Role for dynamin in late endosome dynamics and trafficking of the cation-independent mannose 6-phosphate receptor.
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 tubulo-vesicular appendices on these late endosomes. Upon induction of the dominant-negative dynK44A mutant, confocal microscopy demonstrated a redistribution of the CI-MPR in mutant-expressing 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.
Nicoziani
P
P
Structural Cell Biology Unit, Department of Medical Anatomy, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark.
Vilhardt
F
F
Llorente
A
A
Hilout
L
L
Courtoy
P J
PJ
Sandvig
K
K
van Deurs
B
B
D019943:Amino Acid Substitution; D015703:Antigens, CD / Q000032:analysis; D001692:Biological Transport; D002402:Cathepsin D / Q000737:chemistry / Q000378:metabolism; D002412:Cations / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D003593:Cytoplasm / Q000378:metabolism; D034284:Dynamin I; D034281:Dynamins; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism* / Q000648:ultrastructure; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D005799:Genes, Dominant / Q000235:genetics; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000378:metabolism / Q000648:ultrastructure; D008562:Membrane Glycoproteins / Q000032:analysis; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D011499:Protein Processing, Post-Translational; D017527:Receptor, IGF Type 2 / Q000378:metabolism*
D015703:Antigens, CD; D002412:Cations; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D017527:Receptor, IGF Type 2; D002402:Cathepsin D; D034284:Dynamin I; D020558:GTP Phosphohydrolases; D034281:Dynamins