Session T40: Focus Session: Cytoskeleton and Biomechanics - Forced Dynamics

Dynamics of focals adhesions modulates active cellular response

The cytoskeleton of living cells connects to and senses the extracellular mechanical environment through protein clusters called focal adhesions. We study how the mechanics and dynamics of focal adhesions influence the distribution of mechanical stresses and deformations in a cell which is modeled as an active elastic gel. We carry out our investigations for two types of bond dynamics of focal contacts (a) slip bond dynamics where the bonds are weakened by a tensile mechanical force and (b) catch bond dynamics where they are initially strengthened upon application of a tensile mechanical force, and undergo failure at very large forces. We comment on the effect of different types of focal adhesions on the transmission and regulation of cell traction forces and on cellular mechanosensing.   

3D Neutrophil Tractions in Changing Microenvironments

Neutrophils are well-known as first responders to defend the body against life threatening bacterial diseases, infections and inflammation. The mechanical properties and the local topography of the surrounding microenvironment play a significant role in the regulating neutrophil behavior including cell adhesion, migration and generation of tractions. In navigating to the site of infection, neutrophils are exposed to changing microenvironments that differ in their composition, structure and mechanical properties. Our goal is to investigate neutrophil behavior, specifically migration and cellular tractions in a well-controlled 3D in vitro system. By utilizing an interchangeable 2D-3D sandwich gel structure system with tunable mechanical properties neutrophil migration and cell tractions can be computed as a function of gel stiffness and geometric dimensionality.   

Force transmission through intercellular fluid transfer

Cell force generation and transmission play a vital role in controlling cell-to-cell interactions and cell locomotion. Contraction of the cell's cytoskeletal network generates forces that can be transmitted directly to other cells by cell-cell adherens junctions or through a substrate by traction forces. Within monolayers, cytosolic fluid is transferred between cells through gap junctions. The coupling between intercellular fluid movement and contraction can give rise to a different type of cell-cell force transmission. Here we present preliminary results investigating the role of intercellular force transmission through fluid motion across gap junctions.   

Forcing it on: Cytoskeletal dynamics during lymphocyte activation

Formation of the immune synapse during lymphocyte activation involves cell spreading driven by large scale physical rearrangements of the actin cytoskeleton and the cell membrane. Several recent observations suggest that mechanical forces are important for efficient T cell activation. How forces arise from the dynamics of the cytoskeleton and the membrane during contact formation, and their effect on signaling activation is not well understood. We have imaged membrane topography, actin dynamics and the spatiotemporal localization of signaling clusters during the very early stages of spreading. Formation of signaling clusters was closely correlated with the movement and topography of the membrane in contact with the activating surface. Further, we observed membrane waves driven by actin polymerization originating at these signaling clusters. Actin-driven membrane protrusions likely play an important role in force generation at the immune synapse. In order to study cytoskeletal forces during T-cell activation, we studied cell spreading on elastic gels. We found that gel stiffness influences cell morphology, actin dynamics and receptor activation. Efforts to determine the quantitative relationships between cellular forces and signaling are underway. Our results suggest a role for cytoskeleton driven forces during signaling activation in lymphocytes.   

Aging phenomena in a network model of the cytoskeleton

Motivated by a series of experiments that study the response of the cytoskeleton in living cells to time dependent mechanical forces, we investigate, through Monte Carlo simulations, a three-dimensional network subjected to perturbations. After having prepared the system in a relaxed state, shear is applied and the relaxation processes are monitored. We measure two-time functions of various quantities such as the Euclidean distance between crosslinks and the energy of the system. We discuss possible implications of our results for relaxation processes taking place in the cytoskeleton.   

Mechanical interactions may explain synchronized growth of cytoskeletal actin networks in motile cells

Growing networks of actin fibers are able to organize into compact, stiff two-dimensional structures inside lamellipodia of crawling cells. We examine critically the hypothesis that the growing actin network is a critically self-organized system, in which long-range mechanical stresses arising from the interaction with the plasma membrane provide the selective pressure leading to organization. We show that a simple model based only on this principle leads to stochastic protrusion of lamellipodia (growth periods alternating with fast retractions) and several of the features observed in experiments: a growth velocity initially insensitive to the external force; the capability of the network to organize its orientation; a load-history-dependent growth velocity. Our model predicts that the spectrum of the time series of the height of a growing lamellipodium decays with the inverse of the frequency. This behavior is confirmed by optical tweezer measurements performed in vivo on neuronal growth cones. References L. Cardamone et al.: Cytoskeletal actin networks in motile cells are critically self-organized systems synchronized by mechanical interactions. PNAS, vol. 108, no. 34, pp. 13978-13983   

Investigations of biomechanical activity of macrophages during phagocytosis

Phagocytosis has traditionally been investigated in terms of the relevant biochemical signaling pathways that trigger the process and lead to the deformation of the cell as it engulfs a target. Physical changes in the cell include rearrangement and polymerization of actin in the phagocytic cup, large membrane deformations, increased membrane area via exocytosis, and closure of the phagocytic cup through membrane fusion. Hence, phagocytosis is a fine-tuned balance between biophysical cellular events and chemical signaling, which are responsible for driving these materials and mechanical changes. We present a series of assays designed to probe the physical/mechanical parameters that govern a cell during phagocytosis. Custom built micropipette manipulators are used to manipulate individual cells, facilitating high-resolution microscopy of individual phagocytic events. This work has been supported by NSF PoLS {\#}0848797.   

Collective cell migration during inflammatory response

Wound scratch healing assays of endothelial cell monolayers is a simple model to study collective cell migration as a function of biological signals. A signal of particular interest is the immune response, which after initial wounding in vivo causes the release of various inflammatory factors such as tumor necrosis alpha (TNF-$\alpha$). TNF-$\alpha$ is an innate inflammatory cytokine that can induce cell growth, cell necrosis, and change cell morphology. We studied the effects of TNF-$\alpha$ on collective cell migration using the wound healing assays and measured several migration metrics, such as rate of scratch closure, velocities of leading edge and bulk cells, closure index, and velocity correlation functions between migrating cells. We observed that TNF-$\alpha$ alters all migratory metrics as a function of the size of the scratch and TNF-$\alpha$ content. The changes observed in migration correlate with actin reorganization upon TNF-$\alpha$ exposure.   

Temperature dependence of optically induced cell deformations

The mechanical properties of any material change with temperature, hence this must be true for cellular material. In biology many functions are known to undergo modulations with temperature, like myosin motor activity, mechanical properties of actin filament solutions, CO2 uptake of cultured cells or sex determination of several species. As mechanical properties of living cells are considered to play an important role in many cell functions it is surprising that only little is known on how the rheology of single cells is affected by temperature. We report the systematic temperature dependence of single cell deformations in Optical Stretcher (OS) measurements. The temperature is changed on a scale of about 20 minutes up to hours and compared to defined temperature shocks in the range of milliseconds. Thereby, a strong temperature dependence of the mechanics of single suspended cells is revealed. We conclude that the observable differences arise rather from viscosity changes of the cytosol than from structural changes of the cytoskeleton. These findings have implications for the interpretation of many rheological measurements, especially for laser based approaches in biological studies.   

Biomechanical changes in endothelial cells result from an inflammatory response

During periods of infection and disease, the immune system induces the release of TNF-$\alpha $, an inflammatory cytokine, from a variety of cell types, such as macrophages. TNF-$\alpha $, while circulating in the vasculature, binds to the apical surface of endothelial cells and causes a wide range of biological and mechanical changes to the endothelium. While the biological changes have been widely studied, the biomechanical aspects have been largely unexplored. Here, we investigated the biomechanical changes of the endothelium as a function of TNF-$\alpha $ treatment. First, we studied the traction forces applied by the endothelium, an effect that is much less studied than others. Through the use of traction force microscopy, we found that TNF-$\alpha $ causes an increase in traction forces applied by the endothelial cells as compared to non-treated cells. Then, we investigated cell morphology, cell mechanics, migration, and cytoskeletal dynamics. We found that in addition to increasing applied traction forces, TNF-$\alpha $ causes an increase in cell area and aspect ratio on average, as well as a shift in the organization of F-actin filaments within the cell. Combining these findings together, our results show that an inflammatory response heavily impacts the morphology, cell mechanics, migration, cytoskeletal dynamics, and applied traction forces of endothelial cells.   

Healing of small circular model wounds

We develop a new method to produce numerous circular wounds in an epithelial tissue of MDCK cells in a non-traumatic fashion. The reproducibility of the wounds allows for a quantitative study of the dynamics of healing and for a better understanding of the key processes involved in those collective morphogenetic movements. First, we show different mechanisms of closing depending on the initial size of the wound. We then focus on the healing of the smallest wounds from an experimental and theoretical point of view. At the onset of closure, an actomyosin ring is formed around the wound and small protrusions appear and invade the free surface. Using inhibition and laser ablation experiments, we show the relative contribution of both processes to the dynamics of closing. Finally, we develop a theoretical model of the tissue as a whole, combined with the observed forces, in order to better understand the underlying mechanics of this process. We hope that this qualitative and quantitative description will prove useful in the future for the study of epithelial architecture, collective mechanisms in migrating tissues and, on a broader context, cellular invasion in cancerous tissues.   

Mechanical coupling of smooth muscle cells using local and global stimulations

Mechanical stresses can directly alter many cellular processes, including signal transduction, growth, differentiation, and survival. These stresses, generated primarily by myosin activity within the cytoskeleton, regulate both cell-substrate and cell-cell interactions. We report studies of mechanical cell-cell and cell-substrate interactions using patterned arrays of flexible poly(dimethylsiloxane) (PDMS) microposts combined with application of global stretch or local chemical stimulation. Bovine pulmonary artery smooth muscle cells are patterned onto micropost arrays to create multicellular structures to probe intercellular coupling. Global stimulation is applied by building the micropost arrays on a flexible membrane that can be stretched while allowing simultaneous observation of cell traction forces. Results for triangle wave stretches of single cells show increasing traction forces with increasing strain, and immediate weakening of traction forces as strain is decreased. ``Spritzing,'' a laminar flow technique, is used to expose a single cell within a construct to a drug treatment while cell traction forces are recorded via the microposts. Results will be described showing the response of cells to external stimulation both directly and through intercellular coupling.