Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis

Abstract

Clathrin-mediated endocytosis (CME) proceeds through a series of morphological changes of the plasma membrane induced by a number of protein components. Although the spatiotemporal assembly of these proteins has been elucidated by fluorescence-based techniques, the protein-induced morphological changes of the plasma membrane have not been fully clarified in living cells. Here, we visualize membrane morphology together with protein localizations during CME by utilizing high-speed atomic force microscopy (HS-AFM) combined with a confocal laser scanning unit. The plasma membrane starts to invaginate approximately 30 s after clathrin starts to assemble, and the aperture diameter increases as clathrin accumulates. Actin rapidly accumulates around the pit and induces a small membrane swelling, which, within 30 s, rapidly covers the pit irreversibly. Inhibition of actin turnover abolishes the swelling and induces a reversible open–close motion of the pit, indicating that actin dynamics are necessary for efficient and irreversible pit closure at the end of CME.

Distribution and Co-localization of endosome markers in cells

“…Fluorescence microscopy has been used to visualize the co-localization of cargo with intracellular proteins and membranes [14, 34, 35]. In addition, it has also been used to determine the distribution of various endocytic organelles based on association of Rab proteins with intracellular membranes [36], the dynamics of clathrin recruitment to the cell membrane [37, 38], the dynamics of adapter protein recruitment and removal [39], and the dynamics of dynamin assisted pinching of the invaginated pit [40, 41]. However, no quantitative studies have been performed to determine the extent of co-localization and clustering of Rab5, Rab7, and LAMP-1 markers in cells.…”

Correlative SICM‐FCM reveals changes in morphology and kinetics of endocytic pits induced by disease‐associated mutations in dynamin

“…Using correlative SICM-FCM, the recruitment of DNM2-GFP to topographically resolved CCPs has been demonstrated for the first time (44). This technique has also enabled the discovery and characterization of an alternative mechanism of actin-facilitated CCP closure that was more recently confirmed by high-speed atomic force microscopy combined with confocal laser scanning unit (36). Although temporal resolution of SICM remains slower compared with high-speed atomic force microscopy, STED, and LLSM, its topographical ( i.e.…”

“…Although researchers have demonstrated clear colocalization between AF647-labeled clathrin and CCP structures, the technique is not capable of studying CCP kinetics. The lack of live and dynamic investigations in cell membrane morphologic changes, resulting from the recruitment of specific molecules, is a limitation of current imaging techniques, which has been previously highlighted (36, 37). …”

“…Although researchers have demonstrated clear colocalization between AF647-labeled clathrin and CCP structures, the technique is not capable of studying CCP kinetics. The lack of live and dynamic investigations in cell membrane morphologic changes, resulting from the recruitment of specific molecules, is a limitation of current imaging techniques, which has been previously highlighted (36,37). Previously, we have developed an imaging technique that, in contrast to microscopies described above, enables us to follow morphologic changes of endocytic pits through their entire lifecycle in living cells at close to few nanometers resolution (38).…”

It Takes Two to Tango: Endothelial TGFβ/BMP Signaling Crosstalk with Mechanobiology

“…BMP-SMAD1/5/9 signaling, in particular its turn-over rate, is regulated by endocytosis ( Figure 1 f), which in turn depends on the physical properties of the plasma membrane [ 86 , 87 , 88 ]. Two major endocytic routes were described for membrane-bound BMP-receptor complexes (1) clathrin-mediated endocytosis (CME) involving clathrin-coated pits (CCPs), and (2) caveolin-dependent endocytosis [ 89 , 90 ].…”