Abstract
Clathrin/AP1- and clathrin/AP3-coated vesicular carriers originate from endosomes and the trans-Golgi network. Here, we report the real-time visualization of these structures in living cells reliably tracked by rapid, three-dimensional imaging with the use of 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 s. 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 although 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.
Highlights
Clathrin-based carriers are responsible for a large fraction of the endocytic traffic between the plasma membrane and endosomes, for traffic between endosomes, and for traffic between endosomes and the Trans-Golgi network
The assembly of the endocytic clathrin-based carriers can be followed by expression of clathrin light chains or the σ2 subunit of AP2 fused with EGFP or related fluorescent proteins; suitably designed chimeric proteins do not affect the functional properties of the labeled carriers (Ehrlich et al, 2004)
We find that intracellular, AP-1 containing clathrin carriers are on average slightly shorter-lived than intracellular clathrin/AP3 and endocytic clathrin/AP2 carriers
Summary
Clathrin-based carriers are responsible for a large fraction of the endocytic traffic between the plasma membrane and endosomes, for traffic between endosomes, and for traffic between endosomes and the Trans-Golgi network. The dynamics of AP2-containing clathrin-coated structures at the surface of a cell attached to a glass coverslip have been studied by 2D time-lapse imaging, using confocal or total internal reflection fluorescence microscopy (Cocucci et al, 2012; Ehrlich et al, 2004; Merrifield et al, 2005; Mettlen et al, 2009; Saffarian et al, 2009; Taylor et al, 2011; Yarar et al, 2005) Results from these experiments have shown that clathrin and AP2 adaptors are recruited continuously during coat formation and that the average lifetime of endocytic coats is 40–60 s. These observations, which are consistent with earlier work that demonstrated lack of functional interference with traffic along the secretory pathway by overexpression of dominant negative mutant dynamins, suggest that an alternative budding mechanism, yet to be discovered, is responsible for the scission step of clathrin/AP1 and clathrin/AP3 carriers
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