Fluorescence-coupled micropipette aspiration assay to examine calcium mobilization caused by red blood cell mechanosensing

Abstract

Mechanical stimuli such as tension, compression, and shear stress play critical roles in the physiological functions of red blood cells (RBCs) and their homeostasis, ATP release, and rheological properties. Intracellular calcium (Ca2+) mobilization reflects RBC mechanosensing as they transverse the complex vasculature. Emerging studies have demonstrated the presence of mechanosensitive Ca2+ permeable ion channels and their function has been implicated in the regulation of RBC volume and deformability. However, how these mechanoreceptors trigger Ca2+ influx and subsequent cellular responses are still unclear. Here, we introduce a fluorescence-coupled micropipette aspiration assay to examine RBC mechanosensing at the single-cell level. To achieve a wide range of cell aspirations, we implemented and compared two negative pressure adjusting apparatuses: a homemade water manometer (− 2.94 to 0 mmH2O) and a pneumatic high-speed pressure clamp (− 25 to 0 mmHg). To visualize Ca2+ influx, RBCs were pre-loaded with an intensiometric probe Cal-520 AM, then imaged under a confocal microscope with concurrent bright-field and fluorescent imaging at acquisition rates of 10 frames per second. Remarkably, we observed the related changes in intracellular Ca2+ levels immediately after aspirating individual RBCs in a pressure-dependent manner. The RBC aspirated by the water manometer only displayed 1.1-fold increase in fluorescence intensity, whereas the RBC aspirated by the pneumatic clamp showed up to threefold increase. These results demonstrated the water manometer as a gentle tool for cell manipulation with minimal pre-activation, while the high-speed pneumatic clamp as a much stronger pressure actuator to examine cell mechanosensing directly. Together, this multimodal platform enables us to precisely control aspiration and membrane tension, and subsequently correlate this with intracellular calcium concentration dynamics in a robust and reproducible manner.

Microscale geometrical modulation of PIEZO1 mediated cell mechanosensing via cytoskeletal redistribution buckle

“…1e). This discovery con rms prior assertions about PIEZO1 activity being predominantly modulated by membrane tension 18,26 and adds nuance to our understanding of the variations in calcium fold change observed in different conditions 30,41 .…”

“…Equipped with a high-speed pneumatic pump and a water reservoir, our micropipette holder facilitated precise, steady, and controlled aspiration of targeted RBCs under aspiration pressures ∆p = -5 to -40 mmHg 30 . Before aspiration, we pre-incubated cells with the intensiometric calcium dye, Cal-520 AM (Method).…”

“…To bridge this gap, we utilized an ultrasensitive biomechanical approach-uorescent micropipette aspiration (fMPA) assay 30 , that allows us to visualize intracellular calcium mobilization and cytoskeletal remodeling in living cells while simultaneously manipulating the aspiration mechanics and physical microgeometry, including parameters such as narrow space diameter and inclined angle (Fig. 1).…”

The N-terminal autoinhibitory module of the A1 domain in von Willebrand factor stabilizes the mechanosensor catch bond

“…The stiffness of the RBC ( k RBC ) can be determined by the radii of the orifice ( R p ), the probe bead ( R c ) and RBC ( R 0 ) when the aspirated tongue length ( L p ) of the RBC is equal to R p : 34,72 The pressure applied to the RBC is precisely controlled by our homemade manual water manometer so that the spring constant of the RBC can be modified to 0.3 pN nm −1 by adjusting the height difference between the water level in the reservoir and the tip of the micropipette. 34,73 As a result, the force applied to the probe bead can be interpreted via the deflection of the RBC. During each experiment cycle (Fig.…”

“…The pressure applied to the RBC is precisely controlled by our homemade manual water manometer so that the spring constant of the RBC can be modified to 0.3 pN nm À1 by adjusting the height difference between the water level in the reservoir and the tip of the micropipette. 34,73 As a result, the force applied to the probe bead can be interpreted via the deflection of the RBC. During each experiment cycle (Fig.…”