Using quantitative microscopy approaches to study the formation of clathrin-coated vesicles

Clathrin-coated vesicles (CCVs) are endocytic carriers that mediate the internalization of ligands and their receptors from the plasma membrane. CCVs assembly requires the coordinated recruitment of many proteins that promote receptor sorting and membrane curvature formation. Clathrin triskelia together with the adaptors AP2 and clathrin assembly lymphoid myeloid leukemia protein (CALM) are the most represented components of CCVs. CALM supports the formation of membrane curvature that is essential for the growth of CCVs. Membrane curvature can be reached only by overcoming local plasma membrane tension that, together with the availability of CCV components, determines the type of clathrin structures that forms over the plasma membrane. We hypothesized that CALM recruitment could support the formation of CCVs even under mechanically stringent conditions, such as increased membrane tension. To investigate this, we used knock-in gene editing to obtain cells expressing fluorescently tagged CALM at endogenous levels and spinning-disc and lattice light sheet live-cell microscopy calibrated to the intensity of a single molecule to quantitatively study CALM recruitment during the formation of CCVs in normal conditions, upon membrane tension increase, and/or CALM depletion. Our results demonstrate that CALM promotes CCV completion under membrane tension increase possibly by a mechanism based on protein-protein crowding at the neck of the CCV. We also observed that CALM is differentially expressed across cell types and that given the small footprint of CALM, there is sufficient room on the CCV surface to recruit CALM molecules as needed, depending on the local membrane tension and cellular availability. In conclusion, our data suggest that the competence in clathrin-mediated endocytosis differs across cells and tissues and explain the reason why CALM is essential during embryo development but dispensable in other cellular contexts.