Ultrastructure of Long‐Range Transport Carriers Moving from the trans Golgi Network to Peripheral Endosomes

Abstract

The delivery of mannose 6-phosphate receptors carrying lysosomal hydrolases from the trans-Golgi network (TGN) to the endosomal system is mediated by selective incorporation of the receptor-hydrolase complexes into vesicular transport carriers (TCs) that are coated with clathrin and the adaptor proteins, GGA and AP-1. Previous electron microscopy (EM) and biochemical studies have shown that these TCs consist of spherical coated vesicles with a diameter of 60-100 nm. The use of fluorescent live cell imaging, however, has revealed that at least some of this transport relies on a subset of apparently larger and highly pleiomorphic carriers that detach from the TGN and translocate toward the peripheral cytoplasm until they meet with distally located endosomes. The ultrastructure of such long-range TCs has remained obscure because of the inability to examine by conventional EM the morphological details of rapidly moving organelles. The recent development of correlative light-EM has now allowed us to obtain ultrastructural 'snapshots' of these TCs immediately after their formation from the TGN in live cells. This approach has revealed that such carriers range from typical 60-to 100-nm clathrin-coated vesicles to larger, convoluted tubularvesicular structures displaying several coated buds. We propose that this subset of TCs serve as vehicles for longrange distribution of biosynthetic or recycling cargo from the TGN to the peripheral endosomes.

Clathrin-dependent Association of CVAK104 with Endosomes and theTrans-Golgi Network

“…AP1 is known to be located at the TGN and on tubulovesicular structures that also contain GGA1 and the mannose-6-phosphate receptor (Puertollano et al, 2001;Waguri et al, 2003). These structures are very dynamic, and they undergo transient fusions with peripheral endosomes that allows for the mixing of their contents, e.g., transferrin (Waguri et al, 2003;Polishchuk et al, 2006) and explains the partial colocalization of AP1 with transferrin (Figure 5; Waguri et al, 2003). Our triple-labeling experiments demonstrating colocalization of transferrin with AP1 and CVAK104 ( Figure 5) strongly suggest CVAK104 to be a component of the tubulovesicular structures and the peripheral endosomes.…”

Mophogenesis and Dynamics of Post-Golgi Transport Carriers

“…3B). However, many clathrin-positive PGCs have a grape-like morphology (a tubule with several 'buds'), which suggests that entire chunks of TGN membranes that contain 2-3 clathrin-coated buds can be cleaved from the Golgi complex (Polishchuk et al, 2006). Fission along a budding PGC precursor might also be greatly facilitated by heterogeneous lipid microdomains (Roux et al, 2005).…”

The Unique Endosomal/Lysosomal System of Giardia lamblia

“…The function of the TCs might be to mediate long-range distribution of mannose 6-phosphate receptors (MPR) and their cargo hydrolases to the peripheral cytoplasm (Puertollano et al, 2003). Similar to TCs in HeLa cells (Polishchuk et al, 2006), the tubules might not break down into CCVs en route to PVs in Giardia. In mammalian cells, it has been shown that TCs contain mannose-phosphate receptors, clathrin, Golgi-localizing Gamma-ear containing ARF-binding proteins (GGAs), and/or adaptor protein 1 (AP-1), and it was suggested that these might be uncoated during the TC-endosome fusion or could become integrated into the endosome membrane (Polishchuk et al, 2006).…”

“…It is then possible that, as was observed in vitro (Zhang et al, 2007), clathrin may be organized in a hexagonal array, forming tubes instead of vesicles. Indeed, a different type of clathrin-coated transport carriers (TCs), consisting of larger tubular/vesicular structures having one or more clathrin-coated buds, have been identified (Polishchuk et al, 2006). These TCs travel long distances from the juxtanuclear area of the cell until they fuse with peripheral endosomes.…”

“…Similar to TCs in HeLa cells (Polishchuk et al, 2006), the tubules might not break down into CCVs en route to PVs in Giardia. In mammalian cells, it has been shown that TCs contain mannose-phosphate receptors, clathrin, Golgi-localizing Gamma-ear containing ARF-binding proteins (GGAs), and/or adaptor protein 1 (AP-1), and it was suggested that these might be uncoated during the TC-endosome fusion or could become integrated into the endosome membrane (Polishchuk et al, 2006). Possibly supporting this hypothesis, giardial clathrin and AP-1 were observed not only on ER-exist sites but also in PVs (Marti et al, 2003a;Marti et al, 2003b;Touz et al, 2004;Gaechter et al, 2008).…”