BackgroundEukaryotic cells are large enough to detect signals and then orient to them by differentiating the signal strength across the length and breadth of the cell. Amoebae, fibroblasts, neutrophils and growth cones all behave in this way. Little is known however about cell motion and searching behavior in the absence of a signal. Is individual cell motion best characterized as a random walk? Do individual cells have a search strategy when they are beyond the range of the signal they would otherwise move toward? Here we ask if single, isolated, Dictyostelium and Polysphondylium amoebae bias their motion in the absence of external cues.MethodologyWe placed single well-isolated Dictyostelium and Polysphondylium cells on a nutrient-free agar surface and followed them at 10 sec intervals for ∼10 hr, then analyzed their motion with respect to velocity, turning angle, persistence length, and persistence time, comparing the results to the expectation for a variety of different types of random motion.ConclusionsWe find that amoeboid behavior is well described by a special kind of random motion: Amoebae show a long persistence time (∼10 min) beyond which they start to lose their direction; they move forward in a zig-zag manner; and they make turns every 1–2 min on average. They bias their motion by remembering the last turn and turning away from it. Interpreting the motion as consisting of runs and turns, the duration of a run and the amplitude of a turn are both found to be exponentially distributed. We show that this behavior greatly improves their chances of finding a target relative to performing a random walk. We believe that other eukaryotic cells may employ a strategy similar to Dictyostelium when seeking conditions or signal sources not yet within range of their detection system.
We explain and demonstrate a new method of force-and position-calibration for optical tweezers with back-focal-plane photo detection. The method combines power spectral measurements of thermal motion and the response to a sinusoidal motion of a translation stage. It consequently does not use the drag coefficient of the trapped object as an input. Thus, neither the viscosity, nor the size of the trapped object, nor its distance to nearby surfaces need to be known. The method requires only a low level of instrumentation and can be applied in situ in all spatial dimensions. It is both accurate and precise: true values are returned, with small error-bars. We tested this experimentally, near and far from surfaces. Both position-and force-calibration were accurate to within 3%. To calibrate, we moved the sample with a piezo-electric translation stage, but the laser beam could be moved instead, e.g. by acousto-optic deflectors. Near surfaces, this precision requires an improved formula for the hydrodynamical interaction between an infinite plane and a micro-sphere in non-constant motion parallel to it. We give such a formula.
Optical tweezers are widely used to measure molecular forces in biology. Such measurements are often influenced by a nearby surface that can perturb both the calibration of the tweezers as well as the hydrodynamic forces acting on microspheres to which the biomolecules are attached. In this study, we have used a very stable optical tweezers setup employing a recently developed calibration method (Tolić-Nørrelykke, S. F.; Schäffer, E.; Howard, J.; Pavone, F. S.; Jülicher, F.; Flyvbjerg, H. Rev. Sci. Instrum. 2006, 77 (10), 103101) to determine how the calibration of the tweezers and the forces on the microspheres depend on the height above the surface. We show that the displacement sensitivity of the tweezers is modulated by a standing light wave between the microsphere and the surface. We measured the dependence of the drag coefficient on height and compared it to exact and closed-form solutions to the Navier-Stokes equations. Also, we measured the surface force gradients in different salt solutions and for different surface blocking methods. For a given blocking method, our data suggest that microspheres can experience attractive and/or repulsive forces close to surfaces. For example, a Teflon layer reduces attractive interactions, and the presence of casein can lead to long-range repulsive interactions. These measurements are a prerequisite for the accurate measurement of normal forces with respect to an interface that occur in biological molecules held between surfaces.
Sinusoidal endothelial cells and mesenchymal CXCL12-abundant reticular cells are principal bone marrow stromal components, which critically modulate haematopoiesis at various levels, including haematopoietic stem cell maintenance. These stromal subsets are thought to be scarce and function via highly specific interactions in anatomically confined niches. Yet, knowledge on their abundance, global distribution and spatial associations remains limited. Using three-dimensional quantitative microscopy we show that sinusoidal endothelial and mesenchymal reticular subsets are remarkably more abundant than estimated by conventional flow cytometry. Moreover, both cell types assemble in topologically complex networks, associate to extracellular matrix and pervade marrow tissues. Through spatial statistical methods we challenge previous models and demonstrate that even in the absence of major specific interaction forces, virtually all tissue-resident cells are invariably in physical contact with, or close proximity to, mesenchymal reticular and sinusoidal endothelial cells. We further show that basic structural features of these stromal components are preserved during ageing.
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