Direct characterization of cytoskeletal reorganization during blood platelet spreading

Abstract

Blood platelets are the key cellular players in blood clotting and thus of great biomedical importance. While spreading at the site of injury, they reorganize their cytoskeleton within minutes and assume a flat appearance. As platelets possess no nucleus, many standard methods for visualizing cytoskeletal components by means of fluorescence tags fail. Here we employ silicon-rhodamine actin and tubulin probes for imaging these important proteins in a time-resolved manner. We find two distinct timescales for platelet spread area development and for cytoskeletal reorganization, indicating that although cell spreading is most likely associated with actin polymerization at the cell edges, distinct, stress-fiber-like actin structures within the cell, which may be involved in the generation of contractile forces, form on their own timescale. Following microtubule dynamics allows us to distinguish the role of myosin, microtubules and actin during early spreading.

Human blood platelets contract in perpendicular direction to shear flow

“…The thick actin bundles (purple), including myosin motors (pink), are shown to connect the focal adhesions. This internal organization has previously been reported for studies employing actin stainings 44,45 (compare also Fig. 5c) or integrin force sensors.…”

“…17 It was shown that actin fibers within platelets develop in two phases; first, the actin polymerizes into a scaffold-like actin structure, which is then reinforced and stress-fiber-like bundles appear. 45 Similar time scales were observed for the force development of platelets using force sensors. 17 Here, a lower force regime was observed during actin-polymerization driven spreading, whereas a higher force was observed after initial spreading, coinciding with the reported reinforcement of actin bundles.…”

“…Our results indicate that this response to external forces is gradual and starts at very low physiological shear rates. Future experiments involving the current set-up and fluorescent stainings of the actin cytoskeleton 45 or the usage of integrin force sensors 17 may help to experimentally verify the assumption of a correlation between the internal structures of the platelet and the force alignment. Our single cell study shows that clot stability is already initiated by the settlement of the very first platelet.…”

Dynamics of force generation by spreading platelets

“…These findings are in good agreement with our recent work where we tracked the temporal evolution of the actin cytoskeleton in living platelets, using SiR-actin as a probe. 52 We observed actin polymerization-driven spreading on a time scale of one minute after adhesion and a rapid build-up of stress-fiber-like actin bundles on a time scale of four mins, which are enforced thereafter. Similarly, in the recent paper by Wang et al, 13 a low-tension regime in platelets was reported, Fig.…”

Time-resolved MIET measurements of blood platelet spreading and adhesion

“…Thus, platelets repel other platelets and blood components. 4 Platelets do not divide and lack a microtubule spindle apparatus, however, they do possess an actomyosin cytoskeleton which promotes adhesion, spreading and contraction 5,6 Once a platelet adheres to the substrate, it becomes very flat and its thickness in air-dried conditions reaches from 40 nm for the periphery to 100 nm for the cell center, as measured by scanning electron microscopy (SEM). 7 Due to the flattening during platelet spreading, the cell's surface area increases drastically.…”