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.

Time-resolved MIET measurements of blood platelet spreading and adhesion.

Human blood platelets are non-nucleated fragments of megakaryocytes and of high importance for early hemostasis. To form a blood clot, platelets adhere to the blood vessel wall, spread and attract other platelets. Despite the importance for biomedicine, the exact mechanism of platelet spreading and adhesion to surfaces remains elusive. Here, we employ metal-induced energy transfer (MIET) imaging with a leaflet-specific fluorescent membrane probe to quantitatively determine, with nanometer resolution and in a time-resolved manner, the height profile of the basal and the apical platelet membrane above a rigid substrate during platelet spreading. We observe areas, where the platelet membrane approaches the substrate particularly closely and these areas are stable on a time scale of minutes. Time-resolved MIET measurements reveal distinct behaviors of the outermost rim and the central part of the platelets, respectively. Our findings quantify platelet adhesion and spreading and improve our understanding of early steps in blood clotting. Furthermore, the results of this study demonstrate the potential of MIET for simultaneous imaging of two close-by membranes and thus three-dimensional reconstruction of the cell shape.

Transport and programmed release of nanoscale cargo from cells by using NETosis.

Cells can take up nanoscale materials, which has important implications for understanding cellular functions, biocompatibility as well as biomedical applications. Controlled uptake, transport and triggered release of nanoscale cargo is one of the great challenges in biomedical applications of nanomaterials. Here, we study how human immune cells (neutrophilic granulocytes, neutrophils) take up nanomaterials and program them to release this cargo after a certain time period. For this purpose, we let neutrophils phagocytose DNA-functionalized single-walled carbon nanotubes (SWCNTs) in vitro that fluoresce in the near infrared (980 nm) and serve as sensors for small molecules. Cells still migrate, follow chemical gradients and respond to inflammatory signals after uptake of the cargo. To program release, we make use of neutrophil extracellular trap formation (NETosis), a novel cell death mechanism that leads to chromatin swelling, subsequent rupture of the cellular membrane and release of the cell's whole content. By using the process of NETosis, we can program the time point of cargo release via the initial concentration of stimuli such as phorbol 12-myristate-13-acetate (PMA) or lipopolysaccharide (LPS). At intermediate stimulation, cells continue to migrate, follow gradients and surface cues for around 30 minutes and up to several hundred micrometers until they stop and release the SWCNTs. The transported and released SWCNT sensors are still functional as shown by subsequent detection of the neurotransmitter dopamine and reactive oxygen species (H2O2). In summary, we hijack a biological process (NETosis) and demonstrate how neutrophils transport and release functional nanomaterials.