Analysis of epithelial cell surface polarity with monoclonal antibodies

The hybridoma technique of K?hler and Milstein was utilized to isolate hybrid cell lines secreting monoclonal antibodies against cell surface proteins on the Madin-Darby canine kidney (MDCK) epithelial cell line. These antibodies were employed as high-affinity ligands to study the development and maintenance of epithelial cell polarity in MDCK cells and for the identification of nephron segment-specific proteins. Using standard procedures, we were able to immunoprecipitate glycoproteins with molecular weights of 25,000 ( 25K ), 35,000 ( 35K ), and 50,000 (50K). Immunofluorescence and immunoelectron microscopy of MDCK demonstrated that the 35K and 50K proteins could be localized on both the apical and basolateral membranes of subconfluent cells but primarily on the basolateral membranes of confluent cells. By determining the cell surface distribution of the 35K and 50K proteins on MDCK cells during growth into a confluent monolayer, and after the experimental disruption of tight junctions, evidence was obtained that the polarized distribution of these cell surface glycoproteins required the presence of tight junctions. We propose that confluent MDCK cells have a mechanism that is responsible for the establishment and maintenance of epithelial apical and basolateral membranes as distinct cell surface domains. These monoclonal antibodies were also used to localize the 25K and 35K glycoproteins in the kidney. The distribution of these proteins was mapped by immunofluorescence and immunoelectron microscopy and was determined to be on the basolateral membranes of epithelial cells in only certain tubular segments of the nephron. The possible functional implications of these distributions are discussed.     

In Polarized MDCK Cells Basolateral Vesicles Arise from Clathrin-γ-adaptin–coated Domains on Endosomal Tubules

Human transferrin receptors (TR) and receptors for polymeric immunoglobulins (pIgR) expressed in polarized MDCK cells maintain steady-state, asymmetric distributions on the separate basolateral and apical surfaces even though they are trafficking continuously into and across these cells. The intracellular mechanisms required to maintain these asymmetric distributions have not been located. Here we show that TR and pIgR internalize from both surfaces to a common interconnected endosome compartment that includes tubules with buds coated with clathrin lattices. These buds generate vesicles that carry TR to the basolateral border. The lattices contain γ-adaptin and are dispersed by treatment with brefeldin A (BFA). Since BFA treatment abrogates the vectorial trafficking of TR in polarized MDCK cells, we propose that the clathrin-coated domains of the endosome tubules contain the polarized sorting mechanism responsible for their preferential basolateral distribution.     

Apical and basolateral endosomes of MDCK cells are interconnected and contain a polarized sorting mechanism

We have evaluated transcytotic routes in MDCK cells for their ability to generate a polarized surface distribution of trafficking proteins by following the intracellular sorting of transferrin receptors (TRs). We find that the selective basolateral expression of TRs is maintained in the face of extensive trafficking between the apical and basolateral surfaces. Biochemical studies of receptors loaded with tracer under conditions approaching steady state indicate that TRs internalized from the two surfaces are extensively colocalized within MDCK cells and that both populations of receptors are selectively delivered to the basolateral surface. Tailless TRs in which the cytoplasmic domain has been deleted display an unpolarized cell surface distribution and recycle in an unpolarized fashion. We show by EM that wild-type receptors internalized from each surface are colocalized within endosomal elements distributed throughout the cytoplasm. By preloading endosomal elements directly accessible from the basolateral surface with transferrin (Tf)-HRP, we show that apically internalized TRs rapidly enter the same compartment. We also show that both transcytosing (apically internalized) and recycling (basolaterally internalized) TRs are delivered to the basolateral border by a distinctive subset of exocytotic, 60-nm-diam vesicles. Together, the biochemical and morphological data show that apical and basolateral endosomes of MDCK cells are interconnected and contain a signal- dependent polarized sorting mechanism. We propose a dynamic model of polarized sorting in MDCK cells in which a single endosome-based, signal-dependent sorting step is sufficient to maintain the polarized phenotype.     

Apical and basolateral coated pits of MDCK cells differ in their rates of maturation into coated vesicles, but not in the ability to distinguish between mutant hemagglutinin proteins with different internalization signals

In polarized epithelial MDCK cells, all known endogenous endocytic receptors are found on the basolateral domain. The influenza virus hemagglutinin (HA) which is normally sorted to the apical plasma membrane, can be converted to a basolateral protein by specific mutations in its short cytoplasmic domain that also create internalization signals. For some of these mutations, sorting to the basolateral surface is incomplete, allowing internalization of two proteins that differ by a single amino acid of the internalization signal to be compared at both the apical and basolateral surfaces of MDCK cells. The rates of internalization of HA-Y543 and HA-Y543,R546 from the basolateral surface of polarized MDCK cells resembled those in nonpolarized cells, whereas their rates of internalization from the apical cell surface were fivefold slower. However, HA-Y543,R546 was internalized approximately threefold faster than HA-Y543 at both membrane domains, indicating that apical endocytic pits in polarized MDCK cells retained the ability to discriminate between different internalization signals. Slower internalization from the apical surface could not be explained by a limiting number of coated pits: apical membrane contained 0.7 as many coated pits per cell cross-section as did basolateral membranes. 10-14% of HA-Y543 at the apical surface of polarized MDCK cells was found in coated pits, a percentage not significantly different from that observed in apical coated pits of nonpolarized MDCK cells, where internalization was fivefold faster. Thus, there was no lack of binding sites for HA-Y543 in apical coated pits of polarized cells. However, at the apical surface many more shallow pits, and fewer deep, mature pits, were observed than were seen at the basolateral. These results suggest that the slower internalization at the apical surface is due to slower maturation of coated pits, and not to a difference in recognition of internalization signals.     

Epithelial cell polarity alters Rho-GTPase responses to Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic human pathogen that preferentially infects damaged epithelial tissues. Previous studies have failed to distinguish whether the increased susceptibility of injured epithelium results from the loss of cell polarity or increased access to the basolateral surface. We have used confluent monolayers of Madin-Darby canine kidney (MDCK) cells cultured on porous filter supports for 1-3 d as a model system to investigate whether the differentiation state of a polarized model epithelium affected the response of epithelial cells to this pathogen. Confluent incompletely polarized MDCK cell monolayers (day 1) efficiently internalized apically applied P. aeruginosa via a pathway that required actin polymerization and activation of Rho-family GTPases and was accompanied by an increase in the amount of activated RhoA. In contrast, P. aeruginosa entry into highly polarized MDCK monolayers (day 3) was 10- to 100-fold less efficient and was insensitive to inhibitors of actin polymerization or of Rho-family GTPase activation. There was no activation of RhoA; instead, Cdc42-GTP levels increased significantly. Basolateral infection of highly polarized MDCK monolayers was less efficient and insensitive to Clostridium difficile Toxin B, whereas basolateral infection of incompletely polarized MDCK monolayers was more efficient and required activation of Rho-family GTPases. Together, our findings suggest that as epithelial barrier differentiates and becomes highly polarized, it becomes resistant to P. aeruginosa infection. Nevertheless, polarized epithelial cells still sense the presence of apically infecting P. aeruginosa, but they may do so through a different group of surface proteins and/or downstream signaling pathways than do incompletely polarized cells.     

Viruses budding from either the apical or the basolateral plasma membrane domain of MDCK cells have unique phospholipid compositions.

Influenza virus and vesicular stomatitis virus (VSV) obtain their lipid envelope by budding through the plasma membrane of infected cells. When monolayers of Madin-Darby canine kidney (MDCK) cells, a polarized epithelial cell line, are infected with fowl plague virus (FPV), an avian influenza virus, or with VSV, new FPV buds through the apical plasma membrane whereas VSV progeny is formed by budding through the basolateral plasma membrane. FPV and VSV were isolated from MDCK host cells prelabeled with [32P]orthophosphate and their phospholipid compositions were compared. Infection was carried out at 31 degrees C to delay cytopathic effects of the virus infection, which lead to depolarization of the cell surface. 32P-labeled FPV was isolated from the culture medium, whereas 32P-labeled VSV was released from below the cell monolayer by scraping the cells from the culture dish 8 h after infection. At this time little VSV was found in the culture medium, indicating that the cells were still polarized. The phospholipid composition of the two viruses was distinctly different. FPV was enriched in phosphatidylethanolamine and phosphatidylserine and VSV in phosphatidylcholine, sphingomyelin, and phosphatidylinositol. When MDCK cells were trypsinized after infection and replated, non-infected control cells attached to reform a confluent monolayer within 4 h, whereas infected cells remained in suspension. FPV and VSV could be isolated from the cells in suspension and under these conditions the phospholipid composition of the two viruses was very similar. We conclude that the two viruses obtain their lipids from the plasma membrane in the same way and that the different phospholipid compositions of the viruses from polarized cells reflect differences in the phospholipid composition of the two plasma membrane domains.     

Nonpolarized expression of a secreted murine leukemia virus glycoprotein in polarized epithelial cells

Vaccinia virus recombinants were generated which express the intact gp70/p15E of Friend mink cell focus inducing virus (F-MCFV) or truncated forms of the glycoprotein that lack the transmembrane and cytoplasmic domains. The transport of the intact and truncated envelope glycoproteins to apical or basolateral surfaces was studied in the polarized epithelial MDCK cell line. Infection of MDCK cells with the recombinant expressing the intact F-MCFV envelope glycoprotein resulted in transport exclusively to the basolateral surfaces, whereas the recombinant expressing the truncated glycoprotein was found to be secreted from both the apical and basolateral surfaces. Thus removal of the transmembrane and cytoplasmic domains of the p15E protein results in a loss of directional transport to the basolateral membrane of polarized epithelial cells.     

Polarized endocytosis by Madin-Darby canine kidney cells transfected with functional chicken liver glycoprotein receptor

We have studied the expression of the chicken hepatic glycoprotein receptor (chicken hepatic lectin [CHL]) in Madin-Darby canine kidney (MDCK) cells, by transfection of its cDNA under the control of a retroviral promotor. Transfected cell lines stably express 87,000 surface receptors/cell with a kd = 13 nM. In confluent monolayers, approximately 40% of CHL is localized at the plasma membrane. 98% of the surface CHL is expressed at the basolateral surface where it performs polarized endocytosis and degradation of glycoproteins carrying terminal N-acetylglucosamine at a rate of 50,000 ligand molecules/h. Studies of the half-life of metabolically labeled receptor and of the stability of biotinylated cell surface receptor after internalization indicate that transfected CHL performs several rounds of uptake and recycling before it gets degraded. The successful expression of a functional basolateral receptor in MDCK cells opens the way for the characterization of the mechanisms that control targeting and recycling of proteins to the basolateral membrane of epithelial cells.     

Protein 4.1N is required for translocation of inositol 1,4,5-trisphosphate receptor type 1 to the basolateral membrane domain in polarized Madin-Darby canine kidney cells

Protein 4.1N was identified as a binding molecule for the C-terminal cytoplasmic tail of inositol 1,4,5-trisphosphate receptor type 1 (IP(3)R1) using a yeast two-hybrid system. 4.1N and IP(3)R1 associate in both subconfluent and confluent Madin-Darby canine kidney (MDCK) cells, a well studied tight polarized epithelial cell line. In subconfluent MDCK cells, 4.1N is distributed in the cytoplasm and the nucleus; IP(3)R1 is localized in the cytoplasm. In confluent MDCK cells, both 4.1N and IP(3)R1 are predominantly translocated to the basolateral membrane domain, whereas 4.1R, the prototypical homologue of 4.1N, is localized at the tight junctions (Mattagajasingh, S. N., Huang, S. C., Hartenstein, J. S., and Benz, E. J., Jr. (2000) J. Biol. Chem. 275, 30573-30585), and other endoplasmic reticulum marker proteins are still present in the cytoplasm. Moreover, the 4.1N-binding region of IP(3)R1 is necessary and sufficient for the localization of IP(3)R1 at the basolateral membrane domain. A fragment of the IP(3)R1-binding region of 4.1N blocks the localization of co-expressed IP(3)R1 at the basolateral membrane domain. These data indicate that 4.1N is required for IP(3)R1 translocation to the basolateral membrane domain in polarized MDCK cells.     

TGN38 recycles basolaterally in polarized Madin-Darby canine kidney cells.

Sorting of newly synthesized plasma membrane proteins to the apical or basolateral surface domains of polarized cells is currently thought to take place within the trans-Golgi network (TGN). To explore the relationship between protein localization to the TGN and sorting to the plasma membrane in polarized epithelial cells, we have expressed constructs encoding the TGN marker, TGN38, in Madin-Darby canine kidney (MDCK) cells. We report that TGN38 is predominantly localized to the TGN of these cells and recycles via the basolateral membrane. Analyses of the distribution of Tac-TGN38 chimeric proteins in MDCK cells suggest that the cytoplasmic domain of TGN38 has information leading to both TGN localization and cycling through the basolateral surface. Mutations of the cytoplasmic domain that disrupt TGN localization also lead to nonpolarized delivery of the chimeric proteins to both surface domains. These results demonstrate an apparent equivalence of basolateral and TGN localization determinants and support an evolutionary relationship between TGN and plasma membrane sorting processes.     

Surface expression of viral glycoproteins is polarized in epithelial cells infected with recombinant vaccinia viral vectors.

In polarized epithelial cells, maturation sites of enveloped viruses that form by budding at cell surfaces are restricted to particular membrane domains. Recombinant vaccinia viruses were used to investigate the sites of surface expression in the Madin-Darby canine kidney (MDCK) cell line of the hemagglutinin (HA) of influenza virus, the G glycoprotein of vesicular stomatitis virus (VSV), and gp70/p15E of Friend murine leukemia virus (MuLV). These glycoproteins could be demonstrated by immunofluorescence on the surfaces of MDCK cells as early as 4 h post-infection. In intact MDCK monolayers, vaccinia recombinants expressing HA produced a pattern of surface fluorescence typical of an apically expressed glycoprotein. In contrast, cells infected with vaccinia recombinants expressing VSV-G or MuLV gp70/p15E exhibited surface fluorescence only when monolayers were treated with EGTA to disrupt tight junctions, as expected of glycoproteins expressed on basolateral surfaces. Immunoferritin labeling in conjunction with electron microscopy confirmed that MDCK cells infected with the HA recombinant exhibited specific labeling of the apical surfaces whereas the VSV-G and MuLV recombinants exhibited the respective antigens predominantly on the basolateral membranes. Quantitation of surface expression by [125I]protein A binding assays on intact and EGTA-treated monolayers confirmed the apical localization of the vaccinia-expressed HA and demonstrated that 95% of the VSV-G and 97% of the MuLV gp70/p15E glycoproteins were localized on the basolateral surfaces. These results demonstrate that glycoproteins of viruses that normally mature at basolateral surfaces of polarized epithelial cells contain all of the structural information required for their directional transport to basolateral plasma membranes.     

Identification of a basolateral membrane targeting signal within the cytoplasmic domain of myelin/oligodendrocyte glycoprotein

Oligodendrocytes possess two distinct membrane compartments--uncompacted plasma membrane (cell body, processes) and compact myelin. Specific targeting mechanisms must exist to establish and maintain these membrane domains. Polarized epithelial cells have the best characterized system for targeting components to apical and basolateral compartments. Since oligodendrocytes arise from neuroepithelial cells, we investigated whether they might utilize targeting paradigms similar to polarized epithelial cells. Myelin/oligodendrocyte glycoprotein (MOG) is a transmembrane Ig-like molecule restricted to uncompacted oligodendroglial plasma membrane. We stably expressed MOG in Madin-Darby canine kidney (MDCK) Type II epithelial cells, which have been extensively used in protein-targeting studies. Data from surface biotinylation assays and confocal microscopy revealed that MOG sorts exclusively to the basolateral membrane of MDCK cells. Expression vectors containing progressive truncations of MOG from the cytoplasmic C-terminus were expressed in MDCK cells to localize basolateral sorting signals. A loss of only four C-terminal residues results in some MOG expression at the apical surface. More strikingly, removal of the C-terminal membrane associated hydrophobic domain from MOG results in complete loss of basolateral sorting and specific targeting to the apical membrane. These data suggest that myelinating oligodendrocytes may utilize a sorting mechanism similar to that of polarized epithelia.     

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.     

Basolateral expression of a chimeric protein in which the transmembrane and cytoplasmic domains of vesicular stomatitis virus G protein have been replaced by those of the influenza virus hemagglutinin

Two integral membrane proteins, influenza virus hemagglutinin (HA) and vesicular stomatitis virus G protein, are transported to and accumulated on the apical and basolateral surfaces, respectively, of the plasma membrane of polarized epithelial cells. We have used chimeric constructions to identify the domains of HA and G proteins which contain the signals for polarized transport. Previously, we have shown that a chimeric protein containing the cleavable leader and the ectodomain of HA fused to the anchoring and cytoplasmic domains of G is transported to the apical surface of polarized MDCK cells (McQueen, N.L., Nayak, D.P., Stephens, E.B., and Compans, R.W. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9318-9322). In this report we show that a chimera containing the cleavable leader and ectodomain of G fused to the anchoring and cytoplasmic domains of HA is transported to the basolateral surface of polarized cells. Another chimera which contains the leader sequence of G fused to leader minus HA is transported to the apical surface of polarized cells. These results taken together suggest that the signals for the polarized transport of HA and G proteins may reside in their ectodomains.     

Myosin VI is required for sorting of AP-1B-dependent cargo to the basolateral domain in polarized MDCK cells

In polarized epithelial cells, newly synthesized membrane proteins are delivered on specific pathways to either the apical or basolateral domains, depending on the sorting motifs present in these proteins. Because myosin VI has been shown to facilitate secretory traffic in nonpolarized cells, we investigated its role in biosynthetic trafficking pathways in polarized MDCK cells. We observed that a specific splice isoform of myosin VI with no insert in the tail domain is required for the polarized transport of tyrosine motif containing basolateral membrane proteins. Sorting of other basolateral or apical cargo, however, does not involve myosin VI. Site-directed mutagenesis indicates that a functional complex consisting of myosin VI, optineurin, and probably the GTPase Rab8 plays a role in the basolateral delivery of membrane proteins, whose sorting is mediated by the clathrin adaptor protein complex (AP) AP-1B. Our results suggest that myosin VI is a crucial component in the AP-1B-dependent biosynthetic sorting pathway to the basolateral surface in polarized epithelial cells.     

Carboxypeptidase M, a glycosylphosphatidylinositol-anchored protein, is localized on both the apical and basolateral domains of polarized Madin-Darby canine kidney cells.

Carboxypeptidase M, a glycosylphosphatidylinositol-anchored membrane glycoprotein, is highly expressed in Madin-Darby canine kidney (MDCK) cells, where it was previously shown that the glycosylphosphatidylinositol anchor and N-linked carbohydrate are apical targeting signals. Here, we show that carboxypeptidase M has an unusual, non-polarized distribution, with up to 44% on the basolateral domain of polarized MDCK cells grown on semipermeable inserts. Alkaline phosphatase, as well as five other glycosylphosphatidylinositol-anchored proteins, and transmembrane gamma-glutamyl transpeptidase exhibited the expected apical localization. Basolateral carboxypeptidase M was readily released by exogenous phosphatidylinositol-specific phospholipase C, showing it is glycosylphosphatidylinositol-anchored, whereas apical carboxypeptidase M was more resistant to release. In contrast, the spontaneous release of carboxypeptidase M into the medium was much higher on the apical than the basolateral domain. In pulse-chase studies, newly synthesized carboxypeptidase M arrived in equal amounts within 30 min on both domains, indicating direct sorting. After 4-8 h of chase, the steady-state distribution was attained, possibly due to transcytosis from the basolateral to the apical domain. These data suggest the presence of a unique basolateral targeting signal in carboxypeptidase M that competes with its apical targeting signals, resulting in a non-polarized distribution in MDCK cells.     

Basolateral sorting of syntaxin 4 is dependent on its N-terminal domain and the AP1B clathrin adaptor, and required for the epithelial cell polarity

Generation of epithelial cell polarity requires mechanisms to sort plasma membrane proteins to the apical and basolateral domains. Sorting involves incorporation into specific vesicular carriers and subsequent fusion to the correct target membranes mediated by specific SNARE proteins. In polarized epithelial cells, the SNARE protein syntaxin 4 localizes exclusively to the basolateral plasma membrane and plays an important role in basolateral trafficking pathways. However, the mechanism of basolateral targeting of syntaxin 4 itself has remained poorly understood. Here we show that newly synthesized syntaxin 4 is directly targeted to the basolateral plasma membrane in polarized Madin-Darby canine kidney (MDCK) cells. Basolateral targeting depends on a signal that is centered around residues 24-29 in the N-terminal domain of syntaxin 4. Furthermore, basolateral targeting of syntaxin 4 is dependent on the epithelial cell-specific clathrin adaptor AP1B. Disruption of the basolateral targeting signal of syntaxin 4 leads to non-polarized delivery to both the apical and basolateral surface, as well as partial intercellular retention in the trans-Golgi network. Importantly, disruption of the basolateral targeting signal of syntaxin 4 leads to the inability of MDCK cells to establish a polarized morphology which suggests that restriction of syntaxin 4 to the basolateral domain is required for epithelial cell polarity.     

Tight junction formation is closely linked to the polar redistribution of intramembranous particles in aggregating MDCK epithelia

We have used freeze-fracture electron microscopy to investigate the relationship between the formation of the tight junction in the establishment of a differential distribution of intramembranous particles (IMPs) between the luminal and basolateral membranes of a canine kidney cell line (MDCK). This involves a characterization of the IMP distribution in these membranes in confluent monolayers of MDCK cells, in EGTA-dissociated cells, and in cells at various stages of reassociation. While normal confluent MDCK monolayer cultures exhibit tight junctions and an IMP differential distribution between the luminal and basolateral membranes, cultures dissociated with EGTA lose both formed tight junctional elements and the differential IMP distribution. We have also found that as tight junctions reform between reaggregating MDCK cells, intramembranous particles appear to rapidly redistribute with respect to them. An asymmetric distribution of these particles in the luminal and basolateral membranes is eventually achieved. Tight junction formation appears so closely linked to the genesis of IMP polarity that at early time points when only a string of tight junctional components spans the junctional zone, differential IMP distributions are seen. Thus, our dissociation studies suggest a close relationship between the integrity of the tight junction and the maintenance of IMP polarity between the luminal and basolateral membranes, while cell reassociation studies suggest that the tight junction may be functionally linked to the genesis of IMP polarity.     

Rab10 regulates membrane transport through early endosomes of polarized Madin-Darby canine kidney cells

Rab10, a protein originally isolated from Madin-Darby Canine Kidney (MDCK) epithelial cells, belongs to a family of Rab proteins that includes Rab8 and Rab13. Although both Rab8 and Rab13 have been found to mediate polarized membrane transport, the function of Rab10 in mammalian cells has not yet been established. We have used quantitative confocal microscopy of polarized MDCK cells expressing GFP chimeras of wild-type and mutant forms of Rab10 to analyze the function of Rab10 in polarized cells. These studies demonstrate that Rab10 is specifically associated with the common endosomes of MDCK cells, accessible to endocytic probes internalized from either the apical or basolateral plasma membrane domains. Expression of mutant Rab10 defective for either GTP hydrolysis or GTP binding increased recycling from early compartments on the basolateral endocytic pathway without affecting recycling from later compartments or the apical recycling pathway. These results suggest that Rab10 mediates transport from basolateral sorting endosomes to common endosomes.     

Selective roles for cholesterol and actin in compartmentalization of different proteins in the Golgi and plasma membrane of polarized cells

To determine the roles of cholesterol and the actin cytoskeleton in apical and basolateral protein organization and sorting, we have performed comprehensive confocal fluorescence recovery after photobleaching analyses of apical and basolateral and raft- and non-raft-associated proteins, both at the plasma membrane and in the Golgi apparatus of polarized MDCK cells. We show that at both the apical and basolateral plasma membrane domains, raft-associated proteins diffuse faster than non-raft-associated proteins and that, different from the latter, they become restricted upon depletion of cholesterol. Furthermore, only transmembrane apical proteins are restricted by the actin network. This indicates that cholesterol-dependent domains exist both at the apical and basolateral membranes of polarized cells and that the actin cytoskeleton has a predominant role in the organization of transmembrane proteins independent of their association with rafts at the apical membrane. In the Golgi apparatus apical proteins appear to be segregated from the basolateral ones in a compartment that is sensitive both to cholesterol depletion and actin rearrangements. Furthermore, consistent with the role of actin rearrangements in apical protein sorting, we found that apical proteins exhibit a differential sensitivity to actin depolymerization in the Golgi of polarized and nonpolarized cells.