Sorting Mechanisms Regulating Membrane Protein Traffic in the Apical Transcytotic Pathway of Polarized MDCK Cells

Abstract

The transcytotic pathway followed by the polymeric IgA receptor (pIgR) carrying its bound ligand (dIgA) from the basolateral to the apical surface of polarized MDCK cells has been mapped using morphological tracers. At 20°C dIgA-pIgR internalize to interconnected groups of vacuoles and tubules that comprise the endosomal compartment and in which they codistribute with internalized transferrin receptors (TR) and epidermal growth factor receptors (EGFR). Upon transfer to 37°C the endosome vacuoles develop long tubules that give rise to a distinctive population of 100-nm-diam cup-shaped vesicles containing pIgR. At the same time, the endosome gives rise to multivesicular endosomes (MVB) enriched in EGFR and to 60-nm-diam basolateral vesicles. The cup-shaped vesicles carry the dIgA/pIgR complexes to the apical surface where they exocytose.Using video microscopy and correlative electron microscopy to study cells grown thin and flat we show that endosome vacuoles tubulate in response to dIgA/pIgR but that the tubules contain TR as well as pIgR. However, we show that TR are removed from these dIgA-induced tubules via clathrin-coated buds and, as a result, the cup-shaped vesicles to which the tubules give rise become enriched in dIgA/pIgR.Taken together with the published information available on pIgR trafficking signals, our observations suggest that the steady-state concentrations of TR and unoccupied pIgR on the basolateral surface of polarized MDCK cells are maintained by a signal-dependent, clathrin-based sorting mechanism that operates along the length of the transcytotic pathway. We propose that the differential sorting of occupied receptors within the MDCK endosome is achieved by this clathrin-based mechanism continuously retrieving receptors like TR from the pathways that deliver pIgR to the apical surface and EGFR to the lysosome.

rab4 Regulates Transport to the Apical Plasma Membrane in Madin-Darby Canine Kidney Cells

“…Endocytosed IgA and Tf from the apical or basolateral surface meet in this compartment, hence, its name is the common endosomal compartment. The CE appears to be positioned along microtubules and is oriented toward the apical cytoplasm (35). The third compartment, called the ARE, consists of 100 -150-nm, cup-shaped vesicles distributed immediately below the apical surface.…”

“…This 80-kDa sulfated protein is constitutively secreted in a polarized fashion into the apical medium of filter-grown MDCK cells (34). Transfectants were metabolically labeled for 60 min with 35 S TranS label and chased for 60 min. At the end of the chase time, we immunoprecipitated gp80 from the apical and basolateral media.…”

Dissection of the multifunctional “receptor-recycling” endocytic compartment of hepatocytes

“…29,77 Currently, immunoisolation studies are also in progress in an attempt to understand the dynamic regulation of recycling, transcytosis, and the caveolin cycle. However, in view of the complexity of the recycling endosomal compartment it is presumable that the physical separation of the 3 functional subcompartments from RRC will be complicated, 37,45,78 even questionable, considering the intricacy of tubulo-network membrane structures and the molecular mix up in single clathrin-coated pits or caveolae plasma membrane microdomains.…”

“…[41][42][43] Electron, confocal, and videoenhanced microscopy reveal the complexity of tubularmembrane networks extending throughout the cytoplasm. 44,45 Despite the morphological characterization, the attempts to isolate those structures enriched in recycling-receptors, from liver homogenates, using subcellular fractionation and the subsequent biochemical analysis, are limited. 19,23,24,27,46 When highly purified endosomes from rat liver 27 were used for a comprehensive biochemical and electron microscopic characterization, 28,29,33,47 the morphological and pleiotropic functional complexity of the RRC became evident and, although RRC was first characterized as the endosomal fraction enriched in recycling receptors (transferrin receptor, asialoglycoprotein receptor, LDL receptor-related protein, and LDL receptor), the confirmation that contains molecules that follow the transcytotic pathway such as pIgR-pIgA complexes 48 as well as coated-like vesicles enriched with caveolin-1, 29,49 prompted us to introduce an operational ''functional'' dissection of RRC into 3 subcompartments: RRCr, to those structures predominantly devoted to recycling of receptors; RRCt, to structures involved in transporting molecules to the bile (transcytosis); and RRCc, for those structures enriched in caveolin-1.…”

Cholesterol-sensitive Modulation of Transcytosis

“…At that surface, the ectodomain of pIgR is proteolytically cleaved off to yield the secretory component (SC), which is released together with dIgA into mucosal secretions. In the course of transcytosis, pIgRdIgA complexes traverse various endosomal compartments, among which is an endocytic compartment located immediately underneath the apical surface (Gibson et al, 1998). This apical compartment, defined as apical recycling endosomes (AREs), is enriched in dIgA and is relatively deficient in recycling transferrin (Apodaca et al, 1994;Barroso and Sztul, 1994;Brown et al, 2000).…”