Force generation in human blood platelets by filamentous actomyosin structures.

Blood platelets are central elements of the blood clotting response after wounding. Upon vessel damage, they bind to the surrounding matrix and contract the forming thrombus, thus helping to restore normal blood circulation. The hemostatic function of platelets is directly connected to their mechanics and cytoskeletal organization. The reorganization of the platelet cytoskeleton during spreading occurs within minutes and leads to the formation of contractile actomyosin bundles, but it is not known if there is a direct correlation between the emerging actin structures and the force field that is exerted to the environment. In this study, we combine fluorescence imaging of the actin structures with simultaneous traction force measurements in a time-resolved manner. In addition, we image the final states with superresolution microscopy. We find that both the force fields and the cell shapes have clear geometrical patterns defined by stress fibers. Force generation is localized in a few hotspots, which appear early during spreading, and, in the mature state, anchor stress fibers in focal adhesions. Moreover, we show that, for a gel stiffness in the physiological range, force generation is a very robust mechanism and we observe no systematic dependence on the amount of added thrombin in solution or fibrinogen coverage on the substrate, suggesting that force generation after platelet activation is a threshold phenomenon that ensures reliable thrombus contraction in diverse environments.

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy.

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC). To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Quantifying force transmission through fibroblasts: changes of traction forces under external shearing.

Mammalian cells have evolved complex mechanical connections to their microenvironment, including focal adhesion clusters that physically connect the cytoskeleton and the extracellular matrix. This mechanical link is also part of the cellular machinery to transduce, sense and respond to external forces. Although methods to measure cell attachment and cellular traction forces are well established, these are not capable of quantifying force transmission through the cell body to adhesion sites. We here present a novel approach to quantify intracellular force transmission by combining microneedle shearing at the apical cell surface with traction force microscopy at the basal cell surface. The change of traction forces exerted by fibroblasts to underlying polyacrylamide substrates as a response to a known shear force exerted with a calibrated microneedle reveals that cells redistribute forces dynamically under external shearing and during sequential rupture of their adhesion sites. Our quantitative results demonstrate a transition from dipolar to monopolar traction patterns, an inhomogeneous distribution of the external shear force to the adhesion sites as well as dynamical changes in force loading prior to and after the rupture of single adhesion sites. Our strategy of combining traction force microscopy with external force application opens new perspectives for future studies of force transmission and mechanotransduction in cells.

Extracellular Matrix Geometry and Initial Adhesive Position Determine Stress Fiber Network Organization during Cell Spreading.

Three-dimensional matrices often contain highly structured adhesive tracks that require cells to turn corners and bridge non-adhesive areas. Here, we investigate these complex processes using micropatterned cell adhesive frames. Spreading kinetics on these matrices depend strongly on initial adhesive position and are predicted by a cellular Potts model (CPM), which reflects a balance between adhesion and intracellular tension. As cells spread, new stress fibers (SFs) assemble periodically and parallel to the leading edge, with spatial intervals of ∼2.5 µm, temporal intervals of ∼15 min, and characteristic lifetimes of ∼50 min. By incorporating these rules into the CPM, we can successfully predict SF network architecture. Moreover, we observe broadly similar behavior when we culture cells on arrays of discrete collagen fibers. Our findings show that ECM geometry and initial cell position strongly determine cell spreading and that cells encode a memory of their spreading history through SF network organization.

Mechanical interactions among followers determine the emergence of leaders in migrating epithelial cell collectives.

Regulating the emergence of leaders is a central aspect of collective cell migration, but the underlying mechanisms remain ambiguous. Here we show that the selective emergence of leader cells at the epithelial wound-margin depends on the dynamics of the follower cells and is spatially limited by the length-scale of collective force transduction. Owing to the dynamic heterogeneity of the monolayer, cells behind the prospective leaders manifest locally increased traction and monolayer stresses much before these leaders display any phenotypic traits. Followers, in turn, pull on the future leaders to elect them to their fate. Once formed, the territory of a leader can extend only to the length up-to which forces are correlated, which is similar to the length up-to which leader cells can transmit forces. These findings provide mechanobiological insight into the hierarchy in cell collectives during epithelial wound healing.

Dynamics of force generation by spreading platelets.

In order to gain more insight into the role of human platelets for blood clot formation, here we investigate the dynamics of force generation by platelet spreading onto elastic substrates of variable stiffness. Despite their small size, platelets generate high and rapidly varying traction forces on their extracellular environment, which we reconstruct with adapted implementations of Fourier transform traction cytometry. We find that while the final spread area is reached within a few minutes, the build-up of forces typically takes 10-30 minutes. In addition, we identify two distinct behaviors of individual cells, namely oscillating and non-oscillating platelets. An eigenvalue analysis of the platelet dipole tensor reveals a small anisotropy of the exerted force, which is compatible with a random distribution of a few force transmitting centers, in agreement with the observed shapes and traction patterns. We find a correlation between the maximum force level a platelet reaches and its spread area, which we explain by a thin film model for the actively contracting cell. The model reveals a large internal stress of hundreds of kPa. Experimentally we do not find any statistically relevant relation between the force level reached and the substrate stiffness within the stiffness range from 19 to 83 kPa, which might be related to the high platelet activation level used in our study. In addition, our model suggests that due to the uniquely small thickness of platelets, their mechanosensitivity might be limited to a lower stiffness range.

Optogenetic control of RhoA reveals zyxin-mediated elasticity of stress fibres.

Cytoskeletal mechanics regulates cell morphodynamics and many physiological processes. While contractility is known to be largely RhoA-dependent, the process by which localized biochemical signals are translated into cell-level responses is poorly understood. Here we combine optogenetic control of RhoA, live-cell imaging and traction force microscopy to investigate the dynamics of actomyosin-based force generation. Local activation of RhoA not only stimulates local recruitment of actin and myosin but also increased traction forces that rapidly propagate across the cell via stress fibres and drive increased actin flow. Surprisingly, this flow reverses direction when local RhoA activation stops. We identify zyxin as a regulator of stress fibre mechanics, as stress fibres are fluid-like without flow reversal in its absence. Using a physical model, we demonstrate that stress fibres behave elastic-like, even at timescales exceeding turnover of constituent proteins. Such molecular control of actin mechanics likely plays critical roles in regulating morphodynamic events.

Probabilistic inference in discrete spaces can be implemented into networks of LIF neurons.

The means by which cortical neural networks are able to efficiently solve inference problems remains an open question in computational neuroscience. Recently, abstract models of Bayesian computation in neural circuits have been proposed, but they lack a mechanistic interpretation at the single-cell level. In this article, we describe a complete theoretical framework for building networks of leaky integrate-and-fire neurons that can sample from arbitrary probability distributions over binary random variables. We test our framework for a model inference task based on a psychophysical phenomenon (the Knill-Kersten optical illusion) and further assess its performance when applied to randomly generated distributions. As the local computations performed by the network strongly depend on the interaction between neurons, we compare several types of couplings mediated by either single synapses or interneuron chains. Due to its robustness to substrate imperfections such as parameter noise and background noise correlations, our model is particularly interesting for implementation on novel, neuro-inspired computing architectures, which can thereby serve as a fast, low-power substrate for solving real-world inference problems.