scholarly journals Sorting of Membrane and Fluid at the Apical Pole of Polarized Madin-Darby Canine Kidney Cells

2000 ◽  
Vol 11 (6) ◽  
pp. 2131-2150 ◽  
Author(s):  
Som-Ming Leung ◽  
Wily G. Ruiz ◽  
Gerard Apodaca

When fluid-phase markers are internalized from opposite poles of polarized Madin-Darby canine kidney cells, they accumulate in distinct apical and basolateral early endosomes before meeting in late endosomes. Recent evidence suggests that significant mixing of apically and basolaterally internalized membrane proteins occurs in specialized apical endosomal compartments, including the common recycling endosome and the apical recycling endosome (ARE). The relationship between these latter compartments and the fluid-labeled apical early endosome is unknown at present. We report that when the apical recycling marker, membrane-bound immunoglobulin A (a ligand for the polymeric immunoglobulin receptor), and fluid-phase dextran are cointernalized from the apical poles of Madin-Darby canine kidney cells, they enter a shared apical early endosome (≤2.5 min at 37°C) and are then rapidly segregated from one another. The dextran remains in the large supranuclear EEA1-positive early endosomes while recycling polymeric immunoglobulin receptor–bound immunoglobulin A is delivered to a Rab11-positive subapical recycling compartment. This latter step requires an intact microtubule cytoskeleton. Receptor-bound transferrin, a marker of the basolateral recycling pathway, has limited access to the fluid-rich apical early endosome but is excluded from the subapical elements of the Rab11-positive recycling compartment. We propose that the term ARE be used to describe the subapical Rab11-positive compartment and that the ARE is distinct from both the transferrin-rich common recycling endosome and the fluid-rich apical early endosome.

1999 ◽  
Vol 10 (12) ◽  
pp. 4369-4384 ◽  
Author(s):  
Som-Ming Leung ◽  
Raul Rojas ◽  
Christopher Maples ◽  
Christopher Flynn ◽  
Wily G. Ruiz ◽  
...  

Efficient postendocytic membrane traffic in polarized epithelial cells is thought to be regulated in part by the actin cytoskeleton. RhoA modulates assemblies of actin in the cell, and it has been shown to regulate pinocytosis and phagocytosis; however, its effects on postendocytic traffic are largely unexplored. To this end, we expressed wild-type RhoA (RhoAWT), dominant active RhoA (RhoAV14), and dominant inactive RhoA (RhoAN19) in Madin-Darby canine kidney (MDCK) cells expressing the polymeric immunoglobulin receptor. RhoAV14 expression stimulated the rate of apical and basolateral endocytosis, whereas RhoAN19 expression decreased the rate from both membrane domains. Polarized basolateral recycling of transferrin was disrupted in RhoAV14-expressing cells as a result of increased ligand release at the apical pole of the cell. Degradation of basolaterally internalized epidermal growth factor was slowed in RhoAV14-expressing cells. Although apical recycling of immunoglobulin A (IgA) was largely unaffected in cells expressing RhoAV14, transcytosis of basolaterally internalized IgA was severely impaired. Morphological and biochemical analyses demonstrated that a large proportion of IgA internalized from the basolateral pole of RhoAV14-expressing cells remained within basolateral early endosomes and was slow to exit these compartments. RhoAN19 and RhoAWT expression had little effect on these postendocytic pathways. These results indicate that in polarized MDCK cells activated RhoA may modulate endocytosis from both membrane domains and postendocytic traffic at the basolateral pole of the cell.


2000 ◽  
Vol 11 (8) ◽  
pp. 2775-2791 ◽  
Author(s):  
Raluca Gagescu ◽  
Nicolas Demaurex ◽  
Robert G. Parton ◽  
Walter Hunziker ◽  
Lukas A. Huber ◽  
...  

We present a biochemical and morphological characterization of recycling endosomes containing the transferrin receptor in the epithelial Madin-Darby canine kidney cell line. We find that recycling endosomes are enriched in molecules known to regulate transferrin recycling but lack proteins involved in early endosome membrane dynamics, indicating that recycling endosomes are distinct from conventional early endosomes. We also find that recycling endosomes are less acidic than early endosomes because they lack a functional vacuolar ATPase. Furthermore, we show that recycling endosomes can be reached by apically internalized tracers, confirming that the apical endocytic pathway intersects the transferrin pathway. Strikingly, recycling endosomes are enriched in the raft lipids sphingomyelin and cholesterol as well as in the raft-associated proteins caveolin-1 and flotillin-1. These observations may suggest that a lipid-based sorting mechanism operates along the Madin-Darby canine kidney recycling pathway, contributing to the maintenance of cell polarity. Altogether, our data indicate that recycling endosomes and early endosomes differ functionally and biochemically and thus that different molecular mechanisms regulate protein sorting and membrane traffic at each step of the receptor recycling pathway.


1991 ◽  
Vol 266 (27) ◽  
pp. 17729-17732 ◽  
Author(s):  
S.H. Low ◽  
S.H. Wong ◽  
B.L. Tang ◽  
P. Tan ◽  
V.N. Subramaniam ◽  
...  

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