The fate of mitochondria during platelet activation.

Blood platelets undergo several successive motor-driven reorganizations of the cytoskeleton when they are recruited to an injured part of a vessel. These reorganizations take place during the platelet activation phase, the spreading process on the injured vessel or between fibrin fibers of the forming clot, and during clot retraction. All these steps require a lot of energy, especially the retraction of the clot when platelets develop strong forces similar to those of muscle cells. Platelets can produce energy through glycolysis and mitochondrial respiration. However, although resting platelets have only 5 to 8 individual mitochondria, they produce adenosine triphosphate predominantly via oxidative phosphorylation. Activated, spread platelets show an increase in size compared with resting platelets, and the question arises as to where the few mitochondria are located in these larger platelets. Using expansion microscopy, we show that the number of mitochondria per platelet is increased in spread platelets. Live imaging and focused ion beam-scanning electron microscopy suggest that a mitochondrial fission event takes place during platelet activation. Fission is Drp1 dependent because Drp1-deficient platelets have fused mitochondria. In nucleated cells, mitochondrial fission is associated with a shift to a glycolytic phenotype, and using clot retraction assays, we show that platelets have a more glycolytic energy production during clot retraction and that Drp1-deficient platelets show a defect in clot retraction.

Cell fluorescence photoactivation as a method to select and study cellular subpopulations grown in mechanically heterogeneous environments.

A central challenge to the biology of development and disease is deciphering how individual cells process and respond to numerous biochemical and mechanical signals originating from the environment. Recent advances in genomic studies enabled the acquisition of information about population heterogeneity; however, these so far are poorly linked with the spatial heterogeneity of biochemical and mechanical cues. Whereas in vitro models offer superior control over spatiotemporal distribution of numerous mechanical parameters, researchers are limited by the lack of methods to select subpopulations of cells in order to understand how environmental heterogeneity directs the functional collective response. To circumvent these limitations, we present a method based on the use of photo convertible proteins, which when expressed within cells and activated with light, gives a stable fluorescence fingerprint enabling subsequent sorting and lysis for genomics analysis. Using this technique, we study the spatial distribution of genetic alterations on well-characterized local mechanical stimulation within the epithelial monolayer. Our method is an in vitro alternative to laser microdissection, which so far has found a broad application in ex vivo studies.