Expansion microscopy is a super‐resolution method that allows expanding uniformly biological samples, by increasing the relative distances among fluorescent molecules labeling specific components. One of the main concerns in this approach regards the isotropic behavior at the nanoscale. The present study aims to determine the robustness of such a technique, quantifying the expansion parameters i.e. scale factor, isotropy, uniformity. Our focus is on the nuclear pore complex (NPC), as well‐known nanoscale component endowed of a preserved and symmetrical structure localized on the nuclear envelope. Here, we show that Nup153 is a good reporter to quantitatively address the isotropy of the expansion process. The quantitative analysis carried out on NPCs, at different spatial scales, allows concluding that expansion microscopy can be used at the nanoscale to measure subcellular features with an accuracy from 10 to 5 nm. Therefore, it is an excellent method for structural studies of macromolecular complexes.
Background
Platelets contain a high amount of potentially active A subunit dimer of coagulation factor XIII (cellular FXIII; cFXIII). It is of cytoplasmic localization, not secreted, but becomes translocated to the surface of platelets activated by convulxin and thrombin (CVX+Thr).
Objective
To explore the difference in cFXIII translocation between receptor mediated and non‐receptor mediated platelet activation and if translocation can also be detected on platelet‐derived microparticles. Our aim was also to shed some light on the mechanism of cFXIII translocation.
Methods
Gel‐filtered platelets were activated by CVX+Thr or Ca2+‐ionophore (calcimycin). The translocation of cFXIII and phosphatidylserine (PS) to the surface of activated platelets and platelet‐derived microparticles was investigated by flow cytometry, immunofluorescence, and immune electron microscopy. Fluo‐4‐AM fluorescence was used for the measurement of intracellular Ca2+ concentration.
Results
Receptor mediated activation by CVX+Thr exposed cFXIII to the surface of more than 60% of platelets. Electron microscopy revealed microparticles with preserved membrane structure and microparticles devoid of labeling for membrane glycoprotein CD41a. cFXIII was observed on both types of microparticles but was more abundant in the absence of CD41a. Rhosin, a RhoA inhibitor, significantly decreased cFXIII translocation. Non‐receptor mediated activation of platelets by calcimycin elevated intracellular Ca2+ concentration, induced the translocation of PS to the surface of platelets and microparticles, but failed to expose cFXIII.
Conclusions
The elevation of intracellular Ca2+ concentration is sufficient for the translocation of PS from the internal layer of the membrane, while the translocation of cFXIII from the platelet cytoplasm requires additional receptor mediated mechanism(s).
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