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

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. 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.

High-speed Auto-Polarization Synchronization Modulation Three-dimensional Structured Illumination Microscopy

“…Although 3DSIM can interpret the 3D hollow structure of CCPs, Sparse deconvolution can make the hollow structure clearer. This is in consistent with the physiological structure of CCPs 32 , and similar results were reported with 3D single-molecule localization microscopy 33 . Then we use MRA deconvolution 34 for the processing of tubulin structure of low signal-to-noise ratio in Fig.…”

High-speed autopolarization synchronization modulation three-dimensional structured illumination microscopy

“…Although 3DSIM can interpret the 3D hollow structure of CCPs, sparse deconvolution can make the hollow structure clearer. This is consistent with the physiological structure of CCPs, 32 and similar results were reported with 3D single-molecule localization microscopy. 33 Then, we use MRA deconvolution 34 for the processing of tubulin structure of the low signal-to-noise ratio in Fig.…”

Mechano-regulation by clathrin pit-formation and passive cholesterol-dependent tubules during de-adhesion

“…Processes involved in membrane homeostasis would ensure the maintenance of membrane tension or conservation of the microscopic fluctuations, providing the plasma membrane’s effective tautness or excess area (difference between microscopic area and projected area per unit microscopic area) [ 46 , 64 ]. While enhancing tension can affect endocytosis [ 18 , 29 ] and pit-formation [ 9 ], as displayed for clathrin-mediated endocytosis (CME) [ 2 ], conversely, cells can also alter their endocytosis/exocytosis rates to regulate tension. Studies have addressed the role of membrane trafficking in tension regulation, either in general [ 65 ] or focussing on particular pathways such as the CLIC-GEEC (CG) pathway [ 74 ], CME [ 17 , 39 ] or those using caveolae [ 45 ].…”

“…Depletion of Cholesterol (by MβCD) thus, can arrest the formation of new pits of the CG pathway and caveolae. However, it can also enhance tension [ 7 ], and has been reported to cause a substantial reduction in the formation and budding of deep pits of CME without completely blocking CME [ 2 , 4 , 58 , 73 ]. The use of ATP-depletion can reduce both the pinching-off of a majority of pathways as well as the formation of pits in pathways like CME [ 62 ] and caveolae [ 69 ].…”