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

Cytosolic calcium (Ca2+ ) is a ubiquitous second messenger that influences numerous aspects of cellular function. In many cell types cytosolic Ca2+ concentrations are characterized by periodic pulses whose dynamics can influence downstream signal transduction. Here, we examined the general question of how cells use Ca2+ 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 Ca2+ dynamics in LECs, and that Ca2+ -dependent signaling plays a key role in regulating lymphatic valve formation during embryonic development. However, how fluid flow might influence the Ca2+ pulse dynamics of individual LECs remained, to our knowledge, little explored. We used live-cell imaging to characterize Ca2+ 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 Ca2+ pulses was sensitive to the magnitude of WSS, while the duration of individual Ca2+ pulses increased in the presence of spatial gradients in WSS. These observations reveal an example of how cells can separately modulate Ca2+ pulse frequency and duration to encode distinct forms of information, a phenomenon that could extend to other cell types. Movie S1 Movie S1 HLMVEC Ca2+ dynamics in the IFC, recorded for 30 minutes starting from the onset of flow at t = 50 s. Regions corresponding to Rings 1 and 2 are shown, which have average WSSs of 32 and 65 dyn/cm2 , respectively. The flow direction is radially outward and symmetric about the jet center at the center of Ring 1. Frames were recorded every 5 seconds. Scale bar, 100 μm. Movie S2 Movie S2 HLMVEC Ca2+ dynamics as in Movie 1 for Rings 2 - 6, which have average WSSs of 65, 53, 30, 17 and 11 dyn/cm2 . The flow direction is radially outward and here is roughly from left to right. Frames were recorded every 5 seconds. Scale bar, 100 μm. Movie S3 Movie S3 HLMVEC Ca2+ dynamics for cells exposed to uniform WSS (parallel plate flow), recorded for 30 minutes from the onset of flow at t = 50 s. Here, all HLMVECs experience a WSS of 50 dyn/cm2 . The flow direction is from the bottom of the video to the top. Frames were recorded every 5 seconds. Scale bar, 100 μm. Movie S4 Movie S4 HLMVEC Ca2+ dynamics under no flow conditions, recorded for 30 minutes. Frames were recorded every 5 seconds. Scale bar, 100 μm.