In Situ Measurements of Protein Forces and Intracellular Ca2+ under Fluid Shear Stimuli

Abstract

During traumatic brain injury, brain cells experience transient shear stresses that alter the forces in structural proteins. An increase in intracellular Ca2+ is seen universally in cells subjected to shear forces, but how are mechanical forces coupled to the Ca2+ influx is not well understood. We used a microfluidic chamber driven with a high-speed pressure servo to generate controlled fluid shear stress to cultured astrocytes, and simultaneously measured the intracellular responses using FRET-based force sensors actinin-cpstFRET and Ca2+ probes jRCaMP1h. We found that fluid shear generated non-uniform stresses in actinin. The time dependent force distribution is highly sensitive to the feature of the stimulus such as amplitude, duration rise time, and the frequencies of stimulation. In cells with weaker stress fibers, a rapid shear pulse (23 dyn/cm2, 2 ms rise time, 400 ms duration) produced an immediate and long-lasting increase in actinin stress at the upstream end of the cell and minimal changes at the downstream end. In contrast, a slow ramp to the same amplitude caused a minimal, and a more uniform increase in actinin stresses. Simultaneous Ca2+ imaging showed that the initial Ca2+ rise began at the upstream end of the cell where there was high strain, and it propagated to the entire cell within ∼4 s. Moreover, the Ca2+ peaked much faster (∼4 s) at the front end and slower in the center of cell body (∼10 s). This behavior occurred in ∼40% cells. In cells with abundant strong actin bundles, there was minimal response to shear stress. The actinin stress increased transiently and uniformly over the entire cell during the period of stimulation, and the Ca2+ rise appeared starting from the cell body. These results suggest that the force distribution directly alter the initial Ca2+ dynamics in different cellular domains. This work was funded by NINDS.