過剰浸透圧勾配下における一過性膜透過水流の迅速単一細胞測定

Aquaporins (AQPs) are critical for transmembrane water transport in response to osmotic gradients, but their gating and regulatory mechanisms remain poorly understood. A central challenge is the lack of methods to measure water flow across AQPs from individual cells with the spatiotemporal resolution and sensitivity equivalent to patch-clamp recordings of ion fluxes-a limitation stemming from the electrically silent nature of water flow. Here, we present a novel optical technique-Flow-Induced Fluorescence Increase Velocimetry (FIFIV) based on Laser-Induced Fluorescence Photobleaching Anemometry (LIFPA)-that enables direct, real-time monitoring of cytoplasmic flow induced by transmembrane water transport under osmotic pressure gradients. Using small molecular fluorescent dyes to label cytoplasm in single adherent MDA-MB-231 breast cancer cells, we show detection of instantaneous extremely low transmembrane water flow signals on the order of 1 μm/s following localized hypotonic stimulation. This approach circumvents the limitations of traditional volume-based osmotic permeability assays, achieving single-cell sensitivity and temporal resolution comparable to electrophysiological measurements of ion flux. FIFIV potentially provides a new optical approach for probing transmembrane water-flow-induced intracellular dynamics, and a foundation for future investigations of AQP regulation and gating mechanisms in physiopathological studies and drug discovery.