Genome-edited human stem cells expressing fluorescently labeled endocytic markers allow quantitative analysis of clathrin-mediated endocytosis during differentiation

Abstract

We developed a general approach for investigation of how cellular processes become adapted for specific cell types during differentiation. Previous studies reported substantial differences in the morphology and dynamics of clathrin-mediated endocytosis (CME) sites. However, associating specific CME properties with distinct differentiated cell types and determining how these properties are developmentally specified during differentiation have been elusive. Using genome-edited human embryonic stem cells, and isogenic fibroblasts and neuronal progenitor cells derived from them, we established by live-cell imaging and platinum replica transmission electron microscopy that CME site dynamics and ultrastructure on the plasma membrane are precisely reprogrammed during differentiation. Expression levels for the endocytic adaptor protein AP2μ2 were found to underlie dramatic changes in CME dynamics and structure. Additionally, CME dependency on actin assembly and phosphoinositide-3 kinase activity are distinct for each cell type. Collectively, our results demonstrate that key CME properties are reprogrammed during differentiation at least in part through AP2μ2 expression regulation.

Alternative splicing of clathrin heavy chain contributes to the switch from coated pits to plaques

“…The drastic modification of clathrin assembly occurred independently of a change in clathrin protein levels (both heavy and light chains; Fig. S2 A), although an increase in AP2 protein content was detected in agreement with a recent report showing that an AP2 level increase contributes to the formation of large clathrin structures (Dambournet et al, 2018).…”

Growth factor stimulation promotes multivesicular endosome biogenesis by prolonging recruitment of the late-acting ESCRT machinery

“…With advances in live cell imaging approaches and genome editing technologies, barriers to studying native vesicle-mediated transport processes in mammalian cells are beginning to diminish. The dynamics of clathrin-mediated endocytosis are arguably the best understood, at least in part due to the availability of genome-edited cell lines and facile access to total internal reflection fluorescence microscopy, which is capable of illuminating a narrow, ∼200-nm window at the surface of cells (67)(68)(69)(70)(71). Light sheet imaging with diffraction-limited optics now affords us the opportunity of studying vesicle formation anywhere within mammalian cells, including MVEs that accumulate in the perinuclear region and facilitate the turnover of many integral membrane proteins (60,72).…”

Principles of self-organization and load adaptation by the actin cytoskeleton during clathrin-mediated endocytosis

“…These cells also endogenously express a tagRFP-T fusion with the µ2 subunit of the adaptor protein AP2, allowing us to identify sites of clathrin-mediated endocytosis (Hong et al, 2015) . We determined the relative timing of AP2 and ArpC3 appearance at endocytic sites using time-lapse TIRF imaging and automated two-color particle tracking (Dambournet et al, 2018;Hong et al, 2015) ( Figure 2F). The vast majority (81 ± 10%, n=136) of CME events marked by AP2-RFP culminated in a burst of ArpC3-GFP fluorescence, prior to internalization of the pit, persisting until the pit internalized ( Figure 2G and Movie S4).…”

“…We used stringent tracking parameters with gap size 0 and search radius 0-2.3 pixels (248 nm). GFP and RFP tracks with high variability in the intensity/time profile were automatically rejected (Ferguson et al, 2017) as well as tracks ≤ 3 s in duration (Dambournet et al, 2018) and the remaining tracks were associated spatiotemporally according to a cost matrix (Hong et al, 2015) . We used two track rejection schemes.…”

“…We checked that the manual and automatic track rejection schemes yielded similar results (lifetime distributions and intensity versus time plots) as well as to manual, kymograph-based quantification of lifetimes (below). From the above workflow (Dambournet et al, 2018) we increased throughput by connecting all steps into an automated tracking pipeline requiring minimal user input. For SK-MEL-2 cells expressing CLTA-RFP and DNM2-GFP, we tracked regions that were not near the nucleus (which has a concentration of Golgi-derived clathrin budding) and that did not have large, bright, persistent structures containing invariant RFP and GFP signals ("plaques," which are likely sites of adhesion).…”