Visualisation of macropinosome maturation by the recruitment of sorting nexins.
We report that phosphoinositol-binding sorting nexin 5 (SNX5) associates with newly formed macropinosomes induced by EGF stimulation. We used the recruitment of GFP-SNX5 to macropinosomes to track their maturation. Initially, GFP-SNX5 is sequestered to discrete subdomains of the macropinosome; these subdomains are subsequently incorporated into highly dynamic, often branched, tubular structures. Time-lapse videomicroscopy revealed the highly dynamic extension of SNX5-labelled tubules and their departure from the macropinosome body to follow predefined paths towards the perinuclear region of the cell, before fusing with early endosomal acceptor membranes. The extension and departure of these tubular structures occurs rapidly over 5-10 minutes and is dependent upon intact microtubules. As the tubular structures depart from the macropinosome there is a reduction in the surface area and an increase in tension of the limiting membrane of the macropinosome. In addition to the recruitment of SNX5 to the macropinosome, Rab5, SNX1 and EEA1 are also recruited by newly formed macropinosomes, followed by the accumulation of Rab7. SNX5 forms heterodimers with SNX1 and this interaction is required for endosome association of SNX5. We propose that the departure of SNX5-positive tubules represents a rapid mechanism of recycling components from macropinosomes thereby promoting their maturation into Rab7-positive structures. Collectively these findings provide a detailed real-time characterisation of the maturation process of the macropinocytic endosome.
Kerr
Markus C
MC
Institute for Molecular Bioscience and ARC Centre in Bioinformatics, University of Queensland, St. Lucia, QLD 4072, Australia.
Lindsay
Margaret R
MR
Luetterforst
Robert
R
Hamilton
Nicholas
N
Simpson
Fiona
F
Parton
Robert G
RG
Gleeson
Paul A
PA
Teasdale
Rohan D
RD
D000954:Antigens, Surface / Q000032:analysis; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D002478:Cells, Cultured; D019281:Dimerization; D011992:Endosomes / Q000502:physiology; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D021962:Membrane Microdomains; D008870:Microtubules / Q000378:metabolism; D008954:Models, Biological; D010873:Pinocytosis / Q000502:physiology*; D011485:Protein Binding; D058305:Sorting Nexins; D033921:Vesicular Transport Proteins / Q000378:metabolism / Q000502:physiology*
D000954:Antigens, Surface; D002352:Carrier Proteins; C513978:SNX1 protein, human; C402667:SNX5 protein, human; D058305:Sorting Nexins; D033921:Vesicular Transport Proteins