Andrew R. Harris

The cytoplasm of living cells behaves as a poroelastic material

The cytoplasm is the largest part of the cell by volume and hence its rheology sets the rate at which cellular shape changes can occur. Recent experimental evidence suggests that cytoplasmic rheology can be described by a poroelastic model, in which the cytoplasm is treated as a biphasic material consisting of a porous elastic solid meshwork (cytoskeleton, organelles, macromolecules) bathed in an interstitial fluid (cytosol). In this picture, the rate of cellular deformation is limited by the rate at which intracellular water can redistribute within the cytoplasm. However, direct supporting evidence for the model is lacking. Here we directly validate the poroelastic model to explain cellular rheology at physiologically relevant timescales using microindentation tests in conjunction with mechanical, chemical and genetic treatments. Our results show that water redistribution through the solid phase of the cytoplasm (cytoskeleton and macromolecular crowders) plays a fundamental role in setting cellular rheology.

Characterizing the mechanics of cultured cell monolayers

One-cell-thick monolayers are the simplest tissues in multicellular organisms, yet they fulfill critical roles in development and normal physiology. In early development, embryonic morphogenesis results largely from monolayer rearrangement and deformation due to internally generated forces. Later, monolayers act as physical barriers separating the internal environment from the exterior and must withstand externally applied forces. Though resisting and generating mechanical forces is an essential part of monolayer function, simple experimental methods to characterize monolayer mechanical properties are lacking. Here, we describe a system for tensile testing of freely suspended cultured monolayers that enables the examination of their mechanical behavior at multi-, uni-, and subcellular scales. Using this system, we provide measurements of monolayer elasticity and show that this is two orders of magnitude larger than the elasticity of their isolated cellular components. Monolayers could withstand more than a doubling in length before failing through rupture of intercellular junctions. Measurement of stress at fracture enabled a first estimation of the average force needed to separate cells within truly mature monolayers, approximately ninefold larger than measured in pairs of isolated cells. As in single cells, monolayer mechanical properties were strongly dependent on the integrity of the actin cytoskeleton, myosin, and intercellular adhesions interfacing adjacent cells. High magnification imaging revealed that keratin filaments became progressively stretched during extension, suggesting they participate in monolayer mechanics. This multiscale study of monolayer response to deformation enabled by our device provides the first quantitative investigation of the link between monolayer biology and mechanics.

Experimental validation of atomic force microscopy-based cell elasticity measurements

Atomic force microscopy (AFM) is widely used for measuring the elasticity of living cells yielding values ranging from 100 Pa to 100 kPa, much larger than those obtained using bead-tracking microrheology or micropipette aspiration (100-500 Pa). AFM elasticity measurements appear dependent on tip geometry with pyramidal tips yielding elasticities 2-3 fold larger than spherical tips, an effect generally attributed to the larger contact area of spherical tips. In AFM elasticity measurements, experimental force-indentation curves are analyzed using contact mechanics models that infer the tip-cell contact area from the tip geometry and indentation depth. The validity of these assumptions has never been verified. Here we utilize combined AFM-confocal microscopy of epithelial cells expressing a GFP-tagged membrane marker to directly characterize the indentation geometry and measure the indentation depth. Comparison with data derived from AFM force-indentation curves showed that the experimentally measured contact area for spherical tips agrees well with predicted values, whereas for pyramidal tips, the contact area can be grossly underestimated at forces larger than ∼0.2 nN leading to a greater than two-fold overestimation of elasticity. These data suggest that a re-examination of absolute cellular elasticities reported in the literature may be necessary and we suggest guidelines for avoiding elasticity measurement artefacts introduced by extraneous cantilever-cell contact.

Interaction with surrounding normal epithelial cells influences signalling pathways and behaviour of Src-transformed cells

At the initial stage of carcinogenesis, transformation occurs in a single cell within an epithelial sheet. However, it remains unknown what happens at the boundary between normal and transformed cells. Using Madin-Darby canine kidney (MDCK) cells transformed with temperature-sensitive v-Src, we have examined the interface between normal and Src-transformed epithelial cells. We show that Src-transformed cells are apically extruded when surrounded by normal cells, but not when Src cells alone are cultured, suggesting that apical extrusion occurs in a cell-context-dependent manner. We also observe apical extrusion of Src-transformed cells in the enveloping layer of zebrafish gastrula embryos. When Src-transformed MDCK cells are surrounded by normal MDCK cells, myosin-II and focal adhesion kinase (FAK) are activated in Src cells, which further activate downstream mitogen-activated protein kinase (MAPK). Importantly, activation of these signalling pathways depends on the presence of surrounding normal cells and plays a crucial role in apical extrusion of Src cells. Collectively, these results indicate that interaction with surrounding normal epithelial cells influences the signalling pathways and behaviour of Src-transformed cells.

Statistical evidence links exceptional 1995 Atlantic Hurricane season to record sea warming

Tropical cyclones rank above earthquakes as the major geophysical cause of loss of life and property (Bryant, 1991; Houghton, 1994). In the United States alone, the damage bill from mainland landfalling hurricanes over the last 50 years averages

Cell mechanics: principles, practices, and prospects

2.0 billion per year (Hebert et al., 1996). Years with high numbers of hurricanes provide new insight on the environmental factors influencing interannual variability; hence the interest in the exceptional 1995 Atlantic season which saw 11 hurricanes and a total of 19 tropical storms, double the 50-year average. While most environmental factors in 1995 were favourable for tropical cyclone development, we show that a factor not fully explored before, the sea surface temperature (SST) was the most significant. For the 10 degrees-20 degrees N, 20 degrees-60 degrees W region where 93% of the anomalous 1995 hurricanes developed, similar to 45 year statistical regressions show that SST is the dominating influence, independent of all known other factors, behind the interannual variance in Atlantic hurricance numbers. With this SST experiencing record warm levels in 1995, 0.66 degrees C above the 1946-1995 mean, these regressions indicate that sea warming explains 61+/-34% of the anomalous hurricane activity in 1995 to 95% confidence.

Emergence of homeostatic epithelial packing and stress dissipation through divisions oriented along the long cell axis

Cells generate and sustain mechanical forces within their environment as part of their normal physiology. They are active materials that can detect mechanical stimulation by the activation of mechanosensitive signaling pathways, and respond to physical cues through cytoskeletal re‐organization and force generation. Genetic mutations and pathogens that disrupt the cytoskeletal architecture can result in changes to cell mechanical properties such as elasticity, adhesiveness, and viscosity. On the other hand, perturbations to the mechanical environment can affect cell behavior. These transformations are often a hallmark and symptom of a variety of pathologies. Consequently, there are now a myriad of experimental techniques and theoretical models adapted from soft matter physics and mechanical engineering to characterize cell mechanical properties. Interdisciplinary research combining modern molecular biology with advanced cell mechanical characterization techniques now paves the way for furthering our fundamental understanding of cell mechanics and its role in development, physiology, and disease. We describe a generalized outline for measuring cell mechanical properties including loading protocols, tools, and data interpretation. We summarize recent advances in the field and explain how cell biomechanics research can be adopted by physicists, engineers, biologists, and clinicians alike. WIREs Syst Biol Med 2014, 6:371–388. doi: 10.1002/wsbm.1275

Formation of adherens junctions leads to the emergence of a tissue-level tension in epithelial monolayers

Significance Animal cells undergo a remarkable series of shape changes as they pass through mitosis and divide. In an epithelial tissue, the impact of these morphogenetic processes depends strongly on the orientation of division. However, the cues orienting divisions remain poorly understood. Here, we combine live imaging and mechanical perturbations with computational modeling to investigate the effects of shape changes accompanying mitosis and division in stretched monolayers in the absence of neighbor exchange. We show that divisions orient with the long cell axis rather than with the stress direction, and show how oriented divisions contribute to the restoration of cell packing and stress relaxation. In doing so, we identify a clear role for oriented cell division in morphogenetically active tissues. Cell division plays an important role in animal tissue morphogenesis, which depends, critically, on the orientation of divisions. In isolated adherent cells, the orientation of mitotic spindles is sensitive to interphase cell shape and the direction of extrinsic mechanical forces. In epithelia, the relative importance of these two factors is challenging to assess. To do this, we used suspended monolayers devoid of ECM, where divisions become oriented following a stretch, allowing the regulation and function of epithelial division orientation in stress relaxation to be characterized. Using this system, we found that divisions align better with the long, interphase cell axis than with the monolayer stress axis. Nevertheless, because the application of stretch induces a global realignment of interphase long axes along the direction of extension, this is sufficient to bias the orientation of divisions in the direction of stretch. Each division redistributes the mother cell mass along the axis of division. Thus, the global bias in division orientation enables cells to act collectively to redistribute mass along the axis of stretch, helping to return the monolayer to its resting state. Further, this behavior could be quantitatively reproduced using a model designed to assess the impact of autonomous changes in mitotic cell mechanics within a stretched monolayer. In summary, the propensity of cells to divide along their long axis preserves epithelial homeostasis by facilitating both stress relaxation and isotropic growth without the need for cells to read or transduce mechanical signals.

Stimulation of Cortical Myosin Phosphorylation by p114RhoGEF Drives Cell Migration and Tumor Cell Invasion

ABSTRACT Adherens junctions and desmosomes integrate the cytoskeletons of adjacent cells into a mechanical syncitium. In doing so, intercellular junctions endow tissues with the strength needed to withstand the mechanical stresses encountered in normal physiology and to coordinate tension during morphogenesis. Though much is known about the biological mechanisms underlying junction formation, little is known about how tissue-scale mechanical properties are established. Here, we use deep atomic force microscopy (AFM) indentation to measure the apparent stiffness of epithelial monolayers reforming from dissociated cells and examine which cellular processes give rise to tissue-scale mechanics. We show that the formation of intercellular junctions coincided with an increase in the apparent stiffness of reforming monolayers that reflected the generation of a tissue-level tension. Tension rapidly increased, reaching a maximum after 150 min, before settling to a lower level over the next 3 h as monolayers established homeostasis. The emergence of tissue tension correlated with the formation of adherens junctions but not desmosomes. As a consequence, inhibition of any of the molecular mechanisms participating in adherens junction initiation, remodelling and maturation significantly impeded the emergence of tissue-level tension in monolayers.

Improved sea surface temperature measurements from space

Actinomyosin activity is an important driver of cell locomotion and has been shown to promote collective cell migration of epithelial sheets as well as single cell migration and tumor cell invasion. However, the molecular mechanisms underlying activation of cortical myosin to stimulate single cell movement, and the relationship between the mechanisms that drive single cell locomotion and those that mediate collective cell migration of epithelial sheets are incompletely understood. Here, we demonstrate that p114RhoGEF, an activator of RhoA that associates with non-muscle myosin IIA, regulates collective cell migration of epithelial sheets and tumor cell invasion. Depletion of p114RhoGEF resulted in specific spatial inhibition of myosin activation at cell-cell contacts in migrating epithelial sheets and the cortex of migrating single cells, but only affected double and not single phosphorylation of myosin light chain. In agreement, overall elasticity and contractility of the cells, processes that rely on persistent and more constant forces, were not affected, suggesting that p114RhoGEF mediates process-specific myosin activation. Locomotion was p114RhoGEF-dependent on Matrigel, which favors more roundish cells and amoeboid-like actinomyosin-driven movement, but not on fibronectin, which stimulates flatter cells and lamellipodia-driven, mesenchymal-like migration. Accordingly, depletion of p114RhoGEF led to reduced RhoA, but increased Rac activity. Invasion of 3D matrices was p114RhoGEF-dependent under conditions that do not require metalloproteinase activity, supporting a role of p114RhoGEF in myosin-dependent, amoeboid-like locomotion. Our data demonstrate that p114RhoGEF drives cortical myosin activation by stimulating myosin light chain double phosphorylation and, thereby, collective cell migration of epithelial sheets and amoeboid-like motility of tumor cells.