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Collective and Single Cell Behavior in Epithelial Contact Inhibition 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. arXiv:1112.0465v1 [q-bio.TO] 2 Dec 2011 ∗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 1 Introduction ciated with the AJs at the cell surface [19, 20]. A contact inhibition role has been reported for The precise orchestration of cell division and growth NF2/Merlin, a tumor suppressor gene [21, 22] that is central to morphogenesis and animal develop- encodes a membrane-cytoskeletal scaffolding protein, ment [1, 2]. Complex cellular signaling and regu- which most likely acts via the Hippo kinase path- latory networks are dedicated to growth control and way, controlling nuclear localization of the transcrip- misregulation of cell proliferation leads to tumors and tional activator YAP [23, 24, 13] - itself a known cancer [3]. Epithelial tissue is an important system to regulator of cell proliferation. Contact inhibition study regulation of growth. Normal development of is known to involve the MAPK pathway, which, in epithelial tissue involves a mesenchymal to epithelial turn, promotes cell cycle entry by regulating the ex- transition (MET) [4] associated with the loss of cell pression of cyclinD1 [25, 26, 27]. Also implicated mobility, mitotic arrest and acquisition of epithelial are Nectins [28, 29, 12] - a family of cell adhesion morphology. This transition is reversed in the process molecules that are involved, together with integrins of wound healing [5]. On the other hand, cells that and other proteins, in the regulation of cell motility have undergone oncogenic epithelial to mesenchymal and proliferation. Yet, this accumulated knowledge transition (EMT) typically lose their ability to un- falls far short of a comprehensive picture of contact dergo MET. Hence understanding the normal MET inhibition. The difficulty in achieving a better under- process is of fundamental importance for understand- standing of the molecular mechanism lies in the com- ing oncogenic transformations which disregulate it. plexity of the contact inhibition phenotype, which, as In cultured, non-cancerous epithelial cells, the we describe below, involves the concurrence of many transition from freely proliferating, non-confluent processes. cells to fully differentiated, dense epithelial mono- To facilitate progress in the dissection of the reg- layers is commonly referred to as “contact inhibi- ulatory pathways underlying contact inhibition, we tion” [6, 7, 8, 9]. Contact inhibition in confluent undertook a quantitative reexamination of the spatio- cell cultures is currently defined as i) a dramatic temporal dynamics of an adherent epithelial layer decrease of cell mobility and mitotic rate with in- formed by Madin-Darby Canine Kidney (MDCK) creasing cell density; ii) establishment of a stationary cells. These cells are known for their ability to exhibit post-confluent state which is insensitive to nutrient contact inhibition and achieve characteristic epithe- renewal. It is widely believed that contact inhibition, lial morphology in culture [30, 31] thus providing an as the name suggests, is caused by cell contact. But excellent model system for in vitro study of epithelial despite extensive study, current understanding of the tissue dynamics [32, 33, 34]. Using long-term fluo- mechanism of contact inhibition is far from complete rescence and phase contrast video microscopy in con- (see [10, 11, 12, 13, 14]). junction with image segmentation and cell tracking, we have characterized the temporal progression of Many molecular mechanisms have been proposed contact inhibition in growing MDCK colonies. Quan- to contribute to contact inhibition. It is widely ac- titative analysis of the evolution of cell density, cell cepted that contact inhibition requires establishment motility and cell division rate reveals that contact of E-cadherin mediated cell-cell contacts and subse- inhibition proceeds in three distinct stages: 1) a quent maturation of the adherens junctions (AJs) stage of cell density growth with gradual inhibition of that link E-cadherin and F-actin in a synapse-like motility, but without inhibition of mitosis that is fol- complex involving numerous other proteins [15, 16, lowed by 2) a rapid transition to epithelial cell mor- 17, 18]. However, the nature of the signaling path- phology, followed by 3) continued cell division and way leading to suppression of mitosis remains un- reduction of cell size with a progressively decreasing clear. One possible pathway involves β-catenin, rate of mitosis. Mitotic arrest is achieved once cell a mediator of Wnt signaling, that, in addition to area falls below a certain threshold. Our findings its function as a transcriptional co-factor, is asso- show that contact between cells is not sufficient for 2 inhibition of mitosis in MDCK cells. Instead, inhibi- Of course, exponential increase in colony area can- tion of cell proliferation is a consequence of mechan- not continue indefinitely as it would require the out- ical constraint that causes successive cell divisions to ward motion of peripheral cells to have an exponen- reduce cell area. tially increasing velocity. To support an exponential area increase at a rate 1/τ, the velocity of cells on 2 the boundary must be vb A/4πτ . Comparing this to the observed maximal' velocity of cell motion, 2 Results p vmax 15µm/h, we arrive at an estimate for the crit- ' 6 2 2.1 Large scale analysis ical size of the colony, Ac 2 10 µm . Above this critical area, expansion of the' colony· cannot keep up To separate the effect of cell contact from that of me- with cell proliferation in the bulk without increasing chanical constraint arising upon confluence of prolif- cell density. This estimate is close to the observed erating cells, we first examine the dynamics of iso- area at the time of crossover ( 6 days) from expo- lated, growing colonies of MDCK cells. The colonies, nential to sub-exponential growth∼ of the colony area started from a small initial number of cells, were mon- (Fig.1B). The crossover is indeed coincident with the itored with subcellular resolution by time-lapse video onset of a gradual increase in cell density in the bulk microscopy for up to three weeks until nearly com- of the colony, as shown in Fig. 1C. Single cell anal- plete proliferation arrest (see Materials and Methods ysis (below) confirms that mitotic rate in this ”pre- for details). Fig. 1A shows the large scale dynam- transition” regime does not decrease so that the sub- ics of a growing colony. The boundary of the colony exponential expansion of the colony area is accounted exhibits nontrivial dynamics due to the combined ef- for by increasing density alone. fect of motility and cell division. It moves outward Thus, it appears that increased cell density (and with a non-uniform velocity forming finger-like pro- the associated decrease of average cell size) is a con- trusions [32, 35, 33]. Yet the total area of the colony sequence of mechanical constraint imposed by the in- grows following a simple exponential law (Fig. 1B) ability of the tissue at the periphery to expand fast for up to 5-6 days, reaching over 103 cells. Cell den- enough to accommodate cell proliferation in the bulk. sity in the bulk remains constant during this period As cell density begins to increase, cell motility starts (Fig. 1C). Daughter cells occupy, on average, twice to decrease as shown in Fig. 2C (see also ref. [33]). the area of their mother cell and the rate of colony
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