Role for Dynamin in Late Endosome Dynamics and Trafficking of the Cation-independent Mannose 6-Phosphate Receptor

Abstract

It is well established that dynamin is involved in clathrin-dependent endocytosis, but relatively little is known about possible intracellular functions of this GTPase. Using confocal imaging, we found that endogenous dynamin was associated with the plasma membrane, the trans-Golgi network, and a perinuclear cluster of cation-independent mannose 6-phosphate receptor (CI-MPR)-containing structures. By electron microscopy (EM), it was shown that these structures were late endosomes and that the endogenous dynamin was preferentially localized to tubulovesicular appendices on these late endosomes. Upon induction of the dominant-negative dynK44A mutant, confocal microscopy demonstrated a redistribution of the CI-MPR in mutantexpressing cells. Quantitative EM analysis of the ratio of CI-MPR to lysosome-associated membrane protein-1 in endosome profiles revealed a higher colocalization of the two markers in dynK44A-expressing cells than in control cells. Western blot analysis showed that dynK44A-expressing cells had an increased cellular procathepsin D content. Finally, EM revealed that in dynK44A-expressing cells, endosomal tubules containing CI-MPR were formed. These results are in contrast to recent reports that dynamin-2 is exclusively associated with endocytic structures at the plasma membrane. They suggest instead that endogenous dynamin also plays an important role in the molecular machinery behind the recycling of the CI-MPR from endosomes to the trans-Golgi network, and we propose that dynamin is required for the final scission of vesicles budding from endosome tubules. INTRODUCTIONThe cation-independent mannose 6-phosphate receptor (CI-MPR) transports newly synthesized lysosomal enzymes from the trans-Golgi network (TGN) to endosomes, where the acidic pH causes release of the ligand, making the receptor free to recycle to the TGN (Duncan and Kornfeld, 1988;Goda and Pfeffer, 1988). The CI-MPR recycling from endosomes to the TGN seems to be a strictly controlled event. Rab9 is essential for the transport between endosomes and the TGN (Lombardi et al., 1993;Riederer et al., 1994;Diaz et al., 1997), and ␣-SNAP and NSF stimulate the in vitro transport of CI-MPR from endosomes (Itin et al., 1997). Although budding of clathrin-coated vesicles from endosomes has been reported (Stoorvogel et al., 1996), clathrin does not seem to be required for the transport of CI-MPR from late endosomes to the TGN (Draper et al., 1990;Goda and Pfeffer, 1991) in a reconstituted cell-free system (Goda and Pfeffer, 1988). Recently, a new protein, called TIP47, was found to be important for the endosomal sorting of CI-MPR, probably by acting in cargo selection (Diaz and Pfeffer, 1998). The recruitment of TIP47 is enhanced in the presence of GTP␥S, indicating that a GTPase could be involved in the budding step (Diaz and Pfeffer, 1998). The budding step also seems to require ETF-1, an N-ethylmaleimide-sensitive factor different from NSF (Goda and Pfeffer, 1991). ETF-1 is needed at an early stage of the formation of recycling vesicles, and ...

Clathrin-coated pits with long, dynamin-wrapped necks upon expression of a clathrin antisense RNA

“…Thus, we incubated ultracryosections of CHC antisense RNA-expressing cells with an anti-dynamin antibody (Hudy 1). This antibody not only labeled the clathrin coats as reported (16,18), but also the tubular structures (Fig. 3 C-E).…”

“…The cells were washed with PBS and fixed with 0.1% glutaraldehyde and 2% formaldehyde in 0.1 M phosphate buffer, pH 7.2, followed by dehydration and embedding in Epon. Alternatively, the fixed cells were processed for ultracryosections and subsequent ImmunoGold labeling to localize dynamin (16). In some experiments, cells were fixed in the presence of ruthenium red (0.5 mg͞ml) before they were washed and embedded in Epon (16).…”

“…Alternatively, the fixed cells were processed for ultracryosections and subsequent ImmunoGold labeling to localize dynamin (16). In some experiments, cells were fixed in the presence of ruthenium red (0.5 mg͞ml) before they were washed and embedded in Epon (16). Finally, some cells were incubated for 1 h at 37°C with horseradish peroxidase (5 mg͞ml; Sigma type II) before fixation as described (16).…”

“…In some experiments, cells were fixed in the presence of ruthenium red (0.5 mg͞ml) before they were washed and embedded in Epon (16). Finally, some cells were incubated for 1 h at 37°C with horseradish peroxidase (5 mg͞ml; Sigma type II) before fixation as described (16). Sections were analyzed with a Philips 100 CM electron microscope (Eindhoven, The Netherlands).…”

Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR to lysosomes for degradation

“…Based on the work with the dynamin specific GTP hydrolysis inhibitor dynasore, it was recently proposed that dynamin may in fact act at two different stages in clathrin-vesicle formation, (Macia et al, 2006). Using dynasore, the authors observed the arrest of clathrin-coated vesicle formation at two different stages, both Dynamin has been demonstrated to be involved in recycling from early endosomes to the TGN (Nicoziani et al, 2000) and dynamin has also been shown to be recruited to early endosomal clathrin coats upon overexpression of the endosomal sorting-protein Hrs (Raiborg et al, 2001a). Recycling of the TfR has been proposed to follow two distinct pathways, one pathway where dynamin is involved in transport from the early endosome to the recycling endosome, and another pathway different from that via the recycling endosome (van Dam et al, 2002).…”