Induction of immunity and peripheral tolerance requires contacts between antigen-bearing dendritic cells (DCs) and cognate T cells. Using real-time two-photon microscopy, we have analyzed the dynamics of CD8(+) T cells in lymph nodes during the induction of antigen-specific immunity or tolerance. At 15-20 h after the induction of immunity, T cells stopped moving and established prolonged interactions with DCs. In tolerogenic conditions, despite effective initial T cell activation and proliferation, naive T cells remained motile and established serial brief contacts with multiple DCs. Thus, stable DC-T cell interactions occur during the induction of priming, whereas brief contacts may contribute to the induction of T cell tolerance.
The leading front of a collectively migrating epithelium often destabilizes into multicellular migration fingers where a cell initially similar to the others becomes a leader cell while its neighbours do not alter. The determinants of these leader cells include mechanical and biochemical cues, often under the control of small GTPases. However, an accurate dynamic cartography of both mechanical and biochemical activities remains to be established. Here, by mapping the mechanical traction forces exerted on the surface by MDCK migration fingers, we show that these structures are mechanical global entities with the leader cells exerting a large traction force. Moreover, the spatial distribution of RhoA differential activity at the basal plane strikingly mirrors this force cartography. We propose that RhoA controls the development of these fingers through mechanical cues: the leader cell drags the structure and the peripheral pluricellular acto-myosin cable prevents the initiation of new leader cells.
We use magnetic tweezers to study local viscoelastic response in filamentous actin networks. The choice of magnetic, colloidal particles of varying size allows us to explore properties on the relevant micron and submicron scales. At these scales the mechanical response is determined by the bending properties of individual filaments and described by an anomalous power-law behavior. In the absence of external forces the particles exhibit a subdiffusive motion. [S0031-9007(96)01627-4] Complex molecular systems, such as polymer solutions, polymer melts, gels, (micro)emulsions, and foams, often display a combination of the elastic properties of solids and the viscous properties of fluids. Using classical rheological methods [1], the viscoelastic properties of such materials have been described at scales much larger than the molecular dimensions, and the systems under study have mostly been treated as homogeneous media. In many situations, however, local mechanical properties are of critical importance. For instance, the shape and motility of living cells, as well as cytoplasmic transport, are strongly influenced by the mechanical properties of cytoskeleton networks [2] at submicron and micron scales.Actin filaments ( f-actin), formed upon polymerization of globular actin proteins, are major components of the cytoskeleton and are involved in both transport and motility [3]. Easily purified and polymerized in vitro, actin is a model system for the study of the mechanics and assembly of biopolymers [4,5]. In this paper we show, using the example of actin filaments, how micromechanical measurements can provide information about local viscoelastic properties of the medium.f-actin is a rigid polymer with a persistence length L p of the order of 15 mm [6]. At high enough concentrations, in the so-called semidilute regime, the polymers form a three-dimensional network with a mesh size L. L is typically of the order of a micron and thus much smaller than L p . Viscoelastic properties of this inhomogeneous medium can be locally studied by inserting colloidal magnetic beads and perturbing them with external magnetic forces. In fact, such simple methods have been used for many years to explore the cytoplasm [7]. For beads with diameter, d, much larger than L, the mechanical perturbation is macroscopic. On the other hand, if d is much smaller than the mesh size, the bead is expected to probe only the solvent viscosity and geometrical constraints introduced by polymers. Therefore, the regime that is relevant for exploring the local network mechanics is one for which d is comparable to L. In this case, the bead is moving inside a "cage" of typical linear size L. To move further, it has to perturb the polymers of the cage, either through the influence of an external force or via thermal fluctuations. In both cases, one can study the viscoelastic properties on micron scales by observing the motion of individual beads. We show below that this approach can be made quantitative, and that local mechanical properties of the network can be...
We present a study on the fluctuations of semiflexible actin filaments using fluorescence videomicroscopy, focusing on the end-to-end fluctuations of single filaments. In order to specifically measure the position of the polymer's ends, we developed a novel noninvasive method that consists of annealing short end tags to the filaments. This allows us to probe polymer fluctuations to a very high accuracy. We compared the distribution of the end-to-end distance with recent theoretical results, and found excellent agreement. We also studied the dynamics of the mean-square end-to-end distance deltaR2(t) and orientation of the ends, deltaTheta(2)(t), finding power laws t(3/4) and t(1/4), respectively. Scaling behavior for deltaR2(t) is observed over several decades in relaxation time in agreement with theoretical results.
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