High-resolution, real-time, three-dimensional fluorescence microscopy imaging shows the absence of clathrin-coated pits and vesicles at the ventral surfaces of lamellipodia and lamellae at the front of migrating cells. In addition, the data support the model invoking net membrane deposition at the cell front of migrating cells due to an imbalance between endocytic and exocytic membrane flow.

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. We demonstrate this DHM reflectometry technique by comparing DHM and profilometer measurement of home-made SiO(2)/Si targets and Secondary Ion Mass Spectrometry (SIMS) sputter craters on specimen including different multiple layers.

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. High resolution in both spatial and temporal domains allowed us to detect and characterize distinct classes of clathrin-coated structures. Aside from the clathrin pits and plaques detected in distinct embryonic tissues, we report, for the first time, formation of giant coated pits (GCPs) that can be up to two orders of magnitude larger than the canonical pits. In cultured cells, we show that GCP formation is induced by increased membrane tension. GCPs take longer to grow but their mechanism of curvature generation is the same as the canonical pits. We also demonstrate that GCPs split into smaller fragments during internalization. Considering the supporting roles played by actin filament dynamics under mechanically stringent conditions that slow down completion of clathrin coats, we suggest that local changes in the coat curvature driven by actin machinery can drive splitting and internalization of GCPs.

No abstract

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. High resolution in both spatial and temporal domains allowed us to detect and characterize distinct classes of clathrin-coated structures. For the first time, we show that membrane tension induces formation of giant coated pits (GCPs) that can be up to two orders of magnitude larger than the canonical clathrin-coated pits. GCPs take longer to form but their mechanism of curvature generation is the same as the canonical pits. We also demonstrate that GCPs can split into smaller fragments during internalization. Considering the supporting roles played by actin filament dynamics in clathrin-mediated endocytosis under mechanically stringent conditions, we suggest that local changes in the coat curvature driven by actin machinery can drive splitting and internalization of GCPs.