductive division, determine the average mitotic rate as a function of cell size and demonstrate that complete arrest of mitosis occurs when cell area falls below a critical value. We also present a simple computational model of growth mechanics which captures all aspects of the observed behavior. Our measurements and analysis show that contact inhibition is a consequence of mechanical interaction and constraint rather than interfacial contact alone, and define quantitative phenotypes that can guide future studies of molecular mechanisms underlying contact inhibition.

💡 Analysis

Control of cell proliferation is a fundamental aspect of tissue physiology central to morphogenesis, wound healing and cancer. Although many of the molecular genetic factors are now known, the system level regulation of growth is still poorly understood. A simple form of inhibition of cell proliferation is encountered in vitro in normally differentiating epithelial cell cultures and is known as “contact inhibition”. The study presented here provides a quantitative characterization of contact inhibition dynamics on tissue-wide and single cell levels. Using long-term tracking of cultured MDCK cells we demonstrate that inhibition of cell division in a confluent monolayer follows inhibition of cell motility and sets in when mechanical constraint on local expansion causes divisions to reduce cell area. We quantify cell motility and cell cycle statistics in the low density confluent regime and their change across the transition to epithelial morphology which occurs with increasing cell density. We then study the dynamics of cell area distribution arising through reductive division, determine the average mitotic rate as a function of cell size and demonstrate that complete arrest of mitosis occurs when cell area falls below a critical value. We also present a simple computational model of growth mechanics which captures all aspects of the observed behavior. Our measurements and analysis show that contact inhibition is a consequence of mechanical interaction and constraint rather than interfacial contact alone, and define quantitative phenotypes that can guide future studies of molecular mechanisms underlying contact inhibition.

📄 Content

Collective and single cell behavior in epithelial contact inhibition Alberto Puliafito∗†, Lars Hufnagel‡†, Pierre Neveu∗, Sebastian Streichan‡, Alex Sigal§, Deborah K. Fygenson¶ and Boris I. Shraiman∗¶ October 27, 2018 Abstract Control of cell proliferation is a fundamental aspect of tissue physiology central to morphogenesis, wound healing and cancer. Although many of the molecular genetic factors are now known, the system level regulation of growth is still poorly understood. A simple form of inhibition of cell proliferation is encountered in vitro in normally differentiating epithelial cell cultures and is known as ”contact inhibition”. The study presented here provides a quantitative characterization of contact inhibition dynamics on tissue-wide and single cell levels. Using long-term tracking of cultured MDCK cells we demonstrate that inhibition of cell division in a confluent monolayer follows inhibition of cell motility and sets in when mechanical constraint on local expansion causes divisions to reduce cell area. We quantify cell motility and cell cycle statistics in the low density confluent regime and their change across the transition to epithelial morphology which occurs with increasing cell density. We then study the dynamics of cell area distribution arising through reductive division, determine the average mitotic rate as a function of cell size and demonstrate that complete arrest of mitosis occurs when cell area falls below a critical value. We also present a simple computational model of growth mechanics which captures all aspects of the observed behavior. Our measurements and analysis show that contact inhibition is a consequence of mechanical interaction and constraint rather than interfacial contact alone, and define quantitative phenotypes that can guide future studies of molecular mechanisms underlying contact inhibition. ∗Kavli Institute for Theoretical Physics, UCSB, Santa Barbara, CA, 93106, USA †co-first author ‡European Molecular Biology Laboratory, Meyerhofstraße 1, 69117 Heidelberg, Germany §Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA ¶Department of Physics and Program in Biomolecular Science & Engineering, UCSB, Santa Barbara, CA, 93106, USA 1 arXiv:1112.0465v1 [q-bio.TO] 2 Dec 2011 1 Introduction The precise orchestration of cell division and growth is central to morphogenesis and animal develop- ment [1, 2]. Complex cellular signaling and regu- latory networks are dedicated to growth control and misregulation of cell proliferation leads to tumors and cancer [3]. Epithelial tissue is an important system to study regulation of growth. Normal development of epithelial tissue involves a mesenchymal to epithelial transition (MET) [4] associated with the loss of cell mobility, mitotic arrest and acquisition of epithelial morphology. This transition is reversed in the process of wound healing [5]. On the other hand, cells that have undergone oncogenic epithelial to mesenchymal transition (EMT) typically lose their ability to un- dergo MET. Hence understanding the normal MET process is of fundamental importance for understand- ing oncogenic transformations which disregulate it. In cultured, non-cancerous epithelial cells, the transition from freely proliferating, non-confluent cells to fully differentiated, dense epithelial mono- layers is commonly referred to as “contact inhibi- tion” [6, 7, 8, 9]. Contact inhibition in confluent cell cultures is currently defined as i) a dramatic decrease of cell mobility and mitotic rate with in- creasing cell density; ii) establishment of a stationary post-confluent state which is insensitive to nutrient renewal. It is widely believed that contact inhibition, as the name suggests, is caused by cell contact. But despite extensive study, current understanding of the mechanism of contact inhibition is far from complete (see [10, 11, 12, 13, 14]). Many molecular mechanisms have been proposed to contribute to contact inhibition. It is widely ac- cepted that contact inhibition requires establishment of E-cadherin mediated cell-cell contacts and subse- quent maturation of the adherens junctions (AJs) that link E-cadherin and F-actin in a synapse-like complex involving numerous other proteins [15, 16, 17, 18]. However, the nature of the signaling path- way leading to suppression of mitosis remains un- clear. One possible pathway involves β-catenin, a mediator of Wnt signaling, that, in addition to its function as a transcriptional co-factor, is asso- ciated with the AJs at the cell surface [19, 20]. A contact inhibition role has been reported for NF2/Merlin, a tumor suppressor gene [21, 22] that encodes a membrane-cytoskeletal scaffolding protein, which most likely acts via the Hippo kinase path- way, controlling nuclear localization of the transcrip- tional activator YAP [23, 24, 13] - itself a known regulator of cell proliferation. Contact inhibition is known to involve the MAPK pathway, which, in turn, promotes cell cycle e

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