Vps34p differentially regulates endocytosis from the apical and basolateral domains in polarized hepatic cells

Using a microinjection approach to study apical plasma membrane protein trafficking in hepatic cells, we found that specific inhibition of Vps34p, a class III phosphoinositide 3 (PI-3) kinase, nearly perfectly recapitulated the defects we reported for wortmannin-treated cells (Tuma, P.L., C.M. Finnegan, J.-H Yi, and A.L. Hubbard. 1999. J. Cell Biol. 145:1089-1102). Both wortmannin and injection of inhibitory Vps34p antibodies led to the accumulation of resident apical proteins in enlarged prelysosomes, whereas transcytosing apical proteins and recycling basolateral receptors transiently accumulated in basolateral early endosomes. To understand how the Vps34p catalytic product, PI3P, was differentially regulating endocytosis from the two domains, we examined the PI3P binding protein early endosomal antigen 1 (EEA1). We determined that EEA1 distributed to two biochemically distinct endosomal populations: basolateral early endosomes and subapical endosomes. Both contained rab5, although the latter also contained late endosomal markers but was distinct from the transcytotic intermediate, the subapical compartment. When PI3P was depleted, EEA1 dissociated from basolateral endosomes, whereas it remained on subapical endosomes. From these results, we conclude that PI3P, via EEA1, regulates early steps in endocytosis from the basolateral surface in polarized WIF-B cells. However, PI3P must use different machinery in its regulation of the apical endocytic pathway, since later steps are affected by Vps34p inhibition.     

Clathrin exchange during clathrin-mediated endocytosis

During clathrin-mediated endocytosis, clathrin-coated pits invaginate to form clathrin-coated vesicles (CVs). Since clathrin-coated pits are planar structures, whereas CVs are spherical, there must be a structural rearrangement of clathrin as invagination occurs. This could occur through simple addition of clathrin triskelions to the edges of growing clathrin-coated pits with very little exchange occurring between clathrin in the pits and free clathrin in the cytosol, or it could occur through large scale exchange of free and bound clathrin. In the present study, we investigated this question by studying clathrin exchange both in vitro and in vivo. We found that in vitro clathrin in CVs and clathrin baskets do not exchange with free clathrin even in the presence of Hsc70 and ATP where partial uncoating occurs. However, surprisingly FRAP studies on clathrin-coated pits labeled with green fluorescent protein-clathrin light chains in HeLa cells show that even when endocytosis is blocked by expression of a dynamin mutant or depletion of cholesterol from the membrane, replacement of photobleached clathrin in coated pits on the membrane occurs at almost the same rate and magnitude as when endocytosis is occurring. Furthermore, very little of this replacement is due to dissolution of old pits and reformation of new ones; rather, it is caused by a rapid ATP-dependent exchange of clathrin in the pits with free clathrin in the cytosol. On the other hand, consistent with the in vitro data both potassium depletion and hypertonic sucrose, which have been reported to transform clathrin-coated pits into clathrin cages just below the surface of the plasma membrane, not only block endocytosis but also block exchange of clathrin. Taken together, these data show that ATP-dependent exchange of free and bound clathrin is a fundamental property of clathrin-coated pits, but not clathrin baskets, and may be involved in a structural rearrangement of clathrin as clathrin-coated pits invaginate.     

Tyrosine decaging leads to substantial membrane trafficking during modulation of an inward rectifier potassium channel

Tyrosine side chains participate in several distinct signaling pathways, including phosphorylation and membrane trafficking. A nonsense suppression procedure was used to incorporate a caged tyrosine residue in place of the natural tyrosine at position 242 of the inward rectifier channel Kir2.1 expressed in Xenopus oocytes. When tyrosine kinases were active, flash decaging led both to decreased K(+) currents and also to substantial (15-26%) decreases in capacitance, implying net membrane endocytosis. A dominant negative dynamin mutant completely blocked the decaging-induced endocytosis and partially blocked the decaging-induced K(+) channel inhibition. Thus, decaging of a single tyrosine residue in a single species of membrane protein leads to massive clathrin-mediated endocytosis; in fact, membrane area equivalent to many clathrin-coated vesicles is withdrawn from the oocyte surface for each Kir2.1 channel inhibited. Oocyte membrane proteins were also labeled with the thiol-reactive fluorophore tetramethylrhodamine-5-maleimide, and manipulations that decreased capacitance also decreased surface membrane fluorescence, confirming the net endocytosis. In single-channel studies, tyrosine kinase activation decreased the membrane density of active Kir2.1 channels per patch but did not change channel conductance or open probability, in agreement with the hypothesis that tyrosine phosphorylation results in endocytosis of Kir2.1 channels. Despite the Kir2.1 inhibition and endocytosis stimulated by tyrosine kinase activation, neither Western blotting nor (32)P labeling produced evidence for direct tyrosine phosphorylation of Kir2.1. Therefore, it is likely that tyrosine phosphorylation affects Kir2.1 function indirectly, via interactions between clathrin adaptor proteins and a tyrosine-based sorting motif on Kir2.1 that is revealed by decaging the tyrosine side chain. These interactions inhibit a fraction of the Kir2.1 channels, possibly via direct occlusion of the conduction pathway, and also lead to endocytosis, which further decreases Kir2.1 currents. These data establish that side chain decaging can provide valuable time-resolved data about intracellular signaling systems.     

The relationship between cAMP, Ca(2)+, and transport of CFTR to the plasma membrane

The mechanism whereby cAMP stimulates Cl(-) flux through CFTR ion channels in secretory epithelia remains controversial. It is generally accepted that phosphorylation by cAMP-dependent protein kinase increases the open probability of the CFTR channel. A more controversial hypothesis is that cAMP triggers the translocation of CFTR from an intracellular pool to the cell surface. We have monitored membrane turnover in Calu-3 cells, a cell line derived from human airway submucosal glands that expresses high levels of CFTR using membrane capacitance and FM1-43 fluorescence measurements. Using a conventional capacitance measurement technique, we observe an apparent increase in membrane capacitance in most cells that exhibit an increase in Cl(-) current. However, after we carefully correct our recordings for changes in membrane conductance, the apparent changes in capacitance are eliminated. Measurements using the fluorescent membrane marker FM1-43 also indicate that no changes in membrane turnover accompany the activation of CFTR. Robust membrane insertion can be triggered with photorelease of caged Ca(2)+ in Calu-3 cells. However, no increase in Cl(-) current accompanies Ca(2)+-evoked membrane fusion. We conclude that neither increases in cAMP or Ca(2)+ lead to transport of CFTR to the plasma membrane in Calu-3 cells. In addition, we conclude that membrane capacitance measurements must be interpreted with caution when large changes in membrane conductance occur.     

The eps15 homology (EH) domain-based interaction between eps15 and hrb connects the molecular machinery of endocytosis to that of nucleocytosolic transport

The Eps15 homology (EH) module is a protein-protein interaction domain that establishes a network of connections involved in various aspects of endocytosis and sorting. The finding that EH-containing proteins bind to Hrb (a cellular cofactor of the Rev protein) and to the related protein Hrbl raised the possibility that the EH network might also influence the so-called Rev export pathway, which mediates nucleocytoplasmic transfer of proteins and RNAs. In this study, we demonstrate that Eps15 and Eps15R, two EH-containing proteins, synergize with Hrb and Hrbl to enhance the function of Rev in the export pathway. In addition, the EH-mediated association between Eps15 and Hrb is required for the synergistic effect. The interaction between Eps15 and Hrb occurs in the cytoplasm, thus pointing to an unexpected site of action of Hrb, and to a possible role of the Eps15-Hrb complex in regulating the stability of Rev.     

Functional and morphological correlates of connexin50 expressed in Xenopus laevis oocytes

Electrophysiological and morphological methods were used to study connexin50 (Cx50) expressed in Xenopus laevis oocytes. Oocytes expressing Cx50 exhibited a new population of intramembrane particles (9.0 nm in diameter) in the plasma membrane. The particles represented hemichannels (connexin hexamers) because (a) their cross-sectional area could accommodate 24 +/- 3 helices, (b) when their density reached 300-400/microm2, they formed complete channels (dodecamers) in single oocytes, and assembled into plaques, and (c) their appearance in the plasma membrane was associated with a whole-cell current, which was activated at low external Ca2+ concentration ([Ca2+]o), and was blocked by octanol and by intracellular acidification. The Cx50 hemichannel density was directly proportional to the magnitude of the Cx50 Ca2+-sensitive current. Measurements of hemichannel density and the Ca2+-sensitive current in the same oocytes suggested that at physiological [Ca2+]o (1-2 mM), hemichannels rarely open. In the cytoplasm, hemichannels were present in approximately 0.1-microm diameter "coated" and in larger 0.2-0.5-microm diameter vesicles. The smaller coated vesicles contained endogenous plasma membrane proteins of the oocyte intermingled with 5-40 Cx50 hemichannels, and were observed to fuse with the plasma membrane. The larger vesicles, which contained Cx50 hemichannels, gap junction channels, and endogenous membrane proteins, originated from invaginations of the plasma membrane, as their lumen was labeled with the extracellular marker peroxidase. The insertion rate of hemichannels into the plasma membrane (80, 000/s), suggested that an average of 4,000 small coated vesicles were inserted every second. However, insertion of hemichannels occurred at a constant plasma membrane area, indicating that insertion by vesicle exocytosis (60-500 microm2 membranes/s) was balanced by plasma membrane endocytosis. These exocytotic and endocytotic rates suggest that the entire plasma membrane of the oocyte is replaced in approximately 24 h.     

Degradation of yolk platelets in the early amphibian embryo is regulated by fusion with late endosomes

The eggs of many animal species contain a large store of yolk platelets, lipid droplets and glycogen granules; these are consumed during early embryogenesis. However, the mechanisms by which degradation of these stored materials occurs during early embryogenesis are not clearly understood. The mechanisms underlying yolk degradation in amphibian (newt) embryos were investigated. Electron microscopy using an anion marker, cationic ferritin, revealed that yolk platelets were degraded after fusion with late endosomes containing primary lysosomes. Electron microscopy and the results of experiments using a number of reagents with selective effects on intracellular transport suggested that yolk degradation activity in early amphibian embryos may be regulated at the point of fusion between late endosomes and yolk platelets.     

Gangliosides are Transported from the Plasma Membrane to Intralysosomal Membranes as Revealed by Immuno-Electron Microscopy

A biotin-labeled derivative of the ganglioside GM1 (biotin-GM1) was used to study its transport along the endocytic pathway of cultured fibroblasts by immuno-electron microscopy. Using electron dense endocytic tracers we could demonstrate that late endosomes and lysosomes of these cells are long living organelles with a high content of internal membranes. Our studies show that during endocytosis the biotin-GM1 was transported to these intraendosomal and intralysosomal membranes. These observations support the hypothesis that glycosphingolipids (GSL) are preferentially degraded in intralysosomal vesicles.     

Effects of endocytosis inhibitory drugs on rubella virus entry into VeroE6 cells

It has been suggested that infectious entry of rubella virus (RV) is conducted by receptor mediated endocytosis. To explore the cellular entry mechanism of RV, inhibitory effects of drugs affecting various endocytic pathways on RV entry into VeroE6 cells were analyzed. Results showed that RV infectious entry into VeroE6 cells is mediated by clathrin-dependent endocytosis and not by caveolae-mediated endocytosis. Moreover, chemical inhibition of macropinocytosis such as treatments of amiloride, actin and microtubule-disrupting drug significantly reduced RV infection. Considering that macropinocytosis is inducible endocytosis by cellular stimulations, clathrin-mediated endocytosis is likely to be a major route of RV infectious entry.