It is well accepted that cells in the tissue can be regarded as tiles tessellating space. A number of approaches were developed to find an appropriate mathematical description of such cell tiling. A particularly useful approach is the so called Voronoi tessellation, built from centers of mass of the cell nuclei (CMVT), which is commonly used for estimating the morphology of cells in epithelial tissues. However, a study providing a statistically sound analysis of this method's accuracy is not available in the literature. We addressed this issue here by comparing a number of morphological measures of the cells, including area, perimeter, and elongation obtained from such a tessellation with identical measures extracted from direct imaging acquired by staining the cell membranes. After analyzing the shapes of 15,000 MDCK II epithelial cells under several conditions, we find that CMVT reasonably well reproduces many of the morphological properties of the tissue with an error that is between 10 and 15%. Moreover, cross-correlations between different morphological measures are reproduced qualitatively correctly by this method. However, all of the properties including the cell perimeters, number of neighbors, and anisotropy measures often suffer from systematic or size dependent errors. These discrepancies originate from the polygonal nature of the tessellation which sets the limits of the applicability of CMVT.

It is well established that MDCK II cells grow in circular colonies that densify until contact inhibition takes place. Here, we show that this behavior is only typical for colonies developing on hard substrates and report a new growth phase of MDCK II cells on soft gels. At the onset, the new phase is characterized by small, three-dimensional droplets of cells attached to the substrate. When the contact area between the agglomerate and the substrate becomes sufficiently large, a very dense monolayer nucleates in the center of the colony. This monolayer, surrounded by a belt of three-dimensionally packed cells, has a well-defined structure, independent of time and cluster size, as well as a density that is twice the steady-state density found on hard substrates. To release stress in such dense packing, extrusions of viable cells take place several days after seeding. The extruded cells create second-generation clusters, as evidenced by an archipelago of aggregates found in a vicinity of mother colonies, which points to a mechanically regulated migratory behavior.

Mechanical forces are relevant for many biological processes, from wound healing or tumour formation to cell migration and differentiation. Cytoskeletal actin is largely responsible for responding to forces and transmitting them in cells, while also maintaining cell shape and integrity. Here, we describe a novel approach to employ a FRET-based DNA force sensor in vitro and in cellulo for non-invasive optical monitoring of intracellular mechanical forces. We use fluorescence lifetime imaging to determine the FRET efficiency of the sensor, which makes the measurement robust against intensity variations. We demonstrate the applicability of the sensor by monitoring cross-linking activity in in vitro actin networks by bulk rheology and confocal microscopy. We further demonstrate that the sensor readily attaches to stress fibers in living cells which opens up the possibility of live-cell force measurements.

Actin, a major cytoskeletal biopolymer in eukaryotic cells, is crosslinked into networks of filaments and bundles. These networks are largely responsible for the maintenance of cellular shape, rigidity, and mechanical stability. Other assemblies of actin are involved in a myriad of cellular processes, such as cell migration, division, intracellular transport, and morphogenesis. In these processes, the spatial and temporal regulation of the network structure, their dynamics, and force generation due to myosin motors are crucial. Experimentally, one of the challenges is to measure force transmission across such networks, which is vital to properly understand the function, failure, and repair mechanisms beyond the linear regime. To measure forces across the cytoskeletal network, we have developed a FRET-based, reversible DNA force sensor. We employ these DNA constructs as flexible crosslinkers across semiflexible actin, thereby reconstituting model networks of cytoskeletal structures. Characterization of the rheology and frequency response of these model actin-DNA sensor networks is performed via a macrorheometer and also by utilizing a large bandwidth, high-resolution microrheology set up. DNA force sensors are crosslinked in vitro with actin filaments in order to map force distributions and stress relaxations in the resulting network. We characterize the DNA force sensor in solution and across actin networks through fluorescence lifetime imaging microscopy (FLIM) measurements. From these results, we estimate the FRET efficiency of our DNA sensor. We also test DNA sensors in a cellular environment and describe its preliminary results.