Single-molecule in vitro reconstitution assay for kinesin-1-driven membrane dynamics

Intracellular membrane dynamics, especially the nano-tube formation, plays important roles in vesicle transportation and organelle biogenesis. Regarding the regulation mechanisms, it is well known that during the nano-tube formation, motor proteins act as the driven force moving along the cytoskeleton, lipid composition and its associated proteins serve as the linkers and key mediators, and the vesicle sizes play as one of the important regulators. In this review, we summarized the in vitro reconstitution assay method, which has been applied to reconstitute the nano-tube dynamics during autophagic lysosomal regeneration (ALR) and the morphology dynamics during mitochondria network formation (MNF) in a mimic and pure in vitro system. Combined with the single-molecule microscopy, the advantage of the in vitro reconstitution system is to study the key questions at a single-molecule or single-vesicle level with precisely tuned parameters and conditions, such as the motor mutation, ion concentration, lipid component, ATP/GTP concentration, and even in vitro protein knockout, which cannot easily be achieved by in vivo or intracellular studies.