Dynamics of intracellular clathrin/AP1 and clathrin/AP3 containing carriers

Clathrin/AP1 and clathrin/AP3 coated vesicular carriers originate from endosomes and the TGN. We report here the real-time visualization of these structures in living cells reliably tracked by using rapid, three-dimensional imaging with a spinning-disk confocal microscope. We imaged relatively sparse, diffraction-limited, fluorescent objects containing chimeric fluorescent protein (clathrin light chain, σ adaptor subunits or dynamin2) with a spatial precision of up to ~ 30 nm and a temporal resolution of ~ 1 sec. The dynamic characteristics of the intracellular clathrin/AP1 and clathrin/AP3 carriers are similar to those of endocytic clathrin/AP2 pits and vesicles; the clathrin/AP1 coats are, on average, slightly shorter-lived than their AP2 and AP3 counterparts. We confirmed that while dynamin2 is recruited as a burst to clathrin/AP2 pits immediately before their budding from the plasma membrane, we found no evidence supporting a similar association of dynamin2 with clathrin/AP1 or clathrin/AP3 carriers at any stage during their lifetime. We found no effects of chemical inhibitors of dynamin function or the K44A dominant negative mutant of dynamin on AP1 and AP3 dynamics. This observation suggests that an alternative budding mechanism, yet to be discovered, is responsible for the scission step of clathrin/AP1 and clathrin/ AP3 carriers