Endocytosis at extremes: Formation and internalization of giant clathrin-coated pits under elevated membrane tension.

Internalization of clathrin-coated vesicles from the plasma membrane constitutes the major endocytic route for receptors and their ligands. Dynamic and structural properties of endocytic clathrin coats are regulated by the mechanical properties of the plasma membrane. Here, we used conventional fluorescence imaging and multiple modes of structured illumination microscopy (SIM) to image formation of endocytic clathrin coats within live cells and tissues of developing fruit fly embryos.

Asymmetric formation of coated pits on dorsal and ventral surfaces at the leading edges of motile cells and on protrusions of immobile cells.

Clathrin/AP2-coated vesicles are the principal endocytic carriers originating at the plasma membrane. In the experiments reported here, we used spinning-disk confocal and lattice light-sheet microscopy to study the assembly dynamics of coated pits on the dorsal and ventral membranes of migrating U373 glioblastoma cells stably expressing AP2 tagged with enhanced green fluorescence (AP2-EGFP) and on lateral protrusions from immobile SUM159 breast carcinoma cells, gene-edited to express AP2-EGFP. On U373 cells, coated pits initiated on the dorsal membrane at the front of the lamellipodium and at the approximate boundary between the lamellipodium and lamella and continued to grow as they were swept back toward the cell body; coated pits were absent from the corresponding ventral membrane.

Digital holographic reflectometry.

Digital holographic microscopy (DHM) is an interferometric technique that allows real-time imaging of the entire complex optical wavefront (amplitude and phase) reflected by or transmitted through a sample. To our knowledge, only the quantitative phase is exploited to measure topography, assuming homogeneous material sample and a single reflection on the surface of the sample. In this paper, dual-wavelength DHM measurements are interpreted using a model of reflected wave propagation through a three-interfaces specimen (2 layers deposited on a semi-infinite layer), to measure simultaneously topography, layer thicknesses and refractive indices of micro-structures.