Regulation of cellular contractile force, shape and migration of fibroblasts by oncogenes and Histone deacetylase 6.

The capacity of cells to adhere to, exert forces upon and migrate through their surrounding environment governs tissue regeneration and cancer metastasis. The role of the physical contractile forces that cells exert in this process, and the underlying molecular mechanisms are not fully understood. We, therefore, aimed to clarify if the extracellular forces that cells exert on their environment and/or the intracellular forces that deform the cell nucleus, and the link between these forces, are defective in transformed and invasive fibroblasts, and to indicate the underlying molecular mechanism of control. Confocal, Epifluorescence and Traction force microscopy, followed by computational analysis, showed an increased maximum contractile force that cells apply on their environment and a decreased intracellular force on the cell nucleus in the invasive fibroblasts, as compared to normal control cells. Loss of HDAC6 activity by tubacin-treatment and siRNA-mediated HDAC6 knockdown also reversed the reduced size and more circular shape and defective migration of the transformed and invasive cells to normal. However, only tubacin-mediated, and not siRNA knockdown reversed the increased force of the invasive cells on their surrounding environment to normal, with no effects on nuclear forces. We observed that the forces on the environment and the nucleus were weakly positively correlated, with the exception of HDAC6 siRNA-treated cells, in which the correlation was weakly negative. The transformed and invasive fibroblasts showed an increased number and smaller cell-matrix adhesions than control, and neither tubacin-treatment, nor HDAC6 knockdown reversed this phenotype to normal, but instead increased it further. This highlights the possibility that the control of contractile force requires separate functions of HDAC6, than the control of cell adhesions, spreading and shape. These data are consistent with the possibility that defective force-transduction from the extracellular environment to the nucleus contributes to metastasis, via a mechanism that depends upon HDAC6. To our knowledge, our findings present the first correlation between the cellular forces that deforms the surrounding environment and the nucleus in fibroblasts, and it expands our understanding of how cells generate contractile forces that contribute to cell invasion and metastasis.

Atomic force microscopy reveals the mechanical properties of breast cancer bone metastases.

Mechanically dependent processes are essential in cancer metastases. However, reliable mechanical characterization of metastatic cancer remains challenging whilst maintaining the tissue complexity and an intact sample. Using atomic force microscopy, we quantified the micro-mechanical properties of relatively intact metastatic breast tumours and their surrounding bone microenvironment isolated from mice, and compared with other breast cancer models both ex vivo and in vitro. A mechanical distribution of extremely low elastic modulus and viscosity was identified on metastatic tumours, which were significantly more compliant than both 2D in vitro cultured cancer cells and subcutaneous tumour explants. The presence of mechanically distinct metastatic tumour did not result in alterations of the mechanical properties of the surrounding microenvironment at meso-scale distances (>200 µm). These findings demonstrate the utility of atomic force microscopy in studies of complex tissues and provide new insights into the mechanical properties of cancer metastases in bone.

Calculation of the force field required for nucleus deformation during cell migration through constrictions.

During cell migration in confinement, the nucleus has to deform for a cell to pass through small constrictions. Such nuclear deformations require significant forces. A direct experimental measure of the deformation force field is extremely challenging. However, experimental images of nuclear shape are relatively easy to obtain. Therefore, here we present a method to calculate predictions of the deformation force field based purely on analysis of experimental images of nuclei before and after deformation. Such an inverse calculation is technically non-trivial and relies on a mechanical model for the nucleus. Here we compare two simple continuum elastic models of a cell nucleus undergoing deformation. In the first, we treat the nucleus as a homogeneous elastic solid and, in the second, as an elastic shell. For each of these models we calculate the force field required to produce the deformation given by experimental images of nuclei in dendritic cells migrating in microchannels with constrictions of controlled dimensions. These microfabricated channels provide a simplified confined environment mimicking that experienced by cells in tissues. Our calculations predict the forces felt by a deforming nucleus as a migrating cell encounters a constriction. Since a direct experimental measure of the deformation force field is very challenging and has not yet been achieved, our numerical approaches can make important predictions motivating further experiments, even though all the parameters are not yet available. We demonstrate the power of our method by showing how it predicts lateral forces corresponding to actin polymerisation around the nucleus, providing evidence for actin generated forces squeezing the sides of the nucleus as it enters a constriction. In addition, the algorithm we have developed could be adapted to analyse experimental images of deformation in other situations.

Osteoblast-Derived Paracrine and Juxtacrine Signals Protect Disseminated Breast Cancer Cells from Stress.

Metastatic breast cancer in bone is incurable and there is an urgent need to develop new therapeutic approaches to improve survival. Key to this is understanding the mechanisms governing cancer cell survival and growth in bone, which involves interplay between malignant and accessory cell types. Here, we performed a cellular and molecular comparison of the bone microenvironment in mouse models representing either metastatic indolence or growth, to identify mechanisms regulating cancer cell survival and fate. In vivo, we show that regardless of their fate, breast cancer cells in bone occupy niches rich in osteoblastic cells. As the number of osteoblasts in bone declines, so does the ability to sustain large numbers of breast cancer cells and support metastatic outgrowth. In vitro, osteoblasts protected breast cancer cells from death induced by cell stress and signaling via gap junctions was found to provide important juxtacrine protective mechanisms between osteoblasts and both MDA-MB-231 (TNBC) and MCF7 (ER+) breast cancer cells. Combined with mathematical modelling, these findings indicate that the fate of DTCs is not controlled through the association with specific vessel subtypes. Instead, numbers of osteoblasts dictate availability of protective niches which breast cancer cells can colonize prior to stimulation of metastatic outgrowth.

Modelling cytoskeletal transport by clusters of non-processive molecular motors with limited binding sites.

Molecular motors are responsible for intracellular transport of a variety of biological cargo. We consider the collective behaviour of a finite number of motors attached on a cargo. We extend previous analytical work on processive motors to the case of non-processive motors, which stochastically bind on and off cytoskeletal filaments with a limited number of binding sites available. Physically, motors attached to a cargo cannot bind anywhere along the filaments, so the number of accessible binding sites on the filament should be limited. Thus, we analytically study the distribution and the velocity of a cluster of non-processive motors with limited number of binding sites. To validate our analytical results and to go beyond the level of detail possible analytically, we perform Monte Carlo latticed based stochastic simulations. In particular, in our simulations, we include sequence preservation of motors performing stepping and binding obeying a simple exclusion process. We find that limiting the number of binding sites reduces the probability of non-processive motors binding but has a relatively small effect on force-velocity relations. Our analytical and stochastic simulation results compare well to published data from in vitro and in vivo experiments.

Mechanical Heterogeneity in the Bone Microenvironment as Characterized by Atomic Force Microscopy.

Bones are structurally heterogeneous organs with diverse functions that undergo mechanical stimuli across multiple length scales. Mechanical characterization of the bone microenvironment is important for understanding how bones function in health and disease. Here, we describe the mechanical architecture of cortical bone, the growth plate, metaphysis, and marrow in fresh murine bones, probed using atomic force microscopy in physiological buffer. Both elastic and viscoelastic properties are found to be highly heterogeneous with moduli ranging over three to five orders of magnitude, both within and across regions. All regions include extremely compliant areas, with moduli of a few pascal and viscosities as low as tens of Pa·s. Aging impacts the viscoelasticity of the bone marrow strongly but has a limited effect on the other regions studied. Our approach provides the opportunity to explore the mechanical properties of complex tissues at the length scale relevant to cellular processes and how these impact aging and disease.

Do migrating cells need a nucleus?

How the nucleus affects cell polarity and migration is unclear. In this issue, Graham et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201706097) show that enucleated cells polarize and migrate in two but not three dimensions and propose that the nucleus is a necessary component of the molecular clutch regulating normal mechanical responses.

Immersed Boundary Simulations of Active Fluid Droplets.

We present numerical simulations of active fluid droplets immersed in an external fluid in 2-dimensions using an Immersed Boundary method to simulate the fluid droplet interface as a Lagrangian mesh. We present results from two example systems, firstly an active isotropic fluid boundary consisting of particles that can bind and unbind from the interface and generate surface tension gradients through active contractility. Secondly, a droplet filled with an active polar fluid with homeotropic anchoring at the droplet interface. These two systems demonstrate spontaneous symmetry breaking and steady state dynamics resembling cell motility and division and show complex feedback mechanisms with minimal degrees of freedom. The simulations outlined here will be useful for quantifying the wide range of dynamics observable in these active systems and modelling the effects of confinement in a consistent and adaptable way.

Physical role for the nucleus in cell migration.

Cell migration is important for the function of many eukaryotic cells. Recently the nucleus has been shown to play an important role in cell motility. After giving an overview of cell motility mechanisms we review what is currently known about the mechanical properties of the nucleus and the connections between it and the cytoskeleton. We also discuss connections to the extracellular matrix and mechanotransduction. We identify key physical roles of the nucleus in cell migration.

Stress reorganization and response in active solids.

We present a microscopic model of a disordered viscoelastic active solid, i.e., an active material whose long time behavior is elastic as opposed to viscous. It is composed of filaments, passive cross-links, and molecular motors powered by stored chemical energy, e.g., actomyosin powered by adenosine triphosphate. Our model allows us to study the collective behavior of contractile active elements and how their interaction with each other and the passive elastic elements determines the macroscopic mechanical properties of the active material. As a result of the (un)binding dynamics of the active elements, we find that this system provides a highly responsive material with a dynamic mechanical response strongly dependent on the amount of deformation.

Active polar fluid flow in finite droplets.

We present a continuum level analytical model of a droplet of active contractile fluid consisting of filaments and motors. We calculate the steady state flows that result from a splayed polarisation of the filaments. We account for interaction with the external medium by imposing a viscous friction at the fixed droplet boundary. We then show that the droplet has non-zero force dipole and quadrupole moments, the latter of which is essential for self-propelled motion of the droplet at low Reynolds' number. Therefore, this calculation describes a simple mechanism for the motility of a droplet of active contractile fluid embedded in a three-dimensional environment, which is relevant to cell migration in confinement (for example, embedded within a gel or tissue). Our analytical results predict how the system depends on various parameters such as the effective friction coefficient, the phenomenological activity parameter and the splay of the imposed polarisation.

Evidence of a large-scale mechanosensing mechanism for cellular adaptation to substrate stiffness.

Cell migration plays a major role in many fundamental biological processes, such as morphogenesis, tumor metastasis, and wound healing. As they anchor and pull on their surroundings, adhering cells actively probe the stiffness of their environment. Current understanding is that traction forces exerted by cells arise mainly at mechanotransduction sites, called focal adhesions, whose size seems to be correlated to the force exerted by cells on their underlying substrate, at least during their initial stages. In fact, our data show by direct measurements that the buildup of traction forces is faster for larger substrate stiffness, and that the stress measured at adhesion sites depends on substrate rigidity. Our results, backed by a phenomenological model based on active gel theory, suggest that rigidity-sensing is mediated by a large-scale mechanism originating in the cytoskeleton instead of a local one. We show that large-scale mechanosensing leads to an adaptative response of cell migration to stiffness gradients. In response to a step boundary in rigidity, we observe not only that cells migrate preferentially toward stiffer substrates, but also that this response is optimal in a narrow range of rigidities. Taken together, these findings lead to unique insights into the regulation of cell response to external mechanical cues and provide evidence for a cytoskeleton-based rigidity-sensing mechanism.

Spontaneous contractility-mediated cortical flow generates cell migration in three-dimensional environments.

We present a model of cell motility generated by actomyosin contraction of the cell cortex. We identify, analytically, dynamical instabilities of the cortex and show that they yield steady-state cortical flows, which, in turn, can induce cell migration in three-dimensional environments. This mechanism relies on the regulation of contractility by myosin, whose transport is explicitly taken into account in the model. Theoretical predictions are compared to experimental data of tumor cells migrating in three-dimensional matrigel and suggest that this mechanism could be a general mode of cell migration in three-dimensional environments.

Model for the orientational ordering of the plant microtubule cortical array.

The plant microtubule cortical array is a striking feature of all growing plant cells. It consists of a more or less homogeneously distributed array of highly aligned microtubules connected to the inner side of the plasma membrane and oriented transversely to the cell growth axis. Here, we formulate a continuum model to describe the origin of orientational order in such confined arrays of dynamical microtubules. The model is based on recent experimental observations that show that a growing cortical microtubule can interact through angle dependent collisions with pre-existing microtubules that can lead either to co-alignment of the growth, retraction through catastrophe induction or crossing over the encountered microtubule. We identify a single control parameter, which is fully determined by the nucleation rate and intrinsic dynamics of individual microtubules. We solve the model analytically in the stationary isotropic phase, discuss the limits of stability of this isotropic phase, and explicitly solve for the ordered stationary states in a simplified version of the model.

Survival of the aligned: ordering of the plant cortical microtubule array.

The cortical array is a structure consisting of highly aligned microtubules which plays a crucial role in the characteristic uniaxial expansion of all growing plant cells. Recent experiments have shown polymerization-driven collisions between the membrane-bound cortical microtubules, suggesting a possible mechanism for their alignment. We present both a coarse-grained theoretical model and stochastic particle-based simulations of this mechanism, and we compare the results from these complementary approaches. Our results indicate that collisions that induce depolymerization are sufficient to generate the alignment of microtubules in the cortical array.

Structures of smooth muscle myosin and heavy meromyosin in the folded, shutdown state.

Remodelling the contractile apparatus within smooth muscle cells allows effective contractile activity over a wide range of cell lengths. Thick filaments may be redistributed via depolymerisation into inactive myosin monomers that have been detected in vitro, in which the long tail has a folded conformation. Using negative stain electron microscopy of individual folded myosin molecules from turkey gizzard smooth muscle, we show that they are more compact than previously described, with heads and the three segments of the folded tail closely packed. Heavy meromyosin (HMM), which lacks two-thirds of the tail, closely resembles the equivalent parts of whole myosin. Image processing reveals a characteristic head region morphology for both HMM and myosin, with features identifiable by comparison with less compact molecules. The two heads associate asymmetrically: the tip of one motor domain touches the base of the other, resembling the blocked and free heads of this HMM when it forms 2D crystals on lipid monolayers. The tail of HMM lies between the heads, contacting the blocked motor domain, unlike in the 2D crystal. The tail of whole myosin is bent sharply and consistently close to residues 1175 and 1535. The first bend position correlates with a skip in the coiled coil sequence, the second does not. Tail segments 2 and 3 associate only with the blocked head, such that the second bend is near the C-lobe of the blocked head regulatory light chain. Quantitative analysis of tail flexibility shows that the single coiled coil of HMM has an apparent Young's modulus of about 0.5 GPa. The folded tail of the whole myosin is less flexible, indicating interactions between the segments. The folded tail does not modify the compact head arrangement but stabilises it, indicating a structural mechanism for the very low ATPase activity of the folded molecule.

Dynamic allostery of protein alpha helical coiled-coils.

Alpha helical coiled-coils appear in many important allosteric proteins such as the dynein molecular motor and bacteria chemotaxis transmembrane receptors. As a mechanism for transmitting the information of ligand binding to a distant site across an allosteric protein, an alternative to conformational change in the mean static structure is an induced change in the pattern of the internal dynamics of the protein. We explore how ligand binding may change the intramolecular vibrational free energy of a coiled-coil, using parameterized coarse-grained models, treating the case of dynein in detail. The models predict that coupling of slide, bend and twist modes of the coiled-coil transmits an allosteric free energy of approximately 2kBT, consistent with experimental results. A further prediction is a quantitative increase in the effective stiffness of the coiled-coil without any change in inherent flexibility of the individual helices. The model provides a possible and experimentally testable mechanism for transmission of information through the alpha helical coiled-coil of dynein.

Coupling of global and local vibrational modes in dynamic allostery of proteins.

It is now recognized that internal global protein dynamics play an important role in the allosteric function of many proteins. Alterations of protein flexibility on effector binding affect the entropic cost of binding at a distant site. We present a coarse-grained model for a potential amplification of such entropic allostery due to coupling of fast, localized modes to the slow, global modes. We show how such coupling can give rise to large compensating entropic and enthalpic terms. The model corresponds to the pattern of calorimetry and NMR data from experiments on the Met repressor.

Coarse-grained model of entropic allostery.

Many signaling functions in molecular biology require proteins to bind to substrates such as DNA in response to environmental signals such as the simultaneous binding to a small molecule. Examples are repressor proteins which may transmit information via a conformational change in response to the ligand binding. An alternative entropic mechanism of "allostery" suggests that the inducer ligand changes the intramolecular vibrational entropy, not just the mean static structure. We present a quantitative, coarse-grained model of entropic allostery, which suggests design rules for internal cohesive potentials in proteins employing this effect. It also addresses the issue of how the signal information to bind or unbind is transmitted through the protein. The model may be applicable to a wide range of repressors and also to signaling in trans-membrane proteins.