A proteoglycan undergoes different modifications en route to the apical and basolateral surfaces of Madin-Darby canine kidney cells

We have grown polarized epithelial Madin-Darby canine kidney II (MDCK II) cells on filters in the presence of [(35)S]sulfate, [(3)H]glucosamine, or [(35)S]cysteine/[(35)S]methionine to study proteoglycan (PG) synthesis, sorting, and secretion to the apical and basolateral media. Whereas most of the [(35)S]sulfate label was recovered in basolateral PGs, the [(3)H]glucosamine label was predominantly incorporated into the glycosaminoglycan chains of apical PGs, indicating that basolateral PGs are more intensely sulfated than their apical counterparts. Expression of the PG serglycin with a green fluorescent protein tag (SG-GFP) in MDCK II cells produced a protein core secreted 85% apically, which was largely modified by chondroitin sulfate chains. Surprisingly, the 15% of secreted SG-GFP molecules recovered basolaterally were more heavily sulfated and displayed a different sulfation pattern than the apical counterpart. More detailed studies of the differential modification of apically and basolaterally secreted SG-GFP indicate that the protein cores have been designated to apical and basolateral transport platforms before pathway-specific, post-translational modifications have been completed.     

Dramatic changes of sulfated proteoglycans composition in a tumorigenic SV-40-transformed renal proximal-tubule cell line.

To characterize the sulfated proteoglycans (PGs) alterations associated with malignant transformation of epithelial cells in vitro, the localization, charge, size, and composition of cell-associated and secreted sulfated PGs have been compared in rabbit renal proximal-tubule cells in primary culture (Ronco et al., 1990) and in a derived SV-40 transformed cell line (RC.SV1) exhibiting a proximal phenotype and high tumor-inducing ability (Vandewalle et al., 1989). Both normal and transformed cells incorporated PGs into a thick basement membrane layer as shown by ruthenium red staining and immunodetection with a monoclonal antibody raised against the core protein of the bovine basement membrane heparan sulfate-PG (HS-PG). In primary cultures of normal cells, cell-associated PGs were almost identical to those extracted from renal tubule fractions in vivo by their size (Kav = 0.27 vs. 0.26 on Sepharose CL-6B) and composition characterized by the exclusive presence of heparan sulfate glycosaminoglycan (HS-GAG) chains. In addition, the cells secreted a HS-PG with similar biochemical characteristics (Kav = 0.29; 100% HS-GAG chains). The SV-40-transformed RC.SV1 cells also synthesized and secreted a unique PG with the same charge and Kav values and apparently the same core protein (35 kDa) as in nontransformed cells, but three major differences were observed: (i) an increased proportion of PG-associated [35S]sulfate radioactivity released into the culture medium (36 vs. 21%), (ii) the emergence of free GAG chains unincorporated into PGs and detected only in the cell-associated fraction, and (iii) a dramatic change in the composition of GAG chains in which chondroitin sulfate replaced heparan-sulfate. The latter finding is in keeping with the known chondroitin sulfate increase and heparan-sulfate decrease in epithelial tumors. The alterations of PGs observed in this study may play a role in the acquisition and/or maintenance of the malignant phenotype.     

Caveolin-1 is not essential for biosynthetic apical membrane transport

Caveolin-1 has been implicated in apical transport of glycosylphosphatidylinositol (GPI)-anchored proteins and influenza virus hemagglutinin (HA). Here we have studied the role of caveolin-1 in apical membrane transport by generating caveolin-1-deficient Madin-Darby canine kidney (MDCK) cells using retrovirus-mediated RNA interference. The caveolin-1 knockdown (cav1-KD) MDCK cells were devoid of caveolae. In addition, caveolin-2 was retained in the Golgi apparatus in cav1-KD MDCK cells. However, we found no significant alterations in the apical transport kinetics of GPI-anchored proteins or HA upon depletion of caveolin-1. Similar results were obtained using embryonic fibroblasts from caveolin-1-knockout mice. Thus, we conclude that caveolin-1 does not play a major role in lipid raft-mediated biosynthetic membrane trafficking.     

The MAL proteolipid is necessary for the overall apical delivery of membrane proteins in the polarized epithelial Madin-Darby canine kidney and fischer rat thyroid cell lines

The MAL proteolipid has been recently demonstrated as being necessary for correct apical sorting of the transmembrane influenza virus hemagglutinin (HA) in Madin-Darby canine kidney (MDCK) cells. The fact that, in contrast to MDCK cells, Fischer rat thyroid (FRT) cells target the majority of glycosylphosphatidylinositol (GPI)-anchored proteins to the basolateral membrane provides us with the opportunity to determine the role of MAL in apical transport of membrane proteins under conditions in which the majority of GPI-anchored proteins are (MDCK cells) or are not (FRT cells) targeted to the apical surface. Using an antisense oligonucleotide-based strategy to deplete endogenous MAL, we have observed that correct transport of apical transmembrane proteins associated (HA) or not (exogenous neurotrophin receptor and endogenous dipeptidyl peptidase IV) with lipid rafts, as well as that of the bulk of endogenous apical membrane, takes place in FRT cells by a pathway that requires normal MAL levels. Even transport of placental alkaline phosphatase, a GPI-anchored protein that is targeted apically in FRT cells, was dependent on normal MAL levels. Similarly, in addition to the reported effect of MAL on HA transport, depletion of MAL in MDCK cells caused a dramatic reduction in the apical delivery of the GPI-anchored gD1-DAF protein, neurotrophin receptor, and the bulk of membrane proteins. These results suggest that MAL is necessary for the overall apical transport of membrane proteins in polarized MDCK and FRT cells.     

HGF-Induced Tubulogenesis and Branching of Epithelial Cells Is Modulated by Extracellular Matrix and TGF-β

Beginning with the observation that hepatocyte growth factor (HGF) induces the formation of branching tubular structures in Madin-Darby canine kidney (MDCK) cells cultured in Type I collagen gels but not in basement membrane Matrigel, we examined the individual components within this complex basement membrane extract to determine the effect of these proteins on the morphogenetic changes mediated by HGF. After extraction of several growth factors from Matrigel, HGF was still unable to induce process formation, an early event in tubulogenesis, indicating that one or more of the remaining extracellular matrix (ECM) proteins or growth factors were exerting the inhibitory effect. By individually adding back these components to MDCK cells grown in Type I collagen gels in the presence of HGF, we were able to establish that: (1) certain ECM proteins, such as laminin, entactin, and fibronectln, actually facilitated the formation of branching tubular structures and increased their complexity; (2) other ECM proteins, such as Type IV collagen, heparan sulfate proteoglycan, and vitronectin, caused marked inhibition of HGF-induced morphogenesis; and (3) not only did transforming growth factor-β (TGF-β) inhibit the formation of tubular structures, but those which did form exhibited little branching, thereby suggesting that TGF-β modulates tubulogenesis as well as branching. These results suggest that a tubulogenic morphogen such as HGF and a tubulogenesis-inhibitory morphogen such as TGF-β can, in the context of the dynamic matrix known to exist during epithelial tissue development, modulate the degree of tubule (or ductal) formation, the length of these tubules, and the extent of their arborization. The relevance of these findings to tubulogenesis and branching during kidney development is discussed.     

Pertussis toxin-dependent and -independent hormonal effects on cultured renal epithelioid cells

The present study has been performed to test for the involvement of pertussis toxin-sensitive GTP-binding proteins (G-proteins) in the cellular transduction of hormone-induced activation of potassium channels. In Madin Darby canine kidney (MDCK) cells, a permanent cell line from dog kidney, epinephrine, acetylcholine, bradykinin, serotonin and ATP hyperpolarize the cell membrane by activation of potassium channels. In cells pretreated with pertussis toxin the hyperpolarizations elicited by either acetylcholine or serotonin are completely abolished; that following epinephrine is blunted and only transient. The hyperpolarizing effects of ATP or bradykinin are not affected by pertussis toxin. Thus, in MDCK cells both pertussis toxin-dependent and -independent mechanisms operate in parallel to enhance the potassium conductance of the cell membrane.     

Microporosity of the substratum regulates differentiation of MDCK cells in vitro

Summary We have analyzed the ability of the physical substratum to modulate both the ultrastructural and protein synthetic characteristics of the Madin-Darby canine kidney (MDCK) renal cell line. When MDCK cells were seeded on Millipore Millicell CM microporous membrane cell culture inserts they demonstrated a more columnar organization with an increase in cell density sixfold greater than the same cells seeded on conventional plastic substrata. After 1 wk postseeding on the microporous membrane a partial basal lamina was noted, with a contiguous basement membrane being apparent after 2 wk. One-dimensional sodium dodecyl sulfate gel electrophoresis was used to analyze detergent-solubilized proteins from MDCK cells maintained on plastic substrata vs. microporous membranes. When proteins were pulse-labeled with [³⁵S]methionine, a 55 kDa protein was evident in the cytosolic extract of cells grown on collagen, laminin, and nontreated plastic substrata; but this labeled protein was not evident in similar extracts from cells grown on collagen and laminin-coated microporous membranes. To test if the polarized, basement-membrane secreting phenotype of the MDCK cells could be generated on a microporous membrane without pretreatment with any extracellular matrix (ECM) components, cells were seeded on the Millipore Millicell HA (cellulosic) microporous membrane. This type of substrata does not need a coating of ECM components for cell attachment. A partial basement membrane was formed below cells where the basal surface of the cell was planar, but not in areas where the cell formed large cytoplasmic extensions into the filter. This led us to the conclusion that the microporous nature of the substrata can dictate both ultrastructural and protein synthetic activities of MDCK cells. Furthermore, we suggest that both the planar nature of the basal surface and the microporosity of the substrate are corequisites for the deposition of the basement membrane.     

FSHIP2 regulates epithelial cell polarity through its lipid product,which binds to Dlg1, a pathway subverted by hepatitis C virus core protein

The main targets of hepatitis C virus (HCV) are hepatocytes, the highly polarized cells of the liver, and all the steps of its life cycle are tightly dependent on host lipid metabolism. The interplay between polarity and lipid metabolism in HCV infection has been poorly investigated. Signaling lipids, such as phosphoinositides (PIs), play a vital role in polarity, which depends on the distribution and expression of PI kinases and PI phosphatases. In this study, we report that HCV core protein, expressed in Huh7 and Madin–Darby canine kidney (MDCK) cells, disrupts apicobasal polarity. This is associated with decreased expression of the polarity protein Dlg1 and the PI phosphatase SHIP2, which converts phosphatidylinositol 3,4,5-trisphosphate into phosphatidylinositol 4,5-bisphosphate (PtdIns(3,4)P2). SHIP2 is mainly localized at the basolateral membrane of polarized MDCK cells. In addition, PtdIns(3,4)P2 is able to bind to Dlg1. SHIP2 small interfering RNA or its catalytically dead mutant disrupts apicobasal polarity, similar to HCV core. In core-expressing cells, RhoA activity is inhibited, whereas Rac1 is activated. Of interest, SHIP2 expression rescues polarity, RhoA activation, and restricted core level in MDCK cells. We conclude that SHIP2 is an important regulator of polarity, which is subverted by HCV in epithelial cells. It is suggested that SHIP2 could be a promising target for anti-HCV treatment.     

Immunohistochemical characterization of extracellular matrix components of yolk sac tumors

Proteoglycans (PGs) were isolated from yolk sac tumor and chondroitin sulfate large PG (core molecule with a molecular weight congruent to 200,000) and small PG (core molecule with a molecular weight congruent to 50,000) were detected. Immunohistochemical localization of PGs in three yolk sac tumors was investigated using monoclonal antibodies raised against both small and large PGs, which were purified from human ovarian fibroma capsule and a yolk sac tumor, respectively. The localization of large PG was observed to be distinct from that of small PG. A markedly positive reaction for antibody against large PG was observed in myxomatous areas, perivascular and perivesicular portions; hyaline globules were the most intensely reactive. In the areas showing a polyvesicular vitelline tumor pattern, the compact connective tissue stroma consisted of small PGs. It is conceivable that large PGs are synthesized by immature mesenchymal cells and also by epithelial-like cells as a basement membrane component, whereas small PGs are synthesized by mature fibroblastic cells synthesizing collagen. Immunohistochemical localization of other extracellular matrix components (laminin, fibronectin, type I-IV collagen) was also studied in relation to PG localization.     

Zonula occludens-1 is a scaffolding protein for signaling molecules. Galpha(12) directly binds to the Src homology 3 domain and regulates paracellular permeability in epithelial cells

Zonula occludens proteins are multidomain proteins usually localized at sites of intercellular junctions, yet little is known about their role in regulating junctional properties. Multiple signaling proteins regulate the junctional complex, and several (including G proteins) have been co-localized with zonula occludens-1 (ZO-1) in the tight junction of epithelial cells. However, evidence for direct interactions between signaling proteins and tight junction proteins has been lacking. In these studies, we constructed Galpha-glutathione S-transferase (GST) fusion proteins and tested for interactions with [(35)S]methionine-labeled in vitro translated ZO-1 and ZO-2. Only Galpha(12) directly interacted with in vitro translated ZO-1 and ZO-2. Using a series of ZO-1 domains expressed as GST fusion proteins and in vitro translated [(35)S]methionine-labeled Galpha(12), we found that Galpha(12) and constitutively active (Q229L) alpha(12) (QLalpha(12)) bind to the Src homology 3 (SH3) domain of ZO-1. This binding was not detected with SH3 domains from other proteins. Inducible expression of wild-type alpha(12) and QLalpha(12) in Madin-Darby canine kidney (MDCK) cells was established using the Tet-Off system. In Galpha(12)-expressing cells, we found that ZO-1 and Galpha(12) co-localize by confocal microscopy and co-immunoprecipitate. Galpha(12) from MDCK cell lysates bound to the GST-ZO-1-SH3 domain, and expression of QLalpha(12) in MDCK cells reversibly increased paracellular permeability. These studies indicated that ZO-1 directly interacts with Galpha(12) and that Galpha(12) regulates barrier function of MDCK cells.     

Reovirus-induced apoptosis of MDCK cells is not linked to viral yield and is blocked by Bcl-2.

In this study, we investigated the relationship between reovirus-induced apoptosis and viral growth. Madin-Darby canine kidney (MDCK) epithelial cells infected with prototype reovirus strains type 1 Lang (T1L) or type 3 Dearing (T3D) were found to undergo apoptosis, and T3D induced apoptosis of MDCK cells to a substantially greater extent than T1L. By using T1L x T3D reassortant viruses, we found that differences in the capacities of these strains to induce apoptosis are determined by the viral S1 and M2 gene segments. These genes encode viral outer-capsid proteins that play important roles in viral entry into cells. T1L grew significantly better in MDCK cells than T3D, and these differences in growth segregated with the viral L1 and M1 gene segments. The L1 and M1 genes encode viral core proteins involved in viral RNA synthesis. Bcl-2 overexpression in MDCK cells inhibited reovirus-induced apoptosis but did not substantially affect reovirus growth. These findings indicate that differences in the capacities of reovirus strains to induce apoptosis and grow in MDCK cells are determined by different viral genes and that premature cell death by apoptosis does not limit reovirus growth in MDCK cells.     

A functional assay for proteins involved in establishing an epithelial occluding barrier: identification of a uvomorulin-like polypeptide

A functional assay has been developed to identify cell surface proteins involved in the formation of epithelial tight junctions. Transepithelial electrical resistance was used to measure the presence of intact tight junctions in monolayers of Madin-Darby canine kidney (MDCK) cells cultured on nitrocellulose filters. The strain I MDCK cells used have a transmonolayer resistance greater than 2,000 ohm . cm2. When the monolayers were incubated at 37 degrees C without Ca2+, the intercellular junctions opened and the transmonolayer resistance dropped to the value of a bare filter, i.e., less than 40 ohm . cm2. When Ca2+ was restored, the cell junctions resealed and the resistance recovered rapidly. Polyclonal antibodies raised against intact MDCK cells inhibited the Ca2+-dependent recovery of electrical resistance when applied to monolayers that had been opened by Ca2+ removal. Cross-linking of cell surface molecules was not required because monovalent Fab' fragments also inhibited. In contrast, a variety of other antibodies that recognize specific proteins on the MDCK cell surface failed to inhibit the recovery of resistance. Monoclonal antibodies have been raised and screened for their ability to inhibit resistance recovery. One such monoclonal antibody has been obtained that stained the lateral surface of MDCK cells. This antibody, rr1, recognized a 118-kD polypeptide in MDCK cell extracts and an 81-kD fragment released from the cell surface by trypsinization in the presence of Ca2+. Sequential immunoprecipitation with antibody rr1 and a monoclonal antibody to uvomorulin showed that this polypeptide is related to uvomorulin. The role of uvomorulin-like and liver cell adhesion molecule (L-CAM)-like polypeptides in the establishment of the epithelial occluding barrier is discussed.     

Glycosaminoglycan secretion in xyloside treated polarized human colon carcinoma Caco-2 cells

Polarized epithelial cells like Madin-Darby canine kidney (MDCK) and CaCo-2 cells synthesize and secrete proteoglycans (PGs), mostly of heparan sulphate (HS) type in direction of the basal extracellular matrix, but also some in the apical direction. MDCK cells possess the capacity to synthesize chondroitin sulphate (CS) PGs that are mainly secreted into the apical medium, a process that is enhanced in the presence of hexyl-β-d-xyloside. We have now tested the capacity of several xylosides to enhance glycosaminoglycan (GAG) chain secretion from the human colon carcinoma cell line CaCo-2 in the differentiated and non-differentiated state. In these cells, benzyl-β-d-xyloside was a potent initiator of CS chains, which for these cells were predominantly secreted into the basolateral medium. Xylosides with other aglycone groups mediated only minor changes in GAG secretion. Although benzyl-β-d-xyloside stimulated the basolateral CS-GAG secretion in both differentiated and undifferentiated CaCo-2 cells, basolateral secretion of trypsin-like activity was dramatically enhanced in undifferentiated cells, but not significantly altered in differentiated cells.     

Type I collagen gel induces Madin-Darby canine kidney cells to become fusiform in shape and lose apical-basal polarity

In the embryo, epithelia give rise to mesenchyme at specific times and places. Recently, it has been reported (Greenburg, G., and E. D. Hay. 1986. Dev. Biol. 115:363-379; Greenberg, G., and E. D. Hay. 1988. Development (Camb.). 102:605-622) that definitive epithelia can give rise to fibroblast-like cells when suspended within type I collagen gels. We wanted to know whether Madin-Darby canine kidney (MDCK) cells, an epithelial line, can form mesenchyme under similar conditions. Small explants of MDCK cells on basement membrane were suspended within or placed on top of extracellular matrix gels. MDCK cells on basement membrane gel are tall, columnar in shape, and ultrastructurally resemble epithelia transporting fluid and ions. MDCK explants cultured on type I collagen gel give rise to isolated fusiform-shaped cells that migrate over the gel surface. The fusiform cells extend pseudopodia and filopodia, lose cell membrane specializations, and develop an actin cortex around the entire cell. Unlike true mesenchymal cells, which express vimentin and type I collagen, fusiform cells produce both keratin and vimentin, continue to express laminin, and do not turn on type I collagen. Fusiform cells are not apically-basally polarized, but show mesenchymal cell polarity. Influenza hemagglutinin and virus budding localize to the front end or entire cell surface. Na,K-ATPase occurs intracellularly and also symmetrically distributes on the cell surface. Fodrin becomes diffusely distributed along the plasma membrane, ZO-1 cannot be detected, and desmoplakins distribute randomly in the cytoplasm. The loss of epithelial polarity and acquisition of mesenchymal cell polarity and shape by fusiform MDCK cells on type I collagen gel was previously unsuspected. The phenomenon may offer new opportunities for studying cytoplasmic and nuclear mechanisms regulating cell shape and polarity.     

Retention of differentiated properties in an established dog kidney epithelial cell line (MDCK)

Madin-Darby canine kidney (MDCK) cells grown in tissue culture have the morphological properties of distal tubular epithelial cells, form tight junctions, and lack several proximal tubular enzyme markers. Adenylate cyclase in these cells was stimulated by vasopressin, oxytocin, prostaglandins E1 and E2, glucagon, and cholera toxin. Hormone-stimulated adenylate cyclase activity in isolated membrane preparations was dependent on low concentrations of GTP and had the MgCl2 and pH optima expected for the kidney enzyme. The results, as well as the demonstration of enhanced hemicyst formation induced by cyclic AMP, suggest that the MDCK cell line has retained the differentiated properties of the kidney epithelial cell of origin. When MDCK cells were injected into baby nude mice, continuous nodule growth was observed until adulthood was attained. Histological studies revealed the presence of two cell types: normal mouse fibroblasts which comprise 80--90% of the solid nodule mass, and MDCK cells, which formed epithelial sheets lining internal fluid-filled glands. Electron microscope analysis showed that the mucosal surfaces of the cells were characterized by microvilli which faced the lumen of the glands, that adjacent MDCK cells were joined by tight junctions, and that the serosal surfaces of the epithelial sheets were characterized by smooth plasma membranes which were lined by a continuous basement membrane. These observations lead to the conclusion that the MDCK cells retain regional differentiation of their plasma membranes and the ability to regenerate kidney tubule-like structures in vivo.     

Hepatocyte growth factor rapidly induces the tyrosine phosphorylation of 41-kDa and 43-kDa proteins in mouse keratinocytes.

We have examined the hepatocyte growth factor (HGF)-mediated changes in protein-tyrosine phosphorylation in mouse keratinocytes (PAM-212) and canine kidney epithelial cells (MDCK). In PAM-212 cells HGF and epidermal growth factor, both of which stimulated the DNA synthesis, rapidly induced the tyrosine phosphorylation of two 41-kDa and two 43-kDa proteins: increased tyrosine phosphorylation of those proteins has been commonly observed when quiescent fibroblasts are stimulated with a variety of mitogenic agents. In contrast, HGF did not stimulate the DNA synthesis but induced cell dissociation in MDCK cells; under this condition, increased tyrosine phosphorylation of the 41-kDa and 43-kDa protein was not observed. A possible role of the increased tyrosine phosphorylation of 41-kDa and 43-kDa protein in the signaling pathway of HGF is discussed.     

Surface heat shock protein 90 serves as a potential receptor for calcium oxalate crystal on apical membrane of renal tubular epithelial cells

Adhesion of calcium oxalate monohydrate (COM) crystals on renal tubular epithelial cells is a crucial step in kidney stone formation. Finding potential crystal receptors on the apical membrane of the cells may lead to a novel approach to prevent kidney stone disease. Our previous study identified a large number of crystal-binding proteins on the apical membrane of MDCK cells. However, their functional role as potential crystal receptors had not been validated. The present study aimed to address the potential role of heat shock protein 90 (HSP90) as a COM crystal receptor. The apical membrane was isolated from polarized MDCK cells by the peeling method and recovered proteins were incubated with COM crystals. Western blot analysis confirmed the presence of HSP90 in the apical membrane and the crystal-bound fraction. Immunofluorescence staining without permeabilization and laser-scanning confocal microscopy confirmed the surface HSP90 expression on the apical membrane of the intact cells. Crystal adhesion assay showed that blocking surface HSP90 by specific anti-HSP90 antibody and knockdown of HSP90 by small interfering RNA (siRNA) dramatically reduced crystal binding on the apical surface of MDCK cells (by approximately 1/2 and 2/3, respectively). Additionally, crystal internalization assay revealed the presence of HSP90 on the membrane of endocytic vesicle containing the internalized COM crystal. Moreover, pretreatment of MDCK cells with anti-HSP90 antibody significantly reduced crystal internalization (by approximately 1/3). Taken together, our data indicate that HSP90 serves as a potential receptor for COM crystals on the apical membrane of renal tubular epithelial cells and is involved in endocytosis/internalization of the crystals into the cells.     

Microtubules are involved in the secretion of proteins at the apical cell surface of the polarized epithelial cell, Madin-Darby canine kidney

Microtubule-disrupting drugs (nocodazole, colchicine) and cytochalasin D, which inhibits the polymerization of the actin microfilaments, were used to study the role of the cytoskeleton in protein secretion in the polarized Madin-Darby canine kidney (MDCK) epithelial cells. Two proteins were analyzed. The gp 80 glycoprotein complex, which in untreated cells is sorted into the apical pathway and lysozyme, which is released randomly at both cell surfaces in transfected MDCK cells. Our results show that cytochalasin D has no influence on the transport of the gp 80 complex and lysozyme to either cell surface. However, in the presence of nocodazole or colchicine the secretion of both proteins at the apical cell surface is reduced by 50% with a concomitant increase in the basolateral release. These data suggest that microtubules are necessary for an efficient secretion of proteins at the apical cell surface of MDCK cells. In regard to the yet unresolved discrepancy concerning the involvement of microtubules in the transport of membrane proteins to the apical surface of MDCK cells, our results are consistent with the data of Rindler et al. (Rindler, M. J., Ivanov, I. E., and Sabatini, D. D. (1987) J. Cell. Biol. 104, 231-241) who observed a nonpolarized delivery of the influenza virus hemagglutinin in the presence of nocodazole or colchicine.     

Oligomerization is a specific requirement for apical sorting of glycosyl-phosphatidylinositol-anchored proteins but not for non-raft-associated apical proteins

Protein apical sorting in polarized epithelial cells is mediated by two different mechanisms, raft dependent and raft independent. In Madin-Darby canine kidney (MDCK) cells, an essential step for apical sorting of glycosyl-phosphatidylinositol (GPI)-anchored proteins (GPI-APs) is their coalescence into high-molecular-weight (HMW) oligomers. Here we show that this mechanism is also functional in Fischer rat thyroid cells, which possess a different sorting phenotype compared with MDCK cells. We demonstrate that, as in MDCK cells, both apical and basolateral GPI-APs associate with detergent-resistant microdomains, but that only the apical proteins are able to oligomerize into HMW complexes during their passage through the medial Golgi. We also show that oligomerization is a specific requirement for apical sorting of GPI-APs and is not used by transmembrane, non-raft-associated apical proteins.