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Cytosolic calcium (Ca 2+ ) is a ubiquitous second messenger that influences numerous aspects of cellular function. In many cell types, cytosolic Ca 2+  concentrations are characterized by periodic pulses, whose dynamics can influence downstream signal transduction. Here, we examine the general question of how cells use Ca 2+  pulses to encode input stimuli in the context of the response of lymphatic endothelial cells (LECs) to fluid flow. Previous work shows that fluid flow regulates Ca 2+  dynamics in LECs and that Ca 2+ -dependent signaling plays a key role in regulating lymphatic valve formation during embryonic development. However, how fluid flow might influence the Ca 2+  pulse dynamics of individual LECs has remained, to our knowledge, little explored. We used live-cell imaging to characterize Ca 2+  pulse dynamics in LECs exposed to fluid flow in an in vitro flow device that generates spatial gradients in wall shear stress (WSS), such as are found at sites of valve formation. We found that the frequency of Ca 2+  pulses was sensitive to the magnitude of WSS, while the duration of individual Ca 2+  pulses increased in the presence of spatial gradients in WSS. These observations provide an example of how cells can separately modulate Ca 2+  pulse frequency and duration to encode distinct forms of information, a phenomenon that could extend to other cell types.

Lymphatic endothelial cell calcium pulses are sensitive to spatial gradients in wall shear stress