Membrane tension spatially organizes lysosomal exocytosis

Abstract

Lysosomal exocytosis is involved in many key cellular processes but its spatio-temporal regulation is poorly known. Using total internal reflection fluorescence microscopy (TIRFM) and spatial statistics, we observed that lysosomal exocytosis is not random at the adhesive part of the plasma membrane of RPE1 cells but clustered at different scales. Although the rate of exocytosis is regulated by the actin cytoskeleton, neither interfering with actin or microtubule dynamics by drug treatments alters its spatial organization. Exocytosis events partially co-appear at focal adhesions (FAs) and their clustering is reduced upon removal of FAs. Changes in membrane tension following a hypo-osmotic shock or treatment with methyl-β-cyclodextrin was found to increase clustering. To investigate the link between FAs and membrane tension, cells were cultured on adhesive ring-shaped micropatterns, which allows to control the spatial organization of FAs. By using a combination of TIRFM and fluorescence lifetime imaging microscopy (FLIM) microscopy, we revealed the existence of a radial gradient in membrane tension. By changing the diameter of micropatterned substrates, we further showed that this gradient as well as the extent of exocytosis clustering can be controlled. Together, our data indicate that the spatial clustering of lysosomal exocytosis relies on membrane tension patterning controlled by the spatial organization of FAs.

Technical insights into fluorescence lifetime microscopy of mechanosensitive Flipper probes

“…We have designed, synthesized and characterized small-molecules called FLIPPERS that can report changes of membrane lateral forces due to lipid packing variations through changes of their conformation and hence of their fluorescence properties 7 . The high sensitivity of Flippers allows them to report membrane tension variations by changes of the lifetime of their excited state or fluorescence lifetime 8 , upon osmotic shocks 9,10 or to investigate cellular processes such as lysosomal exocytosis 11 . Over the past six years, we designed various derivatives targeting different organelles 12 (plasma membrane, endoplasmic reticulum, mitochondria and lysosome/late endosome).…”