Intracellular Dynamics of Lysosomes Following the Disruption of Lysosome Associated Membrane Proteins (LAMP)

Abstract

FRET-based genetically encoded metal-ion sensors coupled with fluorescence microscopy provide an important tool for real-time imaging of metal-ion homeostasis. The heterogeneity and in vivo kinetics are important sensor properties, yet are less explored because of lacking available experimental methods. We present a high-throughput microfluidic method capable of characterizing the FRET kinetics of metal-ion sensors in mammalian cells, at a throughput of 15 cells/sec with a time window of a few ms to several seconds. Using this method, we screened the HeLa cell Zn 2þ sensor library and measured the FRET distribution at single cell level. We also report the quantitative highthroughput cellular-level heterogeneity measurement of cell populations, the cluster map of the Zn 2þ sensor FRET change resolves 4 subpopulations with different metal response. Finally, we measured the in vivo sensor kinetics induced by changes in [Zn 2þ ] and [Ca 2þ ]. We observed a > 30fold rate difference between the extracellular and intracellular sensors, and the kinetics are ionophore-dependent.