Abstract
The basolateral recycling and transcytotic pathways of epithelial cells were previously defined using markers such as transferrin (TfR) and polymeric IgA (pIgR) receptors. In contrast, our knowledge of the apical recycling pathway remains fragmentary. Here we utilize quantitative live-imaging and mathematical modelling to outline the recycling pathway of Megalin (LRP-2), an apical receptor with key developmental and renal functions, in MDCK cells. We show that, like TfR, Megalin is a long-lived and fast-recycling receptor. Megalin enters polarized MDCK cells through segregated apical sorting endosomes and subsequently intersects the TfR and pIgR pathways at a perinuclear Rab11-negative compartment termed common recycling endosomes (CRE). Whereas TfR recycles to the basolateral membrane from CRE, Megalin, like pIgR, traffics to subapical Rab11-positive apical recycling endosomes (ARE) and reaches the apical membrane in a microtubule- and Rab11-dependent manner. Hence, Megalin defines the apical recycling pathway of epithelia, with CRE as its apical sorting station.
Highlights
The basolateral recycling and transcytotic pathways of epithelial cells were previously defined using markers such as transferrin (TfR) and polymeric IgA receptors
To measure Megalin endocytosis (Fig. 2a,b), we labelled the plasma membrane (PM) mMeg-HA pool with a mouse anti-HA antibody tagged with SeTau-647 (647-MaHA)[29], allowed internalization for the indicated times and labelled the remaining PM Megalin pool with secondary goat anti-mouse antibodies tagged with Alexa-488 (488-GaM)
The fraction of 647-MaHA that co-localized with 488-GaM plateaued at 19% (CI95 1⁄4 14 À 22%), consistent with a 19% PM/ 81% intracellular steady-state distribution of Megalin, very similar to the 18% PM/80% total ratio observed in surface biotinylation assays in polarized MDCK cells (Fig. 1g)
Summary
The basolateral recycling and transcytotic pathways of epithelial cells were previously defined using markers such as transferrin (TfR) and polymeric IgA (pIgR) receptors. We utilize quantitative live-imaging and mathematical modelling to outline the recycling pathway of Megalin (LRP-2), an apical receptor with key developmental and renal functions, in MDCK cells. Megalin enters polarized MDCK cells through segregated apical sorting endosomes and subsequently intersects the TfR and pIgR pathways at a perinuclear Rab11-negative compartment termed common recycling endosomes (CRE). A 1:1 complex of Megalin and Cubilin (Fig. 1a) on the apical plasma membrane (PM) of proximal tubule (PT) cells binds and mediates endocytosis of a myriad of ultrafiltrate proteins (that is, hormone, vitamin and iron carriers, enzymes and immunoglobulin light chains)[3,4,5], for subsequent lysosomal degradation and retrieval of their ligands and constituent amino acids into the blood[6]. Syndrome[13], Stickler-like syndrome[14] and Imerslund–Grasbeck disease[15,16], mutations in Megalin or Cubilin impair protein absorption in the kidney PT and the affected patients display proteinuria
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