SummaryContraction at the cellular level is vital for living organisms. The most prominent type of contractile cells are heart muscle cells, a lesswell-known example is blood platelets. Blood platelets activate and interlink at injured blood vessel sites, finally contracting to form a compact blood clot. They are ideal model cells to study the mechanisms of cellular contraction, as they are simple, having no nucleus, and their activation can be triggered and synchronized by the addition of thrombin. We have studied contraction using human blood platelets, employing traction force microscopy, a single-cell technique that enables time-resolved measurements of cellular forces on soft substrates with elasticities in the physiological range (,4 kPa). We found that platelet contraction reaches a steady state after 25 min with total forces of ,34 nN. These forces are considerably larger than what was previously reported for platelets in aggregates, demonstrating the importance of a single-cell approach for studies of platelet contraction. Compared with other contractile cells, we find that platelets are unique, because force fields are nearly isotropic, with forces pointing toward the center of the cell area.

The rapid reorganization of the actin cytoskeleton in response to external stimuli is an essential property of many motile eukaryotic cells. Here, we report evidence that the actin machinery of chemotactic Dictyostelium cells operates close to an oscillatory instability. When averaging the actin response of many cells to a short pulse of the chemoattractant cAMP, we observed a transient accumulation of cortical actin reminiscent of a damped oscillation. At the single-cell level, however, the response dynamics ranged from short, strongly damped responses to slowly decaying, weakly damped oscillations. Furthermore, in a small subpopulation, we observed self-sustained oscillations in the cortical F-actin concentration. To substantiate that an oscillatory mechanism governs the actin dynamics in these cells, we systematically exposed a large number of cells to periodic pulse trains of different frequencies. Our results indicate a resonance peak at a stimulation period of around 20 s. We propose a delayed feedback model that explains our experimental findings based on a time-delay in the regulatory network of the actin system. To test the model, we performed stimulation experiments with cells that express GFP-tagged fusion proteins of Coronin and actin-interacting protein 1, as well as knockout mutants that lack Coronin and actin-interacting protein 1. These actin-binding proteins enhance the disassembly of actin filaments and thus allow us to estimate the delay time in the regulatory feedback loop. Based on this independent estimate, our model predicts an intrinsic period of 20 s, which agrees with the resonance observed in our periodic stimulation experiments.Dictyostelium discoideum | microfluidics | caged cAMP | delay-differential equation T he actin cytoskeleton provides the basis for shape dynamics and motility of eukaryotic cells. Essential biological processes like wound healing, embryonic morphogenesis, or cancer metastasis rely on the rapid rearrangement of the actin cytoskeleton in response to external chemical cues (1). Many of the underlying actin-driven processes have been investigated in cells of the social amoeba Dictyostelium discoideum. Under starvation, the singlecelled amoeba expresses a chemotactic signaling system to aggregate into a multicellular structure, mediated by the chemoattractant cAMP. The corresponding receptor signaling pathway and the downstream cytoskeletal machinery show remarkable similarities to motile cells of higher organisms, in particular neutrophils (2), making Dictyostelium one of the most popular models for eukaryotic cell motility and chemotaxis (3, 4).In earlier studies, it was observed that the actin system of chemotactic Dictyostelium cells shows a complex, nonmonotonic response when exposed to a sudden increase in the extracellular chemoattractant concentration (5-7). Between 5 and 10 s after the stimulus, a first maximum in the filamentous actin content is observed, followed by a second, less intense but prolonged maximum that starts about 30 s after the stimulus and...

Blood platelets are instrumental in blood clotting and are thus heavily involved in early wound closure. After adhering to a substrate they spread by forming protrusions like lamellipodia and filopodia. However, the interaction of these protrusions with the physical environment of platelets while spreading is not fully understood. Here we dynamically image platelets during this spreading process and compare their behavior on smooth and on structured substrates. In particular we analyze the temporal evolution of the spread area, the cell morphology and the dynamics of individual filopodia. Interestingly, the topographic cues enable us to distinguish two spreading mechanisms, one that is based on numerous persistent filopodia and one that rather involves lamellipodia. Filopodia-driven spreading coincides with a strong response of platelet morphology to the substrate topography during spreading, whereas lamellipodia-driven spreading does not. Thus, we quantify different degrees of filopodia formation in platelets and the influence of filopodia in spreading on structured substrates.

Injuries in blood vessels are accompanied by disrupted endothelial cell layers. Missing or destroyed endothelial cells lead to rough, structured surfaces on the micrometer scale. The first cells to arrive at the site of injury and to cover the wound are platelets, which subsequently drive blood clot formation. Therefore, investigating the interactions of platelets with structured surfaces is essential for the understanding of blood clotting. Here, we study the effects of underlying topography on platelet spreading using microstructured model substrates with varying area fractions of protein coating. We thereby distinguish the effects of (physical) topography and of (biochemical) protein availability. By analyzing the cell area and morphology, we find that the extent of protrusion formation - but not the total spread area - is determined by the area fractions of coating. The extent of filopodia formation is influenced by the availability of binding sites and the reaction of cells to the substrate's topography. The cells react to the structured substrate by avoiding topographic holes at the cell periphery and thus adapting their outer shape. This finding leads us to the conclusion that both chemically blocked and fibrinogen-coated holes represent "energetic obstacles" to the cells. Thus, the shape of the cell is governed by the interplay between spreading to an optimized area and adaption to the substrate topography.

ContentsMost Important Denitions and Variables patterned substrates chemically patterned substrates structured substrates topographically structured substrates relative perimeter ratio of perimeter of the platelet to perimeter of an ellipse that has the same area, orientation and eccentricity as the platelet v cell-ellipse (β, t) vector between platelet outline and corresponding ellipse, with β being the angle it encloses with the x-axis and t the time point of the image in the time lapse series l cell-ellipse (β, t) signed length of vector v cell−ellipse (β, t) var dir variance between the moving average of signed lengths l cell-ellipse (β, t) in different directions var time variance between the moving average of signed lengths l cell-ellipse (β, t) in time adapted platelets (as defined in chapter 6) platelets on structured substrates that show a mean var dir of larger than 0.18 µm 2 unadapted platelets (as defined in chapter 6) platelets on structured substrates that show a mean var dir of smaller than 0.18 µm 2 vi