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K E Mostov

Publications and source records attributed to K E Mostov.

At least 55 records · Page 3Linked to original sources
PubMed1996

Calmodulin regulates the intracellular trafficking in epithelial cells.

In this study we have demonstrated the role of calmodulin in membrane trafficking. By means of specific calmodulin-antagonist (W13, N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide) the transcytosis, recycling and receptor-mediated endocytosis of IgA were investigated. In W13 treated MDCK cells, exHpressing the polymeric immunoglobulin receptor (pIgR) and plated on polycarbonate filters (transwells), it has been shown that transcytosis of IgA was inhibited, the recycling was concomitantly increased and there were no modification on its internalization. Altogether, point to a role of calmodulin in regulating the function of endocytic compartment in epithelial cells.

Animals↗
PubMed1995

Dimeric and tetrameric IgA are transcytosed equally by the polymeric Ig receptor.

Polymeric IgA (pIgA) is transcytosed across epithelial cells and into external secretions by the polymeric Ig receptor (pIgR). Binding of dimeric IgA (dIgA) to the pIgR stimulates transcytosis of the pIgR. The pIgA in secretions is found as dimers (dIgA) and higher polymers, such as tetramers (tIgA), but little is known of the functional significance of the different sizes. Here we compared the ability of dIgA and tIgA to perform three functions that are essential to their transport into mucosal secretions. 1) Equilibrium binding studies showed that there were twice as many binding sites for tIgA as dIgA at the basolateral cell surface, but that the affinity of these sites for tIgA was one-half of that for dIgA. 2) Both dIgA and tIgA were rapidly transcytosed by the pIgR, although transcytosis of tIgA was slower. 3) Both dIgA and tIgA could stimulate transcytosis of the pIgR, although tIgA was less effective. The possible implications of these findings for the relative biologic roles of dIgA and tIgA are discussed.

Animals↗
PubMed1995

Regulation of protein traffic in polarized epithelial cells.

The plasma membrane of polarized epithelial cells is divided into apical and basolateral surfaces, with different compositions. Proteins can be sent directly from the trans-Golgi network (TGN) to either surface, or can be sent first to one surface and then transcytosed to the other. The glycosyl phosphatidylinositol anchor is a signal for apical targeting. Signals in the cytoplasmic domain containing a beta-turn determine basolateral targeting and retrieval, and are related to other sorting signals. Transcytosed proteins, such as the polymeric immunoglobulin receptor (pIgR), are endocytosed from the basolateral surface and then accumulate in a tubular compartment concentrated underneath the apical surface. This compartment, tentatively termed the apical recycling compartment, may be a central sorting station, as it apparently receives material from both surfaces and sorts them for delivery to the correct surface. Delivery to the apical surface from both the TGN and the apical recycling compartment appears to be regulated by protein kinases A and C, and endocytosis from the apical surface is also regulated by kinases. Transcytosis of the pIgR is additionally regulated by phosphorylation of the pIgR and by ligand binding to the pIgR. Regulation of traffic in polarized epithelial cells plays a central role in cellular homeostasis, response to external signals and differentiation.

Animals↗
PubMed1995

Characterization of Pseudomonas aeruginosa-induced MDCK cell injury: glycosylation-defective host cells are resistant to bacterial killing.

As a model for bacterium-induced epithelial cell injury, we have studied the interaction of Pseudomonas aeruginosa with polarized Madin-Darby canine kidney (MDCK) cells grown on filters. Following an initial period of bacterial adhesion, foci of injured host cells, which consisted of a central region of cell debris, surrounded by cells that were permeable and apparently necrotic, were formed. Host cell death was quantified by measuring the increased permeability of the monolayer to the macromolecular tracer [14C]inulin. Using this MDCK model system, we have identified bacterial and host cell factors necessary for the host cell damage. The ability of P. aeruginosa to cause MDCK cell damage was independent of elastase or exotoxin A production. In contrast, bacteria with a mutation in the regulatory locus exsA (which are deficient in exoenzyme S production) neither bound to nor caused host cell injury. MDCK cells with defects in cell surface glycosylation were resistant to cell injury, indicating that bacteria may require host cell glycolipids and/or glycoproteins as points of adhesion to cause subsequent host cell injury.

Animals↗
PubMed1995

Regulation of protein traffic in polarized epithelial cells.

The plasma membrane of polarized epithelial cells is divided into apical and basolateral surfaces with different compositions. Proteins can be sent directly from the trans Golgi network (TGN) to either surface, or can be sent first to one surface and then transcytosed to the other. The glycosyl phosphatidylinositol anchor is a signal for apical targeting. Signals in the cytoplasmic domain containing a beta-turn determine basolateral targeting and retrieval, and are related to other shorting signals. Transcytosed proteins, such as the polymeric immunoglobulin receptor (pIgR) are endocytosed from the basolateral surface and delivered to the apical recycling compartment underneath the apical surface. This compartment is a central sorting station, as it receives material from both surfaces and sorts them to the correct surface. Delivery to the apical surface from both the TGN and the apical recycling compartment is regulated by protein kinase A and protein kinase C, and endocytosis from the apical surface is also regulated by kinases. Transcytosis of the pIgR is additionally regulated by phosphorylation of the pIgR and by ligand binding to the pIgR. Regulation of traffic in polarized epithelial cells plays a central role in cellular homeostasis, response to external signals, and differentiation.

Animals↗
PubMed1994

The calmodulin antagonist, W-13, alters transcytosis, recycling, and the morphology of the endocytic pathway in Madin-Darby canine kidney cells.

The effect of the calmodulin antagonist N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W-13) on the endosomal system of Madin-Darby canine kidney cells was assessed. W-13 inhibited receptor-mediated IgA transcytosis, recycling of transferrin, and caused all material endocytosed from both surfaces of the cell to be delivered to exceptionally large, novel endosomal structures, which appear to be derived from early endosomes. Treatment with other calmodulin antagonists similarly inhibited transcytosis and caused large endosomes to form. These observations raise the possibility that calmodulin may be involved in regulating membrane trafficking through the endosomes of polarized epithelial cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Rapid internalization of the polymeric immunoglobulin receptor requires phosphorylated serine 726.

S726 of the cytoplasmic domain of the polymeric immunoglobulin receptor (pIgR) resides within a consensus sequence for phosphorylation by protein kinases A, G, and C, and casein kinase II. Mutation of S726 to Ala and expression of this mutant pIgR in Madin-Darby canine kidney cells results in a receptor in which steady-state phosphorylation is reduced to 49% of wild-type levels. This mutant receptor is also significantly impaired in its internalization from the basolateral membrane. During the first minute, internalization of radioiodinated ligands (either dIgA or monovalent anti-pIgR Fabs) by this mutant pIgR is only 35% of that by wild-type pIgR. Internalization of unoccupied mutant receptors is similarly inhibited. Delivery of newly made mutant receptor from the trans-Golgi network to the basolateral surface is completely normal. The only other trafficking step inhibited by this mutation is the transcytosis of radioiodinated dIgA. Within 2 h, the mutant pIgR will transcytose 58% of a preinternalized cohort of dIgA, while the wild-type transcytoses 76%. This inhibition of transcytosis may be an indirect consequence of impaired internalization. The correlation between the loss of phosphorylation and inhibition of internalization suggests that phosphorylation of S726 may represent a novel mechanism for regulation of internalization of the pIgR.

Amino Acid Sequence↗
PubMed1994

Polarized sorting of the polymeric immunoglobulin receptor in the exocytotic and endocytotic pathways is controlled by the same amino acids.

Polarized epithelial cells can sort plasma membrane proteins to the apical or basolateral domain either by direct targeting from the trans-Golgi network (TGN) or by targeting to one surface, followed by endocytosis and transcytosis to the opposite surface. In Madin-Darby canine kidney (MDCK) cells, targeting of the polymeric immunoglobulin receptor (pIgR) to the basolateral surface is controlled by a sorting signal residing in the membrane proximal 17 amino acids of the cytoplasmic domain of this receptor. We have recently found that individual mutations at any of three residues in this signal, His656, Arg657 and Val660, substantially decrease targeting from the TGN to the basolateral surface and correspondingly increase targeting from the TGN to the apical surface. Here we report that these mutations decrease the recycling of basolaterally endocytosed pIgR to that surface, and correspondingly increase its transcytosis to the apical surface. This effect occurred in mutant pIgRs that either contained the full-length cytoplasmic domain or were truncated to contain only the 17-residue basolateral targeting signal, and was independent of phosphorylation of pIgR at Ser664. Our results indicate that polarized sorting of the pIgR in the endocytotic and exocytotic pathways are controlled by the same amino acids.

Amino Acid Sequence↗
PubMed1994

Phorbol myristate acetate-mediated stimulation of transcytosis and apical recycling in MDCK cells.

We observed that phorbol myristate acetate (PMA) stimulates transcytosis of the polymeric immunoglobulin receptor (pIgR) in MDCK cells. Apical release of pre-endocytosed ligand (dimeric IgA) bound to the pIgR can be stimulated twofold within 7 min of addition of PMA while recycling of the ligand from the basal surface is not affected. In addition, apical surface delivery of pIgR and cleavage of its ectodomain to secretory component (SC) is also stimulated by PMA. The recycling of apically internalized ligand back to the apical surface is similarly stimulated. These results suggest that the stimulation of apical delivery is from an apical recycling compartment. The effect of PMA suggests that protein kinase C (PKC) is involved in the regulation of pIgR trafficking in MDCK cells. To test this we down regulated PKC activity by pre-treating cells with PMA for 16 h and observed that transcytosis could no longer be stimulated by PMA. Western blots show that the PKC isozymes alpha and to a lesser extent epsilon, are depleted from MDCK cells which have been pre-treated with PMA for 16 h and that treatment of MDCK cells with PMA for 5 min causes a dramatic translocation of the PKC alpha isozyme and a partial translocation of the epsilon isozyme from the cytosol to the membrane fraction of cell homogenates. This translocation suggests that the alpha and/or epsilon isozymes may be involved in PMA mediated stimulation of transcytosis. A mutant pIgR in which serines 664 and 726, the major sites of phosphorylation, are replaced by alanine is stimulated to transcytose by PMA, suggesting that phosphorylation of pIgR at these sites is not required for the effect of PMA. These results suggest that PMA-mediated stimulation of pIgR transcytosis may involve the activation of PKC alpha and/or epsilon, and that this stimulation occurs independently of the major phosphorylation sites on the pIgR. Finally, PMA stimulates transcytosis of basolaterally internalized transferrin, suggesting that PMA acts to generally stimulate delivery of endocytosed proteins to the apical surface.

Animals↗
PubMed1994

Receptor-mediated transcytosis of IgA in MDCK cells is via apical recycling endosomes.

Classically, the polymeric immunoglobulin receptor and its ligand, IgA, are thought to be sorted from basolateral early endosomes into transcytotic vesicles that directly fuse with the apical plasma membrane. In contrast, we have found that in MDCK cells IgA is delivered from basolateral endosomes to apical endosomes and only then to the apical cell surface. When internalized from the basolateral surface of MDCK cells IgA is found to accumulate under the apical plasma membrane in a compartment that is accessible to two apically added membrane markers: anti-secretory component Fab fragments, and avidin internalized from the biotinylated apical pole of the cell. This accumulation occurs in the presence of apical trypsin, which prevents internalization of the ligand from the apical cell surface. Using a modification of the diaminobenzidine density-shift assay, we estimate that approximately 80% of basolaterally internalized IgA resides in the apical endosomal compartment. In addition, approximately 50% of basolaterally internalized transferrin, a basolateral recycling protein, has access to this apical endosomal compartment and is efficiently recycled back to the basolateral surface. Microtubules are required for the organization of the apical endosomal compartment and it is dispersed in nocodazole-treated cells. Moreover, this compartment is largely inaccessible to fluid-phase markers added to either pole of the cell, and therefore seems analogous to the recycling endosome described in nonpolarized cells. We propose a model in which transcytosis is not a specialized pathway that uses unique transcytotic vesicles, but rather combines portions of pathways used by non-transcytosing molecules.

Animals↗
PubMed1994

The polymeric immunoglobulin receptor accumulates in specialized endosomes but not synaptic vesicles within the neurites of transfected neuroendocrine PC12 cells.

We have expressed in neuroendocrine PC12 cells the polymeric immunoglobulin receptor (pIgR), which is normally targeted from the basolateral to the apical surface of epithelial cells. In the presence of nerve growth factor, PC12 cells extend neurites which contain synaptic vesicle-like structures and regulated secretory granules. By immunofluorescence microscopy, pIgR, like the synaptic vesicle protein synaptophysin, accumulates in both the cell body and the neurites. On the other hand, the transferrin receptor, which normally recycles at the basolateral surface in epithelial cells, and the cation-independent mannose 6-phosphate receptor, a marker of late endosomes, are largely restricted to the cell body. pIgR internalizes ligand into endosomes within the cell body and the neurites, while uptake of ligand by the low density lipoprotein receptor occurs primarily into endosomes within the cell body. We conclude that transport of membrane proteins to PC12 neurites as well as to specialized endosomes within these processes is selective and appears to be governed by similar mechanisms that dictate sorting in epithelial cells. Additionally, two types of endosomes can be identified in polarized PC12 cells by the differential uptake of ligand, a housekeeping type in the cell bodies and a specialized endosome in the neurites. Recent findings suggest that specialized axonal endosomes in neurons are likely to give rise to synaptic vesicles (Mundigl, O., M. Matteoli, L. Daniell, A. Thomas-Reetz, A. Metcalf, R. Jahn, and P. De Camilli. 1993. J. Cell Biol. 122:1207-1221). Although pIgR reaches the specialized endosomes in the neurites of PC12 cells, we find by subcellular fractionation that under a variety of conditions it is efficiently excluded from synaptic vesicle-like structures as well as from secretory granules.

Animals↗
PubMed1994

Transepithelial transport of immunoglobulins.

Immunoglobulins are transported across a variety of epithelial tissues. The best studied example of this is the transport of polymeric IgA and IgM by the polymeric immunoglobulin receptor (pIgR) across many types of epithelial cells. The pIgR binds its ligand at the basolateral surface and is internalized into endosomes. Here it is sorted into vesicles that transcytose it to the apical surface. At the apical surface the pIgR is proteolytically cleaved, and the large extracellular fragment (known as secretory component) is released together with the ligand. The pIgR contains a cytoplasmic domain of 103 amino acids that contains several sorting signals. Targeting from the trans-Golgi network to the basolateral surface is determined by the membrane-proximal 17 residues of this domain. There are two endocytosis signals that contain crucial tyrosine residues. Transcytosis of the pIgR is stimulated by binding of polymeric IgA. Phosphorylation of a cytoplasmic serine promotes transcytosis of the pIgR without ligand bound. Transcytosis may be regulated by the heterotrimeric Gs protein, protein kinase C and calmodulin. IgG is transcytosed from the apical to basolateral surface in several epithelial tissues such as the placenta and the small intestine of newborn rats. The receptor for intestinal transport of IgG is structurally similar to class I MHC molecules.

Animals↗
PubMed1993

Possible role of both the alpha and beta gamma subunits of the heterotrimeric G protein, Gs, in transcytosis of the polymeric immunoglobulin receptor.

In endosomes the polymeric immunoglobulin receptor (pIgR) is sorted into transcytotic vesicles. Transcytosis of the pIgR in polarized Madin-Darby canine kidney (MDCK) cells is regulated both by phosphorylation of Ser664 and by binding of ligand, dimeric IgA (dIgA). Mutation of Ser664 to Ala (pIgR-Ala664) greatly reduces transcytosis of the pIgR without dIgA bound. Here we report that cholera toxin, which specifically activates the heterotrimeric G protein, Gs, stimulates transcytosis of pIgR-Ala664 when dIgA is not bound. However, transcytosis of the wild-type pIgR without dIgA bound was not stimulated by cholera toxin. To further analyze the involvement of Gs in transcytosis, we reconstituted part of the transcytotic pathway. Vesicles containing endocytosed ligand bound to the pIgR could be released from MDCK cells whose apical surface had been mechanically perforated. Release of these vesicles was controlled by phosphorylation of the pIgR and by binding of dIgA. The use of perforated cells allowed us to directly address the involvement of Gs. Our data suggest that both subunits of Gs, Gs alpha and beta gamma, function together to control vesicular release and may therefore be involved in transcytosis in vivo.

Alanine↗
PubMed1993

Transcytosis of placental alkaline phosphatase-polymeric immunoglobulin receptor fusion proteins is regulated by mutations of Ser664.

Two signals are known that regulate the entry of the polymeric immunoglobulin receptor (pIgR) into the transcytotic pathway: binding of the ligand, dimeric IgA, and phosphorylation of Ser664 in the cytoplasmic domain of the receptor. Mutation of Ser664 to an alanine residue generates a receptor that is transcytosed slowly and exhibits increased recycling at the basolateral plasma membrane. In contrast, if Ser664 is mutated to an aspartate (which bears a constant negative charge-like a phosphate group) the receptor is transcytosed at a rate that is greater than the wild-type pIgR. We have constructed fusions of placental alkaline phosphatase (PLAP) with the transmembrane region and cytoplasmic domain of the wild-type or mutant pIgRs described above (PLAP/WT, PLAP/Ala, and PLAP/Asp) in order to study transcytotic signals present in the cytoplasmic domain of the pIgR independently of those present in the lumenal domain. Transcytosis of the fusion proteins was measured by cell surface immunoprecipitation of a single cohort of basolaterally biotinylated fusion proteins as it arrived at the apical plasma membrane. Significantly more of the PLAP/WT fusion (27%) than the PLAP/Ala fusion protein (5%) was transcytosed during a 2-h chase period. In addition, it was found that approximately 10% of a pseudoligand, monovalent 125I-labeled Fabs (derived from affinity-purified anti-PLAP antibodies), was transcyosed by the PLAP/WT fusion, 7% by the PLAP/Ala fusion, and 22% by the PLAP/Asp fusion over a 4-h period. These data are consistent with the hypothesis that the cytoplasmic domain of the pIgR contains information necessary to direct proteins into the transcytotic pathway, albeit inefficiently, and that phosphorylation of Ser664 is at least a partially autonomous signal for transcytosis.

Alkaline Phosphatase↗
PubMed1993

Brefeldin-A inhibits the delivery of the polymeric immunoglobulin receptor to the basolateral surface of MDCK cells.

We have studied the effects of brefeldin A (BFA) on the polarized delivery of the polymeric immunoglobulin receptor to the basolateral surface of MDCK cells. Unlike the delivery of several other basolateral membrane and secretory proteins, the delivery of the polymeric immunoglobulin receptor from the trans-Golgi network to the cell surface was inhibited by BFA. The effect of BFA treatment was apparent at 1.0 microgram/ml (36% inhibition), and maximal inhibition was observed at 10 micrograms/ml (70% inhibition). The delivery of the receptor from the endoplasmic reticulum to the basolateral surface was even more sensitive to the effect of BFA; delivery was inhibited 95% in cells treated with 1 micrograms/ml BFA. The selective action of BFA on the basolateral delivery of the polymeric immunoglobulin receptor suggests that there may be multiple pathways for delivery of proteins to the basolateral cell surface of MDCK cells.

Animals↗
PubMed1993

Apical secretion of a cytosolic protein by Madin-Darby canine kidney cells. Evidence for polarized release of an endogenous lectin by a nonclassical secretory pathway.

In the classical secretory pathway proteins containing a signal peptide are translocated from the cytoplasm of the cell into the lumen of the endoplasmic reticulum (ER). From the ER they are transported to the Golgi apparatus and finally to the plasma membrane (PM) where they are released into the extracellular compartment. However, some proteins are synthesized without a signal peptide and maintain a predominantly cytosolic distribution until they are released from the cell. As a marker for this nonclassical secretory pathway we have chosen L-29, a soluble lectin of M(r) about 29,000, that has affinity for lactose and other beta-galactoside containing glycoconjugates. We were interested in determining if cultured epithelial cells secrete L-29 and if they do so in a polarized fashion. Madin-Darby canine kidney (MDCK)-II cells were found to express large quantities of L-29 (about 1% of the detergent soluble protein). The lectin was diffusely distributed in the cytosol, with little or none in vesicular compartments. The polarity of L-29 secretion, when analyzed in pulse-chase experiments, was selectively into the apical compartment of filter-grown MDCK cells. This secretion was not inhibited by brefeldin A or monensin, drugs that are known to inhibit protein transport through the ER-Golgi-PM pathway. Secretion of L-29 was augmented 3-5-fold by the calcium ionophore A23187 and by increasing the temperature to 42 degrees C, whereas lowering the temperature to 20 degrees C or addition of nocodazole prevented secretion. These results demonstrate the polarized secretion of a cytosolic protein by a nonclassical secretory pathway.

Animals↗