Visualization of TGN to Endosome Trafficking through Fluorescently Labeled MPR and AP-1 in Living Cells

Abstract

We have stably expressed in HeLa cells a chimeric protein made of the green fluorescent protein (GFP) fused to the transmembrane and cytoplasmic domains of the mannose 6-phosphate/insulin like growth factor II receptor in order to study its dynamics in living cells. At steady state, the bulk of this chimeric protein (GFP-CI-MPR) localizes to the trans-Golgi network (TGN), but significant amounts are also detected in peripheral, tubulo-vesicular structures and early endosomes as well as at the plasma membrane. Time-lapse videomicroscopy shows that the GFP-CI-MPR is ubiquitously detected in tubular elements that detach from the TGN and move toward the cell periphery, sometimes breaking into smaller tubular fragments. The formation of the TGN-derived tubules is temperature dependent, requires the presence of intact microtubule and actin networks, and is regulated by the ARF-1 GTPase. The TGN-derived tubules fuse with peripheral, tubulo-vesicular structures also containing the GFP-CI-MPR. These structures are highly dynamic, fusing with each other as well as with early endosomes. Time-lapse videomicroscopy performed on HeLa cells coexpressing the CFP-CI-MPR and the AP-1 complex whose ␥-subunit was fused to YFP shows that AP-1 is present not only on the TGN and peripheral CFP-CI-MPR containing structures but also on TGN-derived tubules containing the CFP-CI-MPR. The data support the notion that tubular elements can mediate MPR transport from the TGN to a peripheral, tubulo-vesicular network dynamically connected with the endocytic pathway and that the AP-1 coat may facilitate MPR sorting in the TGN and endosomes. INTRODUCTIONThe mannose 6-phosphate receptors (MPRs) are essential components for lysosome biogenesis and cellular homeostasis (Kornfeld, 1992;Ludwig et al., 1995). The primary function of the cation-independent and the cation-dependent mannose 6-phosphate receptors (CI-MPR and CD-MPR) is to sort newly synthesized lysosomal enzymes from the secretory pathway for subsequent transport to endosomal/lysosomal compartments. To carry out their function, the MPRs must bind the common mannose 6-phosphate recognition marker on soluble lysosomal enzymes in the trans-Golgi network (TGN), the last sorting station of the secretory pathway, and be packaged into TGN-derived transport intermediates. After budding, these transport intermediates fuse with endosomes where the MPRs unload their bound ligands. Although the lysosomal enzymes are transported to lysosomes, the MPRs are retrieved to the TGN or occasionally to the plasma membrane. Although the precise pathways followed by the MPRs at the exit of the TGN remain to be better defined, it has become clear during the past years that the sorting of MPRs from the compartments they visit, i.e., TGN, endosomes and plasma membrane, is directed by Article published online ahead of print. Mol. Biol. Cell 10.1091/ mbc.E02-06 -0338. Article and publication date are at www. molbiolcell.org/cgi/doi/10.1091/mbc.E02-06 -0338.□ V Online version of this article contains video material. ...

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

“…Outside the juxtanuclear area, the number of CVAK104-positive structures exceeds those of AP1, probably because CVAK104 is also present on AP3 structures. 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).…”

“…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.…”

Clathrin Terminal Domain-Ligand Interactions Regulate Sorting of Mannose 6-Phosphate Receptors Mediated by AP-1 and GGA Adaptors

“…A major cellular function of AP-1-(15) and GGA-containing clathrincoated carriers (16) is the bidirectional traffic of cargo such as MPRs between endosomes and the TGN. GFP-MPRs exit from the TGN via clathrin/AP-1-containing tubules (17) that appear to be distinct from those involved in constitutive secretion of vesicular stomatitis virus G protein (VSVG) (18,19). Acute or sustained loss of AP-1 depletes MPRs from clathrin-coated vesicles and impairs MPR retrieval to the TGN (10,20).…”

“…Acute Interference with Clathrin TD Function Impairs MPR Retrieval to the TGN-MPR sorting of newly synthesized lysosomal enzymes to the endolysosomal system is mediated by carriers coated with clathrin, AP-1, and/or GGAs (15)(16)(17). A hallmark of AP-1 dysfunction is the peripheral dispersion of MPRs caused by defective retrograde sorting to the TGN (10).…”

“…Because Pitstop-2-mediated interference with clathrin TD function stalls AP-1 and GGA dynamics, it seemed likely that it would also cause MPR missorting. To investigate this, we analyzed HeLa cells stably expressing the transmembrane and cytoplasmic domain of the cation-independent (CI) MPR fused to eGFP (GFP-CI-MPR) (17). In control cells, GFP-CI-MPR was concentrated in tubular elements at the TGN that were frequently seen to detach and move toward the cell periphery, sometimes breaking into smaller tubular fragments.…”