In the early days of epithelial cell biology, researchers working with kidney and/or intestinal epithelial cell lines and with hepatocytes described the biosynthetic and recycling routes followed by apical and basolateral plasma membrane (PM) proteins. They identified the trans-Golgi network and recycling endosomes as the compartments that carried out apical-basolateral sorting. They described complex apical sorting signals that promoted association with lipid rafts, and simpler basolateral sorting signals resembling clathrin-coated-pit endocytic motifs. They also noticed that different epithelial cell types routed their apical PM proteins very differently, using either a vectorial (direct) route or a transcytotic (indirect) route. Although these original observations have generally held up, recent studies have revealed interesting complexities in the routes taken by apically destined proteins and have extended our understanding of the machinery required to sustain these elaborate sorting pathways. Here, we critically review the current status of apical trafficking mechanisms and discuss a model in which clustering is required to recruit apical trafficking machineries. Uncovering the mechanisms responsible for polarized trafficking and their epithelial-specific variations will help understand how epithelial functional diversity is generated and the pathogenesis of many human diseases.

Introduction

The characteristic polarity of epithelial cells results from an epithelial polarity program that coordinates the activities of polarity proteins and lipids with those of positional sensors (e.g. E-cadherin and integrins) and GTPase switches to organize tight junctions (TJs), the actin and microtubule (MT) cytoskeletons, and the polarized trafficking machinery, with the ultimate goal of localizing different plasma membrane (PM) proteins to apical and basolateral domains (Bryant and Mostov, 2008; Mellman and Nelson, 2008; Tanos and Rodriguez-Boulan, 2008). For example, Na+-K+ ATPase, certain lactate transporters, neural cell-adhesion molecule (NCAM) and certain integrins are apically localized in retinal pigment epithelium (RPE), whereas they are basolaterally localized in most other body epithelia. This variable polarity of epithelial cells allows them to perform specific functions that are required by their host organs. Different epithelial cells also diverge regarding how they route different apical proteins to their final destination. Whereas Madin-Darby canine kidney (MDCK) cells transport most apical proteins using direct (vectorial) trans-Golgi network (TGN)-to-PM routes (Ellis et al., 2006; Rodriguez-Boulan et al., 2005), liver, intestinal and RPE traffic many apical proteins through an indirect (transcytotic) route via the basolateral membrane (Bonilha, 1997; Hubbard and Stieger, 1989; Le Bivic et al., 1990; Matter et al., 1990). Furthermore, the routing of apical proteins can even change from transcytotic to vectorial during the development of epithelial polarity (Zurzolo et al., 1992).

Thus, the polarized trafficking machinery seems to be exquisitely tailored to the physiology of each individual epithelial cell type. A plethora of recent advances is starting to provide a clearer picture of the mechanisms that underlie this tailoring. This picture is quite different from that suggested by the popular hypothesis of the 1980s that proposed that cells secrete their soluble and PM components using a `bulk-flow' route that does not require transport signals (reviewed by Pfeffer and Rothman, 1987). An extension of this hypothesis to epithelial cells, on the basis of the observation that the basolateral membrane harbors many house-keeping receptors and transporters present in the PM of non-epithelial cells, proposed that the basolateral route operates by bulk flow whereas the apical route requires sorting signals (Simons and Fuller, 1985). Hence, the discovery that both apical and basolateral PM proteins require sorting signals for secretion came as a major surprise (Matter and Mellman, 1994; Mostov et al., 1992; Rodriguez-Boulan and Musch, 2005). An even greater surprise was the finding that basolateral sorting signals were usually dominant over apical sorting signals (Matter and Mellman, 1994; Mostov et al., 1992; Rodriguez-Boulan and Musch, 2005). The complexity of apical targeting mechanisms is turning out to be equally surprising.

The similarity between basolateral signals and endocytic motifs, and the demonstration of a key role for clathrin and clathrin adaptors in basolateral sorting (Deborde et al., 2008; Folsch et al., 2009) has established a solid foundation for the mechanistic understanding of basolateral PM sorting. By contrast, apical PM protein sorting has proven more difficult to elucidate, because it involves information contributed by lipid, carbohydrate or proteinaceous regions that are present in the lumenal, membrane or cytoplasmic domains of the protein (Folsch et al., 2009; Rodriguez-Boulan et al., 2005).

In this Commentary, we highlight our current understanding of apical sorting signals and trafficking mechanisms. Adding to the original description of vectorial and transcytotic routes for apical delivery, recent studies have revealed that most newly synthesized apical PM proteins leaving the TGN traverse early-endosomal compartments before reaching the apical surface. This detour might reflect the requirement to pick up trafficking regulatory machinery at endosomal stations, e.g. MT motors, the actin cytoskeleton, and Rab and Rho GTPases, which have been found to play very specific roles in regulating these routes. Here, we discuss a comprehensive model to explain how diverse targeting signals on apical proteins might enable their segregation via a clustering mechanism that in turn is required to recruit trafficking machinery. We do not cover the mechanisms involved in establishing cell polarity or domain identity; instead, the reader is directed towards recent reviews in these areas (Bryant and Mostov, 2008; Wang and Margolis, 2007).

Apical trafficking machinery and routes

Our knowledge about polarized trafficking is based on a limited set of model PM proteins studied, in most cases under overexpression conditions, in kidney (MDCK, LLCPK), intestinal (Caco-2, HT-29) and liver (native liver, WIF-B, HEPG2) epithelial cells (Table 1). It has been increasingly appreciated, in MDCK cells at least, that different apical proteins emerge from the TGN in different post-Golgi carriers (Guerriero et al., 2008; Jacob et al., 2003) and navigate through distinct subsets of endosomal compartments, which is dependent, at least in part, on their apical sorting signals (Fig. 1, Box 1). This conclusion is partially based on experiments that ablate the function of various early-endosomal compartments that are characteristic of these cells – apical and basal early (sorting) endosomes (AEE/ASEs and BEE/BSEs, respectively), common recycling endosomes (CREs) and apical recycling endosomes [AREs; also called the subapical compartment (SAC)] (Hoekstra et al., 2004). The Rab11-positive ARE is localized to the subapical pole of fully polarized MDCK cells (Apodaca et al., 1994) and maintains a slightly elevated pH compared with the CRE (Wang et al., 2000); however, there remains some debate as to whether the ARE and CRE represent unique compartments or, alternatively, subdomains of a single class of recycling endosome (Mellman and Nelson, 2008).

Table 1.

Sorting mechanisms for constitutive and regulated apical protein delivery

Apical protein Sorting signal Presumed sorting mechanism MT motor References
Constitutive apical transport      
   Influenza HA   TM domain   Lipid-raft-association; raft clustering by MAL1, MAL2 and FAPP2   KIFC3   (Rodriguez-Boulan and Pendergast, 1980; Lin et al., 1998; Puertollano et al., 1999; Scheiffele, 1997)  
   GPI-anchored proteins (e.g. decay-accelerating F factor, folate receptor, GFP-GPI, 5′-nucleotidase, CEA)   GPI   Lipid-raft association and oligomerization; raft clustering by MAL1, MAL2, FAPP2 and galectin-4   ?   (Lisanti et al., 1989a; Brown et al., 1989; Brown and Rose, 1992; de Marco et al., 2006; Delacour et al., 2005; Hannan et al., 1993; Paladino et al., 2008; Paladino et al., 2004; Puertollano et al., 1999; Vieira et al., 2005)  
   p75 neurotrophin receptor   O-glycosylated stalk   Clustering by galectin-3; raft-independent   KIF5B   (Yeaman et al., 1997; Delacour et al., 2007; Jaulin et al., 2007)  
   Lactase-phlorizin hydrolase   O-glycans   Clustering by galectin-3; raft-independent   ?   (Delacour et al., 2006)  
   Sucrase-isomaltase   TM domain   Raft association   ?   (Jacob et al., 2000)  
   Endolyn   N-glycans   Raft-independent   ?   (Potter et al., 2004)  
   Rhodopsin   Cytoplasmic domain   Dynein light chain; raft-independent   Dynein   (Chuang and Sung, 1998; Tai et al., 1999)  
   pIgR   Apical and basolateral signals   Raft-independent; MAL2   ?   (Luton et al., 2009)  
   Syntaxins 1,2,3   FMDE in cytoplasmic domain   ?   ?   (Sharma et al., 2006)  
   Megalin   NPXY-like motifs   Clathrin adaptors?   ?   (Marzolo et al., 2003; Takeda et al., 2003)  
   Prominins 1,2   TM domain?   Lubrol-insoluble rafts?   ?   (Corbeil et al., 2001; Florek et al., 2007)  
   Soluble proteins (e.g. growth hormone, erythropoietin, hepatitis virus antigen)   N-glycans, protein motifs   Receptors?   ?   (Marzolo, 1997; Potter et al., 2006; Rodriguez-Boulan and Gonzalez, 1999; Scheiffele et al., 1995)  
   Na+-K+ ATPase, CD147, MCT1, CAR, apical proteins in renal PCT   Recessive apical signals   Absence of basolateral adaptors   ?   (Gundersen et al., 1991; Deora et al., 2005; Diaz et al., 2009) (Ryan Schreiner and E.R.-B., unpublished data)  
Regulated apical transport      
   Vacuolar H+ ATPase   ?   ? Regulated by metabolic pH, basal-membrane contacts and hensin   ?   (Brown et al., 2009; Schwartz and Al-Awqati, 2005)  
   Gastric H+ ATPase   TM domain   ? Regulated by gastric content and histamine   ?   (Muth and Caplan, 2003)  
   CFTR, Na-Pi cotransporter, GAT3   PDZ-binding motif   PDZ-domain-containing proteins   ?   (Muth and Caplan, 2003)  
   Aquaporin 2   ?   ? Regulated by ADH   ?   (Brown et al., 2009)  
   Na, PiIIa   ?   ? Regulated by PTH   ?   (Muth and Caplan, 2003)  
   Epithelial Na+ channel   ?   ? Regulated by aldosterone, vasopressin    (Muth and Caplan, 2003)  
   ATP transporters (BSEP, MDR1, MDR3, ABCG5/8)   TM domain?   Lubrol-insoluble rafts; regulated by various mechanisms   ?   (Hoekstra et al., 2004; Slimane et al., 2003; Wakabayashi et al., 2006)  
   ATP7B (Menkes) copper transporter   ?   ? Regulated by copper levels   ?   (Kim et al., 2008)  
Apical protein Sorting signal Presumed sorting mechanism MT motor References
Constitutive apical transport      
   Influenza HA   TM domain   Lipid-raft-association; raft clustering by MAL1, MAL2 and FAPP2   KIFC3   (Rodriguez-Boulan and Pendergast, 1980; Lin et al., 1998; Puertollano et al., 1999; Scheiffele, 1997)  
   GPI-anchored proteins (e.g. decay-accelerating F factor, folate receptor, GFP-GPI, 5′-nucleotidase, CEA)   GPI   Lipid-raft association and oligomerization; raft clustering by MAL1, MAL2, FAPP2 and galectin-4   ?   (Lisanti et al., 1989a; Brown et al., 1989; Brown and Rose, 1992; de Marco et al., 2006; Delacour et al., 2005; Hannan et al., 1993; Paladino et al., 2008; Paladino et al., 2004; Puertollano et al., 1999; Vieira et al., 2005)  
   p75 neurotrophin receptor   O-glycosylated stalk   Clustering by galectin-3; raft-independent   KIF5B   (Yeaman et al., 1997; Delacour et al., 2007; Jaulin et al., 2007)  
   Lactase-phlorizin hydrolase   O-glycans   Clustering by galectin-3; raft-independent   ?   (Delacour et al., 2006)  
   Sucrase-isomaltase   TM domain   Raft association   ?   (Jacob et al., 2000)  
   Endolyn   N-glycans   Raft-independent   ?   (Potter et al., 2004)  
   Rhodopsin   Cytoplasmic domain   Dynein light chain; raft-independent   Dynein   (Chuang and Sung, 1998; Tai et al., 1999)  
   pIgR   Apical and basolateral signals   Raft-independent; MAL2   ?   (Luton et al., 2009)  
   Syntaxins 1,2,3   FMDE in cytoplasmic domain   ?   ?   (Sharma et al., 2006)  
   Megalin   NPXY-like motifs   Clathrin adaptors?   ?   (Marzolo et al., 2003; Takeda et al., 2003)  
   Prominins 1,2   TM domain?   Lubrol-insoluble rafts?   ?   (Corbeil et al., 2001; Florek et al., 2007)  
   Soluble proteins (e.g. growth hormone, erythropoietin, hepatitis virus antigen)   N-glycans, protein motifs   Receptors?   ?   (Marzolo, 1997; Potter et al., 2006; Rodriguez-Boulan and Gonzalez, 1999; Scheiffele et al., 1995)  
   Na+-K+ ATPase, CD147, MCT1, CAR, apical proteins in renal PCT   Recessive apical signals   Absence of basolateral adaptors   ?   (Gundersen et al., 1991; Deora et al., 2005; Diaz et al., 2009) (Ryan Schreiner and E.R.-B., unpublished data)  
Regulated apical transport      
   Vacuolar H+ ATPase   ?   ? Regulated by metabolic pH, basal-membrane contacts and hensin   ?   (Brown et al., 2009; Schwartz and Al-Awqati, 2005)  
   Gastric H+ ATPase   TM domain   ? Regulated by gastric content and histamine   ?   (Muth and Caplan, 2003)  
   CFTR, Na-Pi cotransporter, GAT3   PDZ-binding motif   PDZ-domain-containing proteins   ?   (Muth and Caplan, 2003)  
   Aquaporin 2   ?   ? Regulated by ADH   ?   (Brown et al., 2009)  
   Na, PiIIa   ?   ? Regulated by PTH   ?   (Muth and Caplan, 2003)  
   Epithelial Na+ channel   ?   ? Regulated by aldosterone, vasopressin    (Muth and Caplan, 2003)  
   ATP transporters (BSEP, MDR1, MDR3, ABCG5/8)   TM domain?   Lubrol-insoluble rafts; regulated by various mechanisms   ?   (Hoekstra et al., 2004; Slimane et al., 2003; Wakabayashi et al., 2006)  
   ATP7B (Menkes) copper transporter   ?   ? Regulated by copper levels   ?   (Kim et al., 2008)  

ADH, antidiuretic hormone; CAR, coxsackie-adenovirus receptor; CEA, carcinoembryonic antigen; CFTR, cystic fibrosis transmembrane conductance regulator; FAPP, phosphatidylinositol 4-phosphate adaptor protein; GAT, γ-aminobutyric acid transporter; GFP, green fluorescent protein; GPI, glycosylphosphatidylinositol; HA, hemagglutinin; MAL, myelin and lymphocyte protein; MCT, monocarboxylate transporter; PCT, proximal convoluted tubule; PDZ domain, postsynaptic density protein-95/discs large/zonula occludens domain; pIgR, polymeric immunoglobulin receptor; PTH, parathyroid hormone

Fig. 1.

Apical trafficking routes in polarized MDCK cells. Known and suspected routes to and from apical PM domains are shown (blue arrows, blue numbers). For comparison, some of the exocytic and endocytic basolateral routes are shown (red arrows, red numbers). Rab proteins and SNAREs associated with specific routes are indicated. Route 1: lipid-raft-associated proteins, such as influenza HA, GPI-APs, sucrase-isomaltase and dipeptidylpeptidase 4, follow this route. Route 2: rhodopsin is the only protein suspected of following this route, which might be employed to reach the primary cilium. Route 3: hypothetical direct (non-transendosomal) route of raft-associated proteins to the primary cilium. This route might be used by soluble secretory proteins to avoid being targeted to the degradative pathway (Ed; endosome degradation). Route 4: the route followed by the non-raft-associated proteins p75 and sialomucin endolyn. Route 5: an apical variant of the VSVG protein is thought to pass through the CRE upon leaving the TGN. It is unknown whether subsequent transit through the ARE also occurs. Route 6: the pIgR uses a combination of basolateral sorting signals and transcytotic signals to travel first to the basolateral membrane and to then transcytose to the apical membrane via the BEE/BSE, CRE and ARE. In liver and intestinal epithelial cells, the transcytotic route is a major pathway for most apical proteins. Route 7: specific types of exosomes are exocytosed apically, suggesting the existence of apical and basolateral sorting mechanisms at late endosomes. Apical (Er; endosome recycling), degradative (Ed) and basolateral recycling (Br) routes are indicated. E, endosome; LE, late endosome; lys, lysosome; TJ, tight junction; ZA, zonula adherens.

Fig. 1.

Apical trafficking routes in polarized MDCK cells. Known and suspected routes to and from apical PM domains are shown (blue arrows, blue numbers). For comparison, some of the exocytic and endocytic basolateral routes are shown (red arrows, red numbers). Rab proteins and SNAREs associated with specific routes are indicated. Route 1: lipid-raft-associated proteins, such as influenza HA, GPI-APs, sucrase-isomaltase and dipeptidylpeptidase 4, follow this route. Route 2: rhodopsin is the only protein suspected of following this route, which might be employed to reach the primary cilium. Route 3: hypothetical direct (non-transendosomal) route of raft-associated proteins to the primary cilium. This route might be used by soluble secretory proteins to avoid being targeted to the degradative pathway (Ed; endosome degradation). Route 4: the route followed by the non-raft-associated proteins p75 and sialomucin endolyn. Route 5: an apical variant of the VSVG protein is thought to pass through the CRE upon leaving the TGN. It is unknown whether subsequent transit through the ARE also occurs. Route 6: the pIgR uses a combination of basolateral sorting signals and transcytotic signals to travel first to the basolateral membrane and to then transcytose to the apical membrane via the BEE/BSE, CRE and ARE. In liver and intestinal epithelial cells, the transcytotic route is a major pathway for most apical proteins. Route 7: specific types of exosomes are exocytosed apically, suggesting the existence of apical and basolateral sorting mechanisms at late endosomes. Apical (Er; endosome recycling), degradative (Ed) and basolateral recycling (Br) routes are indicated. E, endosome; LE, late endosome; lys, lysosome; TJ, tight junction; ZA, zonula adherens.

Apical trafficking machinery

Rab GTPases play crucial roles in defining apical trafficking routes, but many of the Rab proteins that regulate these routes remain to be identified or characterized in terms of their precise function. Rab11a is present on the ARE, where it interacts with myosin-Vb to modulate protein export to the apical domain (Lapierre et al., 2001; Roland et al., 2007). Rab8 and Rab10 have been proposed to participate in basolateral targeting from the CRE and therefore might be involved in the initial stages of the transcytotic route to the apical surface in MDCK cells (Babbey et al., 2006; Schuck et al., 2007). However, in intestinal cells, Rab8 has been found to regulate apical protein localization (Sato et al., 2007) and, indeed, Rab8 also interacts with myosin-Vb (Roland et al., 2009). Rab13 is involved in apical trafficking through its involvement in transport between the TGN and CRE (Nokes et al., 2008). Rab14 has been postulated to act in the transport of influenza hemagglutin (HA) to the apical surface (Kitt et al., 2008). Apical and basolateral endocytosis presumably depend on the same Rab proteins that have been studied in non-polarized cells, e.g. Rab4, Rab5 (for early stages) and Rab7 (for late stages) (Maxfield and McGraw, 2004; Perret et al., 2005), but this aspect has not been studied in detail in polarized cells.

Apical trafficking routes use specific vesicle-soluble NSF attachment protein (SNAP) receptors (v-SNAREs) or vesicle-associated membrane proteins (VAMPs) in the transport vesicles and target (t)-SNAREs in the PM; these proteins promote fusion by forcing the vesicle and target membranes close together through the formation of a four-helix bundle (reviewed in Sudhof and Rothman, 2009). An early in vitro study suggested that fusion of apical vesicles carrying newly synthesized proteins was independent of the SNARE machinery and was thus radically different from basolateral membrane fusion (Ikonen et al., 1995). However, this study was based, in part, on the lack of sensitivity of apical membrane fusion to N-ethylmaleimide treatment as well as to SNARE cleavage by tetanus and botulinum F neurotoxins, and subsequent experiments revealed that SNARE proteins that are involved in apical fusion are insensitive to these toxins (Galli et al., 1998). Rather, fusion of carriers that contain biosynthetic as well as transcytosing cargo was inhibited by botulinum neurotoxin E, which cleaves SNAP-23 (Apodaca et al., 1996; Low et al., 1998). Disruption of syntaxin-3 function using antibodies or overexpression, or disruption of its apical localization by nocodazole-mediated redistribution or ablation of its apical targeting signal (an FMDE motif in the surface of the helix bundle) disrupted apical delivery of both raft-associated and raft-independent apical markers, suggesting that this represents a general SNARE for apical membrane fusion (Kreitzer et al., 2003; Lafont et al., 1999; Sharma et al., 2006; ter Beest et al., 2005). Syntaxin-2 has been detected in both apical and basolateral PM domains, but its function in apical and basolateral targeting is not yet well defined. Two VAMPs have been identified that can associate with apical membrane SNAREs: VAMP7 [also known as TI-VAMP (so called because of its resistance to tetanus toxin)], and VAMP8 (Steegmaier et al., 2000). How these VAMPs function to regulate the distinct biosynthetic pathways described above in kidney cells is not known; however, in other epithelial cell types, VAMP7 and VAMP8 seem to differentially regulate membrane fusion of apically destined vesicles that originate, respectively, from the vectorial and transcytotic pathways (Pocard et al., 2007). In RPE and in some endocrine epithelia, syntaxin-1A and syntaxin-1B, which are structurally related to syntaxin-3, behave as apical t-SNAREs (Low et al., 2002).

Box 1. Multiple pathways and endosomal stations in apical trafficking routes

What is the physiological significance of having multiple pathways? Multiple pathways provide epithelial cells with the ability to upregulate individual transporters or groups of transporters in response to extracellular signals, which are often mediated by endocytic receptors. For example, regulated apical transporters that perform important functions in liver, intestine, kidney and other epithelia [e.g. vacuolar H+ ATPase, cystic fibrosis transmembrane regulator (CFTR), copper transporter ATP7B (Menkes), gastric H+ ATPase, aquaporin 2, Na+-dependent phosphate transporter IIa and amiloride-dependent epithelial sodium channel] are stored in specialized endosomal compartments until changes in the internal medium (pH, Cl, histamine, osmolarity, phosphate and sodium levels) promote their delivery to the apical surface via specific signaling pathways (Table 1). Different endosomal compartments differ in their lipid and protein composition, providing an opportunity to recruit trafficking machinery that is required to reach more distal compartments. In photoreceptors, targeting of rhodopsin to the AEE/ASE allows the recruitment of SARA (smad anchor for receptor activation), a resident of this compartment through its affinity for phosphatidylinositol 3-phosphate (PI3P) (Chuang et al., 2007). SARA is an adaptor protein that recruits syntaxin-3 and directs vesicular targeting to nascent outer discs, also highly enriched in PI3P. Other experiments indicate that rhodopsin also requires a targeting complex that includes Arf4 and Rab11 (Mazelova et al., 2009). Similarly, experiments in the intestinal cell line HT-29 by Huet and co-workers (Stechly et al., 2009) demonstrate a role for endocytosed galectin-4 in the final stages of trafficking of raft-associated apical proteins in intestinal cells. Polarized sorting in multiple organelles might also enhance the fidelity of sorting and allow cells to dynamically target proteins and lipids to either apical or basolateral surface domains depending on the need. A good example is the sorting of pIgR, which has to be delivered basolaterally in the biosynthetic pathway and in the endosomal system (when not bound to its ligand), and sorted apically in post-endocytic compartments (when bound to its ligand). Finally, the intersection of biosynthetic routes with endosomal compartments might provide a common intracellular depot from which to regulate the delivery of newly synthesized and recycling proteins to enable faster control over protein surface levels (e.g. during epithelial cell migration).

Apical trafficking routes

Figure 1 depicts the known and suspected trafficking routes to the apical surface of polarized MDCK cells. Route 1 is followed by PM proteins, such as glycosylphosphatidylinositol-anchored proteins (GPI-APs) and influenza HA, that are thought to associate with lipid rafts on the basis of their partitioning behavior in cold Triton X-100 (Rodriguez-Boulan et al., 2005). Their surface delivery is inhibited by ablation of the AEE/ASE through horseradish peroxidase (HRP)-catalyzed crosslinking (Cresawn et al., 2007). Fluorescence and pulse-chase analysis detected the passage of two newly synthesized PM proteins, lactase-phlorizin hydrolase and sucrase-isomaltase, successively through Rab4-, Rab8- and Rab11-positive endosomes. Their transport was inhibited by RNA interference against these Rabs, suggesting that these proteins reach the AEE/ASE first after leaving the TGN (Cramm-Behrens et al., 2008). Route 2 is probably used by rhodopsin, the major component of the outer segments of retinal photoreceptors, which constitute an enlarged version of the primary cilium that is present in most vertebrate cells (Singla and Reiter, 2006). The cilium represents a distinct subdomain of the apical surface that sequesters a unique complement of proteins and lipids. Biogenesis of the cilium requires the polarity complex comprising PDZ (postsynaptic density protein-95/discs large/zonula occludens)-domain-containing proteins Par3, Par6 and atypical protein kinase C; this complex also plays an important role in establishing the apical membrane during cell polarization and in apical delivery (Fan et al., 2004; Wang and Margolis, 2007). All resident proteins at the primary cilium and photoreceptor outer segments, which lack protein biosynthetic machinery, must be synthesized and sorted at the cytoplasm. On the basis of recent experiments in photoreceptors, rhodopsin, a non-lipid-raft-associated protein that is targeted apically in MDCK cells (Tai et al., 2001), is believed to reach the AEE/ASE (Chuang et al., 2007). Recent reports showed that transport to the cilium requires the BBsome, a multiprotein complex, and functional Rab8 (Loktev et al., 2008; Omori et al., 2008; Yoshimura et al., 2007). For transport to the primary cilium, the polarity protein Crumbs 3A interacts with PDZ domains in Par3 (Fan et al., 2004) (Sfakianos et al., 2007), which also interacts with kinesin-2, a motor protein that is involved in intraciliary transport (see also below). The exocyst, a multisubunit complex with myriad cellular functions, has also been implicated in cilia formation, as well as in apical trafficking (Oztan et al., 2007; Zuo et al., 2009). Moreover, lipid rafts have been shown to be involved in protein and lipid delivery to the cilium (Vieira et al., 2006). Thus, transport to the primary cilium might involve several of the apical routes discussed in this section. This includes a hypothetical direct pathway (route 3), which might also be used by apically secreted soluble proteins that must avoid the endocytic degradative pathway.

Route 4 is used by several proteins for which apical delivery is raft independent but glycosylation dependent, including endolyn (Cresawn et al., 2007) and p75 (Kerry Cresawn, Beth Potter, and O.A.W., unpublished data). The conclusion that these proteins traffic through the subapical ARE is based in part on the observation that a dominant-negative mutant of the motor protein myosin-Vb, which inhibits ARE-to-apical-membrane traffic (Lapierre et al., 2001), disrupts apical delivery of these proteins. Additionally, newly synthesized endolyn released from the TGN was found to colocalize with Rab11a, a marker of the ARE (Brown et al., 2000), and myosin-Vb in polarized MDCK cells. Importantly, conversion of endolyn to a GPI-anchored protein altered its biosynthetic itinerary into the AEE/ASE (route 1), because apical delivery of the protein was inhibited by ablation of these endosomes. In liver cells, route 4 is probably followed by ABC transporters, a group of cyclic-AMP-regulated multispan proteins that recycle back and forth between the bile canalicular (apical) membrane and the subapical compartment (Hoekstra et al., 2004; Wakabayashi et al., 2006). The subapical compartment, as defined by these studies, might include Rab11-positive AREs and Rab11-negative CREs. Route 5 traverses the CRE, as concluded on the basis of both CRE-ablation experiments with transferrin-HRP (the transferrin receptor is a marker of the CRE) and experiments using the expression of Rab8 and Rab10 mutants that disrupt exit from this compartment (Ang et al., 2004; Schuck et al., 2007). Because these Rab proteins regulate the basolateral recycling route (see Fig. 1), the single protein that represents this group, an apically targeted variant of vesicular stomatitis virus G (VSVG) protein, might be reaching the apical surface by transcytosis. Route 6 is the transcytotic route, which is followed by many apical proteins. The best-studied transcytotic pathway in MDCK cells is that used by the polymeric IgA receptor (pIgR), a key component of mucosal immunological defense (Mostov, 1994). The pIgR is believed to be targeted from the TGN to the basolateral membrane directly or via the BEE/BSE via a basolateral signal (Fig. 1, red arrows) (Orzech et al., 2000) and then traverse the CRE and ARE before arriving at the apical surface (Brown et al., 2000), guided by specific transcytosis signals (Luton et al., 2009). A less-well-characterized apical-to-basolateral transcytotic route also exists in these cells (Tzaban et al., 2009). Route 7 is used by apically released exosomes that result from the fusion of late endosomes with the apical surface. This route is suggested by recent evidence that shows that epithelial cells can release different exosomes from apical and basolateral PM domains (Lakkaraju and Rodriguez-Boulan, 2008); the underlying mechanisms, however, are currently unknown.

Apical sorting signals and mechanisms

Lipid rafts in apical targeting

Lipid rafts are tightly linked to the history of epithelial polarity. The lipid-raft hypothesis was initially proposed to explain the sorting of apical proteins in epithelial cells. On the basis of previous observations that showed that cholesterol and glycosphingolipids were enriched in the apical domain, and that membrane domains enriched in these lipids have a tendency to self-segregate themselves as `rafts' in the plane of the membrane, it was proposed that lipid rafts could act as primary sorters of apical proteins at the Golgi complex by promoting their incorporation into apically directed carrier vesicles (van Meer and Simons, 1988). Recently, in their work in budding yeast, Simons and co-workers (Klemm et al., 2009) provided a missing link – the first proof that sorting of lipid rafts can occur intracellularly at the level of the TGN. However, this has yet to be shown for epithelial cells.

Early support for the raft hypothesis was provided by experiments showing that all endogenous GPI-APs expressed by polarized MDCK cells were apically localized (Lisanti et al., 1988), that chimeric GPI-APs were directed to the apical membrane (Brown et al., 1989; Lisanti et al., 1989b) and that GPI-APs were incorporated into detergent-resistant membrane (DRM) domains as they reached the Golgi complex (Brown and Rose, 1992). Additional support for the raft hypothesis was provided by the finding that the transmembrane domain of the envelope glycoprotein HA of the apically budding influenza virus (Rodriguez-Boulan and Pendergast, 1980) seemed to specifically promote apical targeting through its affinity for DRMs (Lin et al., 1998; Scheiffele, 1997). Unlike in MDCK cells, in which GPI-APs are delivered vectorially to the apical surface (Lisanti et al., 1990; Paladino et al., 2006), in hepatocytes most GPI-APs are targeted first to the basolateral surface and subsequently transcytosed to the apical domain (Schell et al., 1992; Slimane et al., 2003). The underlying mechanisms are starting to emerge and are discussed later in this Commentary.

Subsequent experiments revealed that GPI anchors are not all the same and do not contribute to apical sorting in every cell. In Fischer rat thyroid (FRT) cells, most GPI-APs were found to be basolateral (Zurzolo et al., 1993); the molecular mechanism remains unknown. In MDCK cells, the prion protein, a GPI-AP, is targeted basolaterally (Sarnataro et al., 2002). Furthermore, the GPI anchor by itself (i.e. without any attached protein) was detected at similar levels at both apical and basolateral domains of the MDCK PM (Van't Hof et al., 1995). This might be explained by the fact that free GPI anchors dwell in the cytoplasmic leaflet of the PM, and TJs do not function as a fence at this level (van Meer et al., 1986). Additional studies in both MDCK and FRT cells showed that both apical and basolateral GPI-APs can associate with DRMs, further indicating that lipid-raft association by itself is not sufficient for apical delivery (Paladino et al., 2004; Paladino et al., 2007). Early experiments with a clonal MDCK cell line resistant to killing by the lectin concanavalin A (ConA) (Meiss et al., 1982) suggested an explanation. ConA-resistant MDCK cells were unable to correctly sort GPI-APs to the apical surface, and fluorescence recovery after photobleaching (FRAP) experiments showed that, unlike wild-type MDCK cells, they were unable to `cluster' GPI-APs into immobile aggregates, suggesting that “correctly sorted GPI-APs are clustered before arrival at the apical surface” (Hannan et al., 1993). More recent experiments by Zurzolo and co-workers (Paladino et al., 2004) provided additional evidence for a key clustering event in apical targeting of GPI-APs. GPI-anchored green fluorescent protein (GFP-GPI) was found to be apically polarized only when the intrinsic dimerizing activity of GFP was preserved; sedimentation experiments further supported the concept that GPI-anchored proteins reaching the apical membrane are present as larger oligomers than those reaching the basolateral membrane.

Recent results by Kinoshita et al. (Kinoshita et al., 2008) add a new level of complexity, as they demonstrated that GPI anchors experience considerable remodeling of their fatty-acid chains after they are linked to the protein in the endoplasmic reticulum (ER). Remodeling involves the replacement of the unsaturated fatty-acid chains that are present in free GPIs by saturated fatty-acid chains, which is carried out by specialized enzymes present in the Golgi complex [post-GPI attachment protein 2 (PGPA2) and PGPA3] following deacylation from inositol by PGPA1 in the ER (Maeda et al., 2007). This remodeling is required for the attachment of GPI-APs to rafts, thus providing an alternative explanation to the hypothesis that GPI-APs become detergent insoluble in the Golgi complex because lipid rafts are assembled there (Brown and Rose, 1992). Variations in the sensitivity of GPI anchors to these enzymes might determine variations in the strength of raft association between different GPI-APs. Indeed, the GPI attachment sequences have recently been demonstrated to play important roles in directing protein fate. GFP reporter constructs fused to the GPI attachment signals of apical and basolateral GPI-APs (from the folate receptor and prion protein, respectively) were differentially targeted in polarized MDCK cells and also exhibited differences in their ability to form high-molecular-weight oligomers (Paladino et al., 2008). Moreover, loading MDCK cells with cholesterol enabled basolaterally directed GPI-APs to form oligomers, altered their diffusional mobility in the Golgi complex and caused their partial rerouting to the apical surface (Paladino et al., 2008) (Lebreton et al., 2008). Thus, there is some synergy between the lipid environment that is accessible to a GPI-AP and the propensity to form oligomers that is clearly important for apical targeting.

Carbohydrates as apical sorting determinants

Both N- and O-linked glycans have been implicated in biosynthetic sorting of a subset of apical proteins (reviewed in Potter et al., 2006) (Table 1). A role for glycosylation in polarized sorting was first suggested by observations that some membrane and secretory proteins are missorted in lectin-resistant MDCK cells that display altered glycosylation profiles (Le Bivic et al., 1993; Meiss et al., 1982; Parczyk and Koch-Brandt, 1991). Subsequent experiments showed that introducing N-glycosylation consensus sites into the rat growth hormone (rGH) sequence changed its sorting pattern (Scheiffele et al., 1995). Whereas nonglycosylated (wild type) rGH expressed in polarized MDCK cells was secreted with a slight preference into the basolateral medium, the addition of one glycosylation site reversed the polarity of secretion, and the addition of a second site resulted in an apical:basolateral secretion ratio of ∼9:1.

O-glycosylation can also apparently mediate biosynthetic apical sorting, although the lack of selective perturbants of O-glycosylation has made this difficult to confirm directly. Truncation of the juxtamembrane O-glycosylated stalk of the apical p75 neurotrophin receptor results in decreased polarity of surface delivery (Yeaman et al., 1997). This stalk region was also required for apical secretion of a truncated form of p75, suggesting that a membrane tether is not required for recognition of the sorting information in this portion of the protein. Similarly, the appendage of an O-glycosylated stalk, important for apical sorting of sucrase-isomaltase to the secreted protein rGH, caused the chimera to be secreted predominantly into the apical medium (Alfalah et al., 1999; Spodsberg et al., 2001). In this case, O-glycosylation led to the association of rGH with detergent-resistant microdomains, as also observed for sucrase-isomaltase.

In many cases, glycosylation of specific sites on a protein is required to mediate apical sorting. For example, efficient apical delivery of the sialomucin endolyn along both the biosynthetic and recycling pathways requires glycosylation at two of its eight N-glycosylation sites (Potter et al., 2006; Potter et al., 2004). In some instances, the core region of the N-glycan is required (Wagner et al., 1995), whereas sorting of other proteins depends on terminal processing of the glycans (Potter et al., 2006). Whereas the endolyn extracellular domain is highly O-glycosylated, these two key N-glycans are located on a disulfide-bonded loop in the protein that lacks O-glycosylation sites. Similarly, one of the three N-glycans on erythropoietin is required for its efficient apical secretion (Kitagawa et al., 1994). In the case of O-glycosylation, specific amino acid sequences within the stalk region might also play a role in the apical sorting signal, as suggested by the observations of Jacob et al. (Jacob et al., 2000), who found that the N-terminal but not the C-terminal region of the O-glycosylated stalk of sucrase-isomaltase was sufficient to direct apical delivery.

Two models have been proposed to explain the role of carbohydrates in apical sorting (Rodriguez-Boulan and Gonzalez, 1999). The first proposes that carbohydrates are necessary to place proteins in a transport-competent conformation that is required for their forward progress along the biosynthetic pathway. Evidence for this idea comes from the observation that inhibiting glycosylation of some apical proteins results in their retention in the TGN (Gut et al., 1998). Alternatively, the existence of a sorting receptor that recognizes carbohydrates or carbohydrate-dependent conformations of some proteins has been proposed. An early candidate for this role was vesicular integral membrane protein 36 (VIP36), a transmembrane lectin originally identified in detergent-insoluble extracts of post-Golgi carriers (Fiedler et al., 1994). However, later studies suggested that this protein is localized to the early biosynthetic pathway rather than the TGN (Fullekrug et al., 1999).

More recently, galectin-3 and galectin-4 have been proposed to participate in the apical localization of apical proteins in renal and intestinal cells. Galectins are cytosolic proteins that are released from cells via a nonclassical secretion mechanism that then bind to cell-surface proteins and can be internalized into endosomal compartments (Hughes, 2004). Knockdown or knockout of the apically secreted galectin-3 inhibits the delivery of the raft-independent protein p75 (Delacour et al., 2006; Delacour et al., 2008; Lindstedt et al., 1993), whereas knockdown of galectin-4 in intestinal cells inhibits lipid-raft association of raft-associated proteins [the GPI-APs carcinoembryonic antigen (CEA) and CD59, and the transmembrane protein dipeptidyl peptidase IV (DPPIV)] and prevents their apical delivery (Delacour et al., 2005; Stechly et al., 2009). Interestingly, the apical addition of galectin-4 to intestinal cells depleted of this protein reconstituted apical delivery of DPPIV, which otherwise was accumulated in a post-Golgi endosomal compartment (Stechly et al., 2009). The experiments from the Huet laboratory support a model in which apically secreted and internalized galectin-4 interacts with raft-associated proteins in a post-Golgi early-endosomal compartment and, furthermore, that this interaction is required for apical delivery. This laboratory also showed that galectin-4 interacted with high affinity with both lipid-raft glycosphingolipids and with bi-, tri- and tetra-antennary complex N-linked carbohydrates containing N-acetyl-lactosamine (Morelle et al., 2009; Stechly et al., 2009). These carbohydrates were found to be enriched in the apical membrane, which provides important support for the hypothesis that galectin-4 is involved in the sorting of apical membrane proteins and lipids.

Other mechanisms that result in apical protein localization

Some epithelial cell types lack clathrin adaptors that are involved in basolateral protein targeting, such as the epithelial-specific adaptor AP1B (Ohno et al., 1999). This results in the apical localization of subsets of cognate basolateral proteins (Fig. 2; Table 1). Such is the case with epithelia that lack this adaptor: the kidney cell line LLC-PK1 (Folsch et al., 1999), the neuroepithelium RPE, which is responsible for the blood-retinal barrier and provides crucial support functions for photoreceptors (Diaz et al., 2009), and the kidney's proximal convoluted tubule (Ryan Schreiner and E.R.-B., unpublished data). Knockdown of AP1B in MDCK cells results in the apical localization of the basolateral protein coxsackie-adenovirus receptor (CAR), leading to increased apical infectivity by adenoviruses, which use CAR as a receptor to invade epithelial cells (Diaz et al., 2009). Apical localization of the basolateral proteins results from missorting at the level of recycling endosomes, where AP1B localizes preferentially (Gan et al., 2002; Gravotta et al., 2007). Other cognate basolateral proteins are also expressed apically in the RPE, such as Na+-K+ ATPase (Gundersen et al., 1991), and monocarboxylate transporter 1 (MCT1) and its chaperone CD147 (Deora et al., 2005), although the lack of AP1B does not seem to be responsible for their reversed apical polarity. In hepatocytes, however, which also lack AP1B expression, basolateral protein sorting is preserved. This might in part reflect the paucity of vectorial biosynthetic trafficking pathways to the apical surface; however, in these cells it is not clear how the fidelity of basolateral recycling of cargos that are AP1B-dependent in other cell types (e.g. transferrin receptor) is maintained.

Fig. 2.

Apical localization resulting from absence of a basolateral clathrin adaptor. (A) In epithelial cells that express the clathrin adaptor AP1B (e.g. MDCK cells), basolateral proteins are recycled efficiently back to the basolateral membrane owing to the presence of this adaptor in the CRE. In these cells, basolateral proteins never reach the Rab11-positive ARE. The exception to this rule is E-cadherin, which seems to transit through a Rab11-positive compartment in its biosynthetic route (Desclozeaux et al., 2008). (B) The situation is different in non-polarized cells, in which the transferrin receptor recycles to the PM from Rab11-positive recycling endosomes. When AP1B is missing, transferrin receptors are missorted to the apical surface, probably through the ARE. This probably also happens in some native epithelia that lack AP1B, such as the RPE (Diaz et al., 2009) and renal proximal tubule (Ryan Schreiner and E.R.-B., unpublished data), although the transferrin receptor is basolaterally targeted in hepatocytes (Levine and Woods, 1990). Red arrows represent basolateral routes. Blue arrows represent an apical route that develops when AP1B is not expressed. Br, basolateral recycling route.

Fig. 2.

Apical localization resulting from absence of a basolateral clathrin adaptor. (A) In epithelial cells that express the clathrin adaptor AP1B (e.g. MDCK cells), basolateral proteins are recycled efficiently back to the basolateral membrane owing to the presence of this adaptor in the CRE. In these cells, basolateral proteins never reach the Rab11-positive ARE. The exception to this rule is E-cadherin, which seems to transit through a Rab11-positive compartment in its biosynthetic route (Desclozeaux et al., 2008). (B) The situation is different in non-polarized cells, in which the transferrin receptor recycles to the PM from Rab11-positive recycling endosomes. When AP1B is missing, transferrin receptors are missorted to the apical surface, probably through the ARE. This probably also happens in some native epithelia that lack AP1B, such as the RPE (Diaz et al., 2009) and renal proximal tubule (Ryan Schreiner and E.R.-B., unpublished data), although the transferrin receptor is basolaterally targeted in hepatocytes (Levine and Woods, 1990). Red arrows represent basolateral routes. Blue arrows represent an apical route that develops when AP1B is not expressed. Br, basolateral recycling route.

A mysterious apical protein (in terms of apical sorting mechanisms) is megalin, a `velcro' receptor in the proximal nephron that uses endocytosis to recover proteins that reach the kidney space through the glomerular filtration membrane, thus preventing their loss in the urine. Megalin resembles basolateral proteins in that it recycles quickly between the PM and endosomal compartments, dependent on NPXY-type tyrosine sorting signals that resemble PM endocytic motifs. Interestingly, its apical sorting might also be guided by a cytoplasmic sequence that resembles the NPXY tyrosine motif (Marzolo et al., 2003; Takeda et al., 2003), a puzzling finding that requires further exploration.

Other apical PM proteins also use cytoplasmic determinants as apical signals but the sorting mechanisms involved are poorly understood (Muth and Caplan, 2003). The ABC transporters MDR1, MDR2, Mrp2 and the sister of P-glycoprotein are multispan proteins that are targeted vectorially from the TGN to the apical membrane of liver hepatocytes via a route that probably involves the ARE and TX-100-soluble, Lubrol-insensitive lipid rafts (Slimane et al., 2003; Wakabayashi et al., 2006). Two other proteins that have been shown to associate with such lipid rafts are prominin-1 and prominin-2, penta-spanning proteins that associate with microvilli (Florek et al., 2007). Prominin-1 is targeted apically whereas prominin-2 is not sorted by epithelial cells. Much more work is necessary to elucidate the role of Lubrol-resistant lipid rafts in apical trafficking.

Clustering and apical trafficking

Growing evidence supports the concept that correct sorting of both raft-associated and raft-independent apical PM proteins also requires clustering of the cargo proteins (Fig. 3). The mechanisms and molecular purpose of clustering, however, remain poorly understood. Early evidence for clustering as a requirement for apical targeting of GPI-anchored proteins was obtained in MDCK cells resistant to killing by the lectin ConA (Hannan et al., 1993). In these cells, GPI-APs lose their apical polarity and are unable to cluster before reaching the PM. The carbohydrate changes in ConA-resistant MDCK cells have not yet been investigated. Recent data from the Zurzolo laboratory show that genetic deletion of O-glycans and N-glycans from apical GPI-APs does not disrupt their oligomerization and apical targeting; however, treatment of the host cells (wild-type MDCK) with tunicamycin does (Catino et al., 2008). The authors' interpretation of this finding is that carbohydrates are required not as a sorting signal on the GPI-AP cargo, but rather for the sorting function of a putative sorter molecule.

Fig. 3.

Clustering events in apical trafficking routes. Trafficking routes for the indicated cargo proteins are diagrammed. Various candidate molecules or complexes that might contribute to clustering in MDCK (A), intestinal (B) and liver (C) epithelial cells are noted within each cell type. Routes 2 (rhodopsin) and 5 (VSVG-apical variant) are hypothetical in intestinal cells. Br, basolateral recycling route; E, endocytic route; TJ, tight junction; ZA, zonula adherens.

Fig. 3.

Clustering events in apical trafficking routes. Trafficking routes for the indicated cargo proteins are diagrammed. Various candidate molecules or complexes that might contribute to clustering in MDCK (A), intestinal (B) and liver (C) epithelial cells are noted within each cell type. Routes 2 (rhodopsin) and 5 (VSVG-apical variant) are hypothetical in intestinal cells. Br, basolateral recycling route; E, endocytic route; TJ, tight junction; ZA, zonula adherens.

Several molecules have emerged that might participate in the lipid-raft clustering events that are involved in apical trafficking. The myelin and lymphocyte (MAL) proteolipids are ∼20 kD tetraspanning raft-associated membrane proteins that have been implicated as potent regulators of apical delivery. MAL1 is expressed in MDCK cells and recycles between the Golgi complex and the apical membrane (Puertollano and Alonso, 1999), and its knockdown results in decreased apical transport and basolateral missorting of influenza HA and GPI-APs (Cheong et al., 1999; Puertollano et al., 1999). MAL2 recycles between the ARE and the apical membrane, and has been implicated in transcytotic transport of GPI-APs and single-pass transmembrane apical proteins in hepatocytes (de Marco et al., 2006). Interestingly, expression of MAL1 in hepatocytes (which normally lack it) promotes vectorial delivery of GPI-APs and single-pass transmembrane proteins from the TGN to the apical surface, suggesting that the MAL1:MAL2 ratio of different epithelia might control the use of the direct and indirect routes by apical proteins (Ramnarayanan et al., 2007). The transcytotic mechanism might also be regulated by the levels of surface E-cadherin (Theard et al., 2007).

Another candidate lipid-raft clustering agent is phosphatidylinositol 4-phosphate adaptor protein 2 (FAPP2), a glucosylceramide-transfer protein that has a pivotal role in the synthesis of complex glycosphingolipids and localizes to the TGN through the binding of phosphatidylinositol 4-phosphate and the small GTPase ARF1 (D'Angelo et al., 2007). Knockdown of FAPP2 with siRNA inhibits apical trafficking of YFP-GPI (Vieira et al., 2005) and disrupts the apical polarity of Forsmann antigen, an apical glycolipid (Vieira et al., 2006). Yet another lipid-raft clustering agent is galectin-4, which can interact with glycosphingolipids and N-linked complex carbohydrates through multiple binding sites (Delacour et al., 2005; Stechly et al., 2009). Knockdown of galectin-4 with siRNA in intestinal enterocytes results in disruption of the apical polarity of CEA, a GPI-anchored protein, and the transmembrane protein DPPIV. Galectin-4, but not MAL1, is expressed by intestinal cells; raft clustering, therefore, might be performed by different agents in different epithelial cells.

Clustering is also required for correct apical sorting of raft-independent apical proteins, a process that might involve galectin-3 (Delacour et al., 2006; Delacour et al., 2007; Delacour et al., 2008). Whereas galectin-4 is believed to meet apical proteins in a post-Golgi endosomal compartment (Stechly et al., 2009), the exact site where galectin-3 meets apical cargo has not been established. Whether clustering is important for apical sorting of secreted proteins is also not known.

Apical trafficking and the cytoskeleton

Key roles of MTs and MT motors

Subconfluent MDCK cells display a centrosomal arrangement of MTs, similar to that found in migrating cells, with very dynamic MTs extending their plus ends towards the cell cortex. By contrast, confluent MDCK and Caco-2 (human intestinal) cells display an epithelial-specific arrangement of MTs, with a large population of stable MTs organized vertically along the cell cortex, with their minus ends facing the apical membrane and their plus ends facing the basal membrane (Bacallao et al., 1989; Gilbert et al., 1991) (Fig. 4). Musch and collaborators (Cohen et al., 2004a; Cohen et al., 2004b) have shown that the polarity protein kinase Par1b (also known as EMK1) controls the epithelial-specific reorganization of MTs. Interestingly, whereas knockdown of Par1b or blocking its kinase activity prevents the reorganization of MTs that are associated with polarization, Par-1 overexpression promotes a dramatic repolarization of MDCK cells into a liver-like phenotype, with bile-canaliculi-like lumina at the lateral membrane and liver-specific MT arrays. Moreover, these cells now adopt transcytotic routes to deliver newly synthesized apical membrane proteins (Cohen et al., 2004b). Thus, MT organization clearly modulates the biosynthetic routing.

Recently, Kreitzer and co-workers (Jaulin et al., 2007) have shown that polarized MDCK cells have a small but very dynamic population of MTs emerging from the MT-organizing center (MTOC) that preferentially grow their plus ends towards the apical surface (Fig. 4). It is likely that the dynamic MT population originates the stable MTs through capture of their negative ends by the junctional regions of the lateral PM. This is suggested by the observation that expression of E-cadherin and/or membrane-bound α-catenin in fibroblasts stabilize non-centrosomal MTs (Chausovsky et al., 2000; Shtutman et al., 2008).

Fig. 4.

MT motors and myosins in apical trafficking. MTs are organized into two populations in polarized epithelial cells: stable cortical MTs with the minus ends facing apically, and dynamic MTs that originate at the MTOC, with the plus ends facing apically. Actin filaments have very different organizations under the apical surface (apical actin), under the lateral surface (lateral actin) and at the perinuclear region (peri-Golgi actin). The minus-end kinesin KIFC3 has been implicated in the transport of influenza HA, a lipid-raft-associated protein (route 1). Dynein participates in the transport of rhodopsin from the TGN (route 2). The plus-end kinesin KIF5B participates in the transport of p75 (route 4). Myosin-II and myosin-VI have been implicated in basolateral transport from the TGN and CRE, respectively. Myosin-IA has been implicated in post-Golgi transport, transcytosis and transport across the apical actin. E, endocytic route; Ed, endocytic degradative route; Er, endocytic recycling route.

Fig. 4.

MT motors and myosins in apical trafficking. MTs are organized into two populations in polarized epithelial cells: stable cortical MTs with the minus ends facing apically, and dynamic MTs that originate at the MTOC, with the plus ends facing apically. Actin filaments have very different organizations under the apical surface (apical actin), under the lateral surface (lateral actin) and at the perinuclear region (peri-Golgi actin). The minus-end kinesin KIFC3 has been implicated in the transport of influenza HA, a lipid-raft-associated protein (route 1). Dynein participates in the transport of rhodopsin from the TGN (route 2). The plus-end kinesin KIF5B participates in the transport of p75 (route 4). Myosin-II and myosin-VI have been implicated in basolateral transport from the TGN and CRE, respectively. Myosin-IA has been implicated in post-Golgi transport, transcytosis and transport across the apical actin. E, endocytic route; Ed, endocytic degradative route; Er, endocytic recycling route.

Early pharmacologic studies on the participation of MTs in apical-basolateral trafficking yielded conflicting results (reviewed in Musch, 2004). Rindler et al. first showed that pharmacologic disassembly of MTs in MDCK cells resulted in the loss of apical budding polarity of influenza virus, whereas the basolateral budding polarity of VSVG was not affected (Rindler et al., 1987). Subsequent pharmacologic experiments suggested that both dynein and kinesin were involved in the apical delivery of the apical viral glycoprotein influenza HA (Lafont et al., 1994; Tai et al., 2001). However, other reports found no effect of MT disassembly on virus budding polarity, or found a slight retardation in the apical trafficking of influenza HA without disruption of its polarized delivery (Salas et al., 1986; van Zeijl and Matlin, 1990). In retrospect, these apparently contradictory findings might be explained by the existence of both stable and dynamic MTs (Fig. 4), which might have been disrupted to different extents by the protocols used by different laboratories.

More recent studies demonstrate that different MT motors are remarkably selective for the transport of specific apical cargos. Chuang and Sung provided the first demonstration of this important point by showing that the apical transport of rhodopsin in MDCK cells depends on cytoplasmic determinants that interact with dynein light chains (Chuang and Sung, 1998) (Fig. 4, route 2). Hirokawa and co-workers later showed that KIFC3, a minus-end kinesin, targets influenza HA and annexin XIIIb to the apical surface (Noda et al., 2001) (Fig. 4, route 1). Kreitzer et al. showed that exit from the TGN of the apical protein p75 can be blocked by anti-kinesin antibodies (Kreitzer et al., 2000) and, more recently, identified KIF5B as the plus-end kinesin involved (Jaulin et al., 2007). It is likely that transport of p75 by KIF5B uses dynamic MTs that are emerging from the MTOC (Fig. 4, route 4). Interestingly, a different kinesin is required for p75 transport to the PM in non-polarized MDCK cells (Geri Kreitzer, personal communication). The diversity of MT motors that participate in apical trafficking might explain puzzling observations in MDCK cell variants and hepatocytes that fail to reorganize their MTs during polarization but nonetheless can polarize normally under such conditions (Grindstaff et al., 1998; Wojtal et al., 2007)

Cooperation of MT motors and the actin cytoskeleton in apical trafficking

An emergent theme in cell biology is the cooperation between MTs and actin cytoskeletons in cell migration, endocytosis, exocytosis and other cellular functions. Apical trafficking provides a growing number of examples of such cooperation. Plus-end and minus-end kinesins, as well as cytoplasmic dynein, play important and very selective roles in the transport of different apical cargo molecules. So too do the actin cytoskeleton and cytoplasmic myosins, several of which have been involved in apical trafficking (Fig. 4). Recent work has highlighted the cooperation of the actin cytoskeleton and MT motors in the exit of apical cargos from the TGN (Fig. 5). Given that mammalian cells might express over 50 kinesins (Jaulin et al., 2007) and over 35 myosins (Woolner and Bement, 2009), it is likely that many more examples of this cooperation will be found in the future. We propose the hypothesis that the cargo-clustering event required for apical trafficking might facilitate the recruitment of specific MT motors and/or myosin motors required for exit from sorting compartments or delivery to the PM. Some MT motors (e.g. KIF1A) are thought to acquire the ability to interact with lipid rafts upon dimerization (Klopfenstein et al., 2002). Similarly, some myosin motors, such as myosins I, V and VI, have the ability to oligomerize upon physiological stimuli and perform a variety of `unconventional' functions, such as tethering vesicles, organizing actin to bend membranes or regulating the activity of MT motors (Woolner and Bement, 2009). These activities could contribute to the assembly of functional sorting rafts, recruitment of sorting machinery and/or the formation of specific transport vesicles in the TGN or CRE (Fig. 5).

Fig. 5.

Cooperation between the actin and MT cytoskeletons in TGN exit. Recent experiments suggest that a specialized organization of the peri-Golgi actin is regulated by LIMK1, a Golgi-resident enzyme that inactivates the actin-severing function of cofilin by phosphorylation. The short and branched actin filaments, bound to the Golgi membrane via ARF1, might be used by a Golgi myosin to bend the membrane and initiate the formation of short tubules carrying p75. The tubules are elongated by kinesin 5B, a plus-end motor, moving on the MTs. Dynamin 2, recruited via syndapin II and cortactin, participates in the fission of p75-containing vesicles and tubules that then move towards the apical surface. Modified from Salvarezza et al. (Salvarezza et al., 2008).

Fig. 5.

Cooperation between the actin and MT cytoskeletons in TGN exit. Recent experiments suggest that a specialized organization of the peri-Golgi actin is regulated by LIMK1, a Golgi-resident enzyme that inactivates the actin-severing function of cofilin by phosphorylation. The short and branched actin filaments, bound to the Golgi membrane via ARF1, might be used by a Golgi myosin to bend the membrane and initiate the formation of short tubules carrying p75. The tubules are elongated by kinesin 5B, a plus-end motor, moving on the MTs. Dynamin 2, recruited via syndapin II and cortactin, participates in the fission of p75-containing vesicles and tubules that then move towards the apical surface. Modified from Salvarezza et al. (Salvarezza et al., 2008).

Rho GTPases and actin dynamics in apical delivery

Beneath the apical and lateral surfaces of epithelial cells is a dense network of actin filaments that must be penetrated by transport carriers destined for these domains. Their different organization is regulated by a variety of proteins, such as ezrin, villin and a CD317 (tetherin)-Rich2 complex for the subapical network and the E-cadherin system for the lateral network (Fievet et al., 2007; Rollason et al., 2009). A specialized organization of the actin cytoskeleton is also found in the peri-Golgi region (Percival et al., 2004). Thus, it is not surprising that regulation of actin dynamics can profoundly affect apical and basolateral protein trafficking. The small G proteins of the mammalian Rho GTPase family (Rac, Rho and Cdc42) are key regulators of actin dynamics at various intracellular sites (Ridley, 2006). The expression of constitutively active but not dominant-negative Rac1 in polarized MDCK cells selectively inhibited the delivery of newly synthesized and transcytosing proteins to the apical surface, which was due to retention of apically destined proteins in a subapical aggregate that also contained various endocytic markers (Jou et al., 2000). Additionally, the expression of Cdc42 mutants in polarized MDCK cells stimulated export of apical proteins from the TGN and surface delivery of apical proteins (Musch et al., 2001; Rojas et al., 2001), but inhibited TGN export, biosynthetic delivery and polarity of basolateral proteins (Cohen et al., 2001; Kroschewski et al., 1999; Musch et al., 2001).

The differential regulation of apical and basolateral routes by Rho GTPases might be explained by the use of different downstream effectors. For example, Rho isoforms regulate the activation of LIM kinases, which phosphorylate and inhibit the actin-severing protein cofilin, whereas Cdc42 is known to stimulate N-WASP–Arp2/3-mediated actin comet formation. Apical delivery kinetics of raft-associated influenza HA were found to be enhanced under conditions that stimulate formation of N-WASP–Arp2/3-dependent actin comets and slowed by inhibitors of comet formation (Guerriero et al., 2006) (Fig. 1, route 1). The non-raft-associated apical protein p75 is not regulated by this mechanism but, instead, is regulated by LIM kinase 1 (LIMK1) and cofilin, which probably regulate p75 by promoting the organization of a specific actin organization in the perinuclear region (Salvarezza et al., 2009) (Fig. 1, route 4). This organization is required for the segregation of p75 from other TGN proteins and for the dynamin-dependent fission of p75-containing tubules (Salvarezza et al., 2009). Interestingly, TGN export of GPI-anchored proteins does not seem to be regulated by LIMK1 and, unlike p75, is not inhibited by actin-depolymerizing drugs (Jacob et al., 2003; Lazaro-Dieguez et al., 2007). However, these studies were performed in non-polarized MDCK cells and the actin dependence of these routes might be different in polarized cells (Lebreton et al., 2008). It is also of interest to point out that, whereas at the PM dynamin is required for both clathrin-dependent and clathrin-independent endocytosis, in exocytic routes it is only required by some apical routes and not by basolateral routes, which are in most cases clathrin dependent (Bonazzi et al., 2005; Kreitzer et al., 2000; Liu et al., 2008). An unresolved issue is whether cofilin at the TGN regulates actin polymerization via severing and Arp2/3-dependent polymerization of branched filaments, as is thought to occur in lamellipodia (DesMarais et al., 2005).

Lastly, growing evidence indicates that myosins play specific roles in apical and basolateral trafficking, although the exact mechanism might involve non-conventional functions of myosins as tethers, regulators of MT motors and/or regulators of filament organization (Woolner and Bement, 2009). As discussed above, myosin-Vb is involved in trafficking between the ARE and the apical membrane, and is regulated by Rab11. Recently, it has been shown that mutations in the gene encoding myosin-Vb are a major cause of microvillus inclusion disease, a lethal disease that is characterized by the absence of surface microvilli and the accumulation of large intracellular vacuoles in enterocytes (Muller et al., 2008). A similar phenotype is observed in Rab8-knockout mice (Sato et al., 2007), and previous work has shown evidence of interactions between Rab8 and myosin-Vb (Roland et al., 2007). Intracellular vacuoles, also called intracellular lumens (Remy, 1986) or VACs (vacuolar apical compartments) (Vega-Salas et al., 1988), are often observed in transformed epithelial cells and in epithelial cells deprived of cell-cell contacts. They are thought to be normal intermediates in the biogenesis of the apical surface in epithelial and endothelial cells, and often result from the fusion of apical-transport vesicles under conditions of reduced delivery to the apical PM (Verges et al., 2007).

Another myosin that has been implicated in apical transport at the Golgi and post-Golgi levels is myosin-I. Cell-fractionation assays demonstrated its presence in post-Golgi vesicles in developing intestinal cells (Fath and Burgess, 1993), suggesting a role in apical transport. Expression of a truncated form of myosin-I in intestinal cells inhibited transcytosis of an apical protein (Durrbach et al., 2000), and recent indirect evidence, based on depletion of a kinase for myosin-IA, suggests a role in apical trafficking of sucrase-isomaltase (Heine et al., 2005).

Myosins have also been involved in basolateral transport (Fig. 4). Myosin-IIA associates with the Golgi complex and was shown to selectively promote the exit of basolateral proteins (Musch et al., 1997), whereas myosin-VI was shown to participate in the transport of basolateral cargo proteins that require the clathrin adaptor AP1B (Au et al., 2007).

Future directions

Studies over the past two decades have revealed an amazing diversity of signals in apical cargo proteins, and a puzzling complexity in their preferences for different endosomal compartments and MT and actin motors. Rather than a single compartment carrying out all apical-basolateral sorting, it seems that apical sorting occurs as a succession of events at pre- and post-Golgi levels. An important task for the future is to understand the details of how the sorting machineries are assembled and how their variations contribute to functionally different epithelial phenotypes. The mechanisms underlying clustering and the participation of Rabs, kinesins and myosins in specific apical routes need to be established. The precise roles of glycans, lectins and GPI-anchor remodeling in promoting cargo recognition by different MT motors and by different components of the actin cytoskeleton need to be clarified. Given the emerging fundamental roles of exosomes in cell-cell communication, it will be important to study the nature of the apical-basolateral sorting mechanisms operating at the level of late endosomes and how they resemble or differ from those operating at the level of the TGN and early endosomes.

We thank Anant Menon, Ching-Hwa Sung and members of our laboratories for useful comments on this manuscript. Studies on polarized membrane traffic in our laboratories are supported by NIH R01 grants GM34107 and EY08538 (to E.R.-B.) and DK54407 and DK064613 (to O.A.W.). E.R.-B. was also supported by the Dyson Foundation and the Research to Prevent Blindness Foundation. Deposited in PMC for release after 12 months.

References

Alfalah, M., Jacob, R., Preuss, U., Zimmer, K. P., Naim, H. and Naim, H. Y. (
1999
). O-linked glycans mediate apical sorting of human intestinal sucrase-isomaltase through association with lipid rafts.
Curr. Biol.
9
,
593
-596.
Ang, A. L., Taguchi, T., Francis, S., Folsch, H., Murrells, L. J., Pypaert, M., Warren, G. and Mellman, I. (
2004
). Recycling endosomes can serve as intermediates during transport from the Golgi to the plasma membrane of MDCK cells.
J. Cell Biol.
167
,
531
-543.
Apodaca, G., Katz, L. A. and Mostov, K. E. (
1994
). Receptor-mediated transcytosis of IgA in MDCK cells is via apical recycling endosomes.
J. Cell Biol.
125
,
67
-86.
Apodaca, G., Cardone, M. H., Whiteheart, S. W., DasGupta, B. R. and Mostov, K. E. (
1996
). Reconstitution of transcytosis in SLO-permeabilized MDCK cells: existence of an NSF-dependent fusion mechanism with the apical surface of MDCK cells.
EMBO J.
15
,
1471
-1481.
Au, J. S., Puri, C., Ihrke, G., Kendrick-Jones, J. and Buss, F. (
2007
). Myosin VI is required for sorting of AP-1B-dependent cargo to the basolateral domain in polarized MDCK cells.
J. Cell Biol.
177
,
103
-114.
Babbey, C. M., Ahktar, N., Wang, E., Chen, C. C., Grant, B. D. and Dunn, K. W. (
2006
). Rab10 regulates membrane transport through early endosomes of polarized Madin-Darby canine kidney cells.
Mol. Biol. Cell
17
,
3156
-3175.
Bacallao, R., Antony, C., Dotti, C., Karsenti, E., Stelzer, E. H. and Simons, K. (
1989
). The subcellular organization of Madin-Darby canine kidney cells during the formation of a polarized epithelium.
J. Cell Biol.
109
,
2817
-2832.
Bonazzi, M., Spano, S., Turacchio, G., Cericola, C., Valente, C., Colanzi, A., Kweon, H. S., Hsu, V. W., Polishchuck, E. V., Polishchuck, R. S. et al. (
2005
). CtBP3/BARS drives membrane fission in dynamin-independent transport pathways.
Nat. Cell Biol.
7
,
570
-580.
Bonilha, V. L., Marmorstein, A. D., Cohen-Gould, L. and Rodriguez-Boulan, E. (
1997
). Apical sorting of hemaglutinin by transcytosis in retinal pigment epithelium.
J. Cell Sci.
110
,
1717
-1727.
Brown, D., Breton, S., Ausiello, D. A. and Marshansky, V. (
2009
). Sensing, signaling and sorting events in kidney epithelial cell physiology.
Traffic
10
,
275
-284.
Brown, D. A. and Rose, J. K. (
1992
). Sorting of GPI-anchored proteins to glycolipid enriched membrane subdomains during transport to the apical cell surface.
Cell
68
,
533
-544.
Brown, D. A., Crise, B. and Rose, J. K. (
1989
). Mechanism of membrane anchoring affects polarized expression of two proteins in MDCK cells.
Science
245
,
1499
-1501.
Brown, P. S., Wang, E., Aroeti, B., Chapin, S. J., Mostov, K. E. and Dunn, K. W. (
2000
). Definition of distinct compartments in polarized Madin-Darby canine kidney (MDCK) cells for membrane-volume sorting, polarized sorting and apical recycling.
Traffic
1
,
124
-140.
Bryant, D. M. and Mostov, K. E. (
2008
). From cells to organs: building polarized tissue.
Nat. Rev. Mol. Cell. Biol.
9
,
887
-901.
Catino, M. A., Paladino, S., Tivodar, S., Pocard, T. and Zurzolo, C. (
2008
). N- and O-glycans are not directly involved in the oligomerization and apical sorting of GPI proteins.
Traffic
9
,
2141
-2150.
Chausovsky, A., Bershadsky, A. D. and Borisy, G. G. (
2000
). Cadherin-mediated regulation of microtubule dynamics.
Nat. Cell Biol.
2
,
797
-804.
Cheong, K. H., Zacchetti, D., Schneeberger, E. E. and Simons, K. (
1999
). VIP17/MAL, a lipid raft-associated protein, is involved in apical transport in MDCK cells.
Proc. Natl. Acad. Sci. USA
96
,
6241
-6248.
Chuang, J. Z. and Sung, C. H. (
1998
). The cytoplasmic tail of rhodopsin acts as a novel apical sorting signal in polarized MDCK cells.
J. Cell Biol.
142
,
1245
-1256.
Chuang, J. Z., Zhao, Y. and Sung, C. H. (
2007
). SARA-regulated vesicular targeting underlies formation of the light-sensing organelle in mammalian rods.
Cell
130
,
535
-547.
Cohen, D., Musch, A. and Rodriguez-Boulan, E. (
2001
). Selective control of basolateral membrane protein polarity by cdc42.
Traffic
2
,
556
-564.
Cohen, D., Brennwald, P., Rodriguez-Boulan, E. and Musch, A. (
2004a
). Mammalian PAR-1 determines epithelial lumen polarity by organizing the microtubule cytoskeleton.
J. Cell Biol.
164
,
717
-728.
Cohen, D., Rodriguez-Boulan, E. and Musch, A. (
2004b
). Par-1 promotes a hepatic mode of apical protein trafficking in MDCK cells.
Proc. Natl. Acad. Sci. USA
101
,
13792
-13797.
Corbeil, D., Roper, K., Fargeas, C. A., Joester, A. and Huttner, W. B. (
2001
). Prominin: a story of cholesterol, plasma membrane protrusions and human pathology.
Traffic
2
,
82
-91.
Cramm-Behrens, C. I., Dienst, M. and Jacob, R. (
2008
). Apical cargo traverses endosomal compartments on the passage to the cell surface.
Traffic
9
,
2206
-2220.
Cresawn, K. O., Potter, B. A., Oztan, A., Guerriero, C. J., Ihrke, G., Goldenring, J. R., Apodaca, G. and Weisz, O. A. (
2007
). Differential involvement of endocytic compartments in the biosynthetic traffic of apical proteins.
EMBO J.
26
,
3737
-3748.
D'Angelo, G., Polishchuk, E., Di Tullio, G., Santoro, M., Di Campli, A., Godi, A., West, G., Bielawski, J., Chuang, C. C., van der Spoel, A. C. et al. (
2007
). Glycosphingolipid synthesis requires FAPP2 transfer of glucosylceramide.
Nature
449
,
62
-67.
de Marco, M. C., Puertollano, R., Martinez-Menarguez, J. A. and Alonso, M. A. (
2006
). Dynamics of MAL2 during glycosylphosphatidylinositol-anchored protein transcytotic transport to the apical surface of hepatoma HepG2 cells.
Traffic
7
,
61
-73.
Deborde, S., Perret, E., Gravotta, D., Deora, A., Salvarezza, S., Schreiner, R. and Rodriguez-Boulan, E. (
2008
). Clathrin is a key regulator of basolateral polarity.
Nature
452
,
719
-723.
Delacour, D., Gouyer, V., Zanetta, J. P., Drobecq, H., Leteurtre, E., Grard, G., Moreau-Hannedouche, O., Maes, E., Pons, A., Andre, S. et al. (
2005
). Galectin-4 and sulfatides in apical membrane trafficking in enterocyte-like cells.
J. Cell Biol.
169
,
491
-501.
Delacour, D., Cramm-Behrens, C. I., Drobecq, H., Le Bivic, A., Naim, H. Y. and Jacob, R. (
2006
). Requirement for galectin-3 in apical protein sorting.
Curr. Biol.
16
,
408
-414.
Delacour, D., Greb, C., Koch, A., Salomonsson, E., Leffler, H., Le Bivic, A. and Jacob, R. (
2007
). Apical sorting by galectin-3-dependent glycoprotein clustering.
Traffic
8
,
379
-388.
Delacour, D., Koch, A., Ackermann, W., Eude-Le Parco, I., Elsasser, H. P., Poirier, F. and Jacob, R. (
2008
). Loss of galectin-3 impairs membrane polarisation of mouse enterocytes in vivo.
J. Cell Sci.
121
,
458
-465.
Deora, A. A., Philp, N., Hu, J., Bok, D. and Rodriguez-Boulan, E. (
2005
). Mechanisms regulating tissue-specific polarity of monocarboxylate transporters and their chaperone CD147 in kidney and retinal epithelia.
Proc. Natl. Acad. Sci. USA
102
,
16245
-16250.
Desclozeaux, M., Venturato, J., Wylie, F. G., Kay, J. G., Joseph, S. R., Le, H. T. and Stow, J. L. (
2008
). Active Rab11 and functional recycling endosome are required for E-cadherin trafficking and lumen formation during epithelial morphogenesis.
Am. J. Physiol. Cell Physiol.
295
,
C545
-C556.
DesMarais, V., Ghosh, M., Eddy, R. and Condeelis, J. (
2005
). Cofilin takes the lead.
J. Cell Sci.
118
,
19
-26.
Diaz, F., Gravotta, D., Deora, A., Schreiner, R., Schoggins, J., Falck-Pedersen, E. and Rodriguez-Boulan, E. (
2009
). Clathrin adaptor AP1B controls adenovirus infectivity of epithelial cells.
Proc. Natl. Acad. Sci. USA
106
,
11143
-11148.
Durrbach, A., Raposo, G., Tenza, D., Louvard, D. and Coudrier, E. (
2000
). Truncated brush border myosin I affects membrane traffic in polarized epithelial cells.
Traffic
1
,
411
-424.
Ellis, M. A., Potter, B. A., Cresawn, K. O. and Weisz, O. A. (
2006
). Polarized biosynthetic traffic in renal epithelial cells: sorting, sorting, everywhere.
Am. J. Physiol. Renal. Physiol.
291
,
F707
-F713.
Fan, S., Hurd, T. W., Liu, C. J., Straight, S. W., Weimbs, T., Hurd, E. A., Domino, S. E. and Margolis, B. (
2004
). Polarity proteins control ciliogenesis via kinesin motor interactions.
Curr. Biol.
14
,
1451
-1461.
Fath, K. R. and Burgess, D. R. (
1993
). Golgi-derived vesicles from developing epithelial cells bind actin filaments and possess myosin-I as a cytoplasmically oriented peripheral membrane protein.
J. Cell Biol.
120
,
117
-127.
Fiedler, K., Parton, R. G., Kellner, R., Etzold, T. and Simons, K. (
1994
). VIP36, A novel component of glycolipid rafts and exocytic carrier vesicles in epithelial cells.
EMBO J.
13
,
1729
-1740.
Fievet, B., Louvard, D. and Arpin, M. (
2007
). ERM proteins in epithelial cell organization and functions.
Biochim. Biophys. Acta
1773
,
653
-660.
Florek, M., Bauer, N., Janich, P., Wilsch-Braeuninger, M., Fargeas, C. A., Marzesco, A. M., Ehninger, G., Thiele, C., Huttner, W. B. and Corbeil, D. (
2007
). Prominin-2 is a cholesterol-binding protein associated with apical and basolateral plasmalemmal protrusions in polarized epithelial cells and released into urine.
Cell Tissue Res.
328
,
31
-47.
Folsch, H., Ohno, H., Bonifacino, J. S. and Mellman, I. (
1999
). A novel clathrin adaptor complex mediates basolateral targeting in polarized epithelial cells.
Cell
99
,
189
-198.
Folsch, H., Mattila, P. E. and Weisz, O. A. (
2009
). Taking the scenic route: biosynthetic traffic to the plasma membrane in polarized epithelial cells.
Traffic
.
8
,
972
-981.
Fullekrug, J., Scheiffele, P. and Simons, K. (
1999
). VIP36 localisation to the early secretory pathway.
J. Cell Sci.
112
,
2813
-2821.
Galli, T., Zahraoui, A., Vaidyanathan, V. V., Raposo, G., Tian, J. M., Karin, M., Niemann, H. and Louvard, D. (
1998
). A novel tetanus neurotoxin-insensitive vesicle-associated membrane protein in SNARE complexes of the apical plasma membrane of epithelial cells.
Mol. Biol. Cell
9
,
1437
-1448.
Gan, Y., McGraw, T. E. and Rodriguez-Boulan, E. (
2002
). The epithelial-specific adaptor AP1B mediates post-endocytic recycling to the basolateral membrane.
Nat. Cell Biol.
4
,
605
-609.
Gilbert, T., Le Bivic, A., Quaroni, A. and Rodriguez-Boulan, E. (
1991
). Microtubular organization and its involvement in the biogenetic pathways of plasma membrane proteins in Caco-2 intestinal epithelial cells.
J. Cell Biol.
113
,
275
-288.
Gravotta, D., Deora, A., Perret, E., Oyanadel, C., Soza, A., Schreiner, R., Gonzalez, A. and Rodriguez-Boulan, E. (
2007
). AP1B sorts basolateral proteins in recycling and biosynthetic routes of MDCK cells.
Proc. Natl. Acad. Sci. USA
104
,
1564
-1569.
Grindstaff, K. K., Bacallao, R. L. and Nelson, W. J. (
1998
). Apiconuclear organization of microtubules does not specify protein delivery from the trans-Golgi network to different membrane domains in polarized epithelial cells.
Mol. Biol. Cell
9
,
685
-699.
Guerriero, C. J., Weixel, K. M., Bruns, J. R. and Weisz, O. A. (
2006
). Phosphatidylinositol 5-kinase stimulates apical biosynthetic delivery via an Arp2/3-dependent mechanism.
J. Biol. Chem.
281
,
15376
-15384.
Guerriero, C. J., Lai, Y. and Weisz, O. A. (
2008
). Differential sorting and Golgi export requirements for raft-associated and raft-independent apical proteins along the biosynthetic pathway.
J. Biol. Chem.
283
,
18040
-18047.
Gundersen, D., Orlowski, J. and Rodriguez-Boulan, E. (
1991
). Apical polarity of Na,K-ATPase in retinal pigment epithelium is linked to a reversal of the ankyrin-fodrin submembrane cytoskeleton.
J. Cell Biol.
112
,
863
-872.
Gut, A., Kappeler, F., Hyka, N., Balda, M. S., Hauri, H. P. and Matter, K. (
1998
). Carbohydrate-mediated Golgi to cell surface transport and apical targeting of membrane proteins.
EMBO J.
17
,
1919
-1929.
Hannan, L. A., Lisanti, M. P., Rodriguez-Boulan, E. and Edidin, M. (
1993
). Correctly sorted molecules of a GPI-anchored protein are clustered and immobile when they arrive at the apical surface of MDCK cells.
J. Cell Biol.
120
,
353
-358.
Heine, M., Cramm-Behrens, C. I., Ansari, A., Chu, H. P., Ryazanov, A. G., Naim, H. Y. and Jacob, R. (
2005
). Alpha-kinase 1, a new component in apical protein transport.
J. Biol. Chem.
280
,
25637
-25643.
Hoekstra, D., Tyteca, D. and van IJzendoorn, S. C. D. (
2004
). The subapical compartment: a traffic center in membrane polarity development.
J. Cell Sci.
117
,
2183
-2192.
Hubbard, A. L. and Stieger, B. (
1989
). Biogenesis of endogenous plasma membrane proteins in epithelial cells.
Annu. Rev. Physiol.
51
,
755
-770.
Hughes, R. C. (
2004
). Galectins in kidney development.
Glycoconj. J.
19
,
621
-629.
Ikonen, E., Tagaya, M., Ullrich, O., Montecucco, C. and Simons, K. (
1995
). Different requirements for NSF, SNAP, and Rab proteins in apical and basolateral transport in MDCK cells.
Cell
81
,
571
-580.
Jacob, R., Alfalah, M., Grunberg, J., Obendorf, M. and Naim, H. Y. (
2000
). Structural determinants required for apical sorting of an intestinal brush-border membrane protein.
J. Biol. Chem.
275
,
6566
-6572.
Jacob, R., Heine, M., Alfalah, M. and Naim, H. Y. (
2003
). Distinct cytoskeletal tracks direct individual vesicle populations to the apical membrane of epithelial cells.
Curr. Biol.
13
,
607
-612.
Jaulin, F., Xue, X., Rodriguez-Boulan, E. and Kreitzer, G. (
2007
). Polarization-dependent selective transport to the apical membrane by KIF5B in MDCK cells.
Dev. Cell
13
,
511
-522.
Jou, T. S., Leung, S. M., Fung, L. M., Ruiz, W. G., Nelson, W. J. and Apodaca, G. (
2000
). Selective alterations in biosynthetic and endocytic protein traffic in Madin-Darby canine kidney epithelial cells expressing mutants of the small GTPase Rac1.
Mol. Biol. Cell
11
,
287
-304.
Kim, B. E., Nevitt, T. and Thiele, D. J. (
2008
). Mechanisms for copper acquisition, distribution and regulation.
Nat. Chem. Biol.
4
,
176
-185.
Kinoshita, T., Fujita, M. and Maeda, Y. (
2008
). Biosynthesis, remodelling and functions of mammalian GPI-anchored proteins: recent progress.
J. Biochem.
144
,
287
-294.
Kitagawa, Y., Sano, Y., Ueda, M., Higashio, K., Narita, H., Okano, M., Matsumoto, S. I. and Sasaki, R. (
1994
). N-Glycosylation of erythroipoietin is critical for apical secretion by Madin-Darby canine kidney cells.
Experimental Cell Research
213
,
449
-457.
Kitt, K. N., Hernandez-Deviez, D., Ballantyne, S. D., Spiliotis, E. T., Casanova, J. E. and Wilson, J. M. (
2008
). Rab14 regulates apical targeting in polarized epithelial cells.
Traffic
9
,
1218
-1231.
Klemm, R. W., Ejsing, C. S., Surma, M. A., Kaiser, H. J., Gerl, M. J., Sampaio, J. L., de Robillard, Q., Ferguson, C., Proszynski, T. J., Shevchenko, A. et al. (
2009
). Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network.
J. Cell Biol.
185
,
601
-612.
Klopfenstein, D. R., Tomishige, M., Stuurman, N. and Vale, R. D. (
2002
). Role of phosphatidylinositol(4,5)bisphosphate organization in membrane transport by the Unc104 kinesin motor.
Cell
109
,
347
-358.
Kreitzer, G., Marmorstein, A., Okamoto, P., Vallee, R. and Rodriguez-Boulan, E. (
2000
). Kinesin and dynamin are required for post-Golgi transport of a plasma-membrane protein.
Nat. Cell Biol.
2
,
125
-127.
Kreitzer, G., Schmoranzer, J., Low, S.-H., Li, X., Gan, Y., Weimbs, T., Simon, S. and Rodriguez-Boulan, E. (
2003
). Three-dimensional analysis of post-Golgi carrier exocytosis in epithelial cells.
Nat. Cell Biol.
5
,
126
-136.
Kroschewski, R., Hall, A. and Mellman, I. (
1999
). Cdc42 controls secretory and endocytic transport to the basolateral plasma membrane of MDCK cells.
Nat. Cell. Biol.
1
,
8
-13.
Lafont, F., Burkhardt, J. and Simons, K. (
1994
). Involvement of microtubule motors in basolateral and apical transport in kidney cells.
Nature
372
,
801
-803.
Lafont, F., Verkade, P., Galli, T., Wimmer, C., Louvard, D. and Simons, K. (
1999
). Raft association of SNAP receptors acting in apical trafficking in Madin-Darby canine kidney cells.
Proc. Natl. Acad. Sci. USA
96
,
3734
-3738.
Lakkaraju, A. and Rodriguez-Boulan, E. (
2008
). Itinerant exosomes: emerging roles in cell and tissue polarity.
Trends Cell Biol.
18
,
199
-209.
Lapierre, L. A., Kumar, R., Hales, C. M., Navarre, J., Bhartur, S. G., Burnette, J. O., Provance, D. W., Jr., Mercer, J. A., Bahler, M. and Goldenring, J. R. (
2001
). Myosin vb is associated with plasma membrane recycling systems.
Mol. Biol. Cell
12
,
1843
-1857.
Lazaro-Dieguez, F., Colonna, C., Cortegano, M., Calvo, M., Martinez, S. E. and Egea, G. (
2007
). Variable actin dynamics requirement for the exit of different cargo from the trans-Golgi network.
FEBS Lett.
581
,
3875
-3881.
Le Bivic, A., Quaroni, A., Nichols, B. and Rodriguez-Boulan, E. (
1990
). Biogenetic pathways of plasma membrane proteins in Caco-2, a human intestinal epithelial cell line.
J. Cell Biol.
111
,
1351
-1361.
Le Bivic, A., Garcia, M. and Rodriguez-Boulan, E. (
1993
). Ricin resistant Madin-Darby canine kidney cells missort a major endogenous apical sialoglycoprotein.
J. Biol. Chem.
268
,
6909
-6916.
Lebreton, S., Paladino, S. and Zurzolo, C. (
2008
). Selective roles for cholesterol and actin in compartmentalization of different proteins in the Golgi and plasma membrane of polarized cells.
J. Biol. Chem.
283
,
29545
-29553.
Levine, D. S. and Woods, J. W. (
1990
). Immunolocalization of transferrin and transferrin receptor in mouse small intestinal absorptive cells.
J. Histochem. Cytochem.
38
,
851
-858.
Lin, S., Naim, H. Y., Rodriguez, A. C. and Roth, M. G. (
1998
). Mutations in the middle of the transmembrane domain reverse the polarity of transport of the influenza virus hemagglutinin in MDCK epithelial cells.
J. Cell Biol.
142
,
51
-57.
Lindstedt, R., Apodaca, G., Barondes, S. H., Mostov, K. E. and Leffler, H. (
1993
). 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.
J. Biol. Chem.
268
,
11750
-11757.
Lisanti, M., Sargiacomo, M., Graeve, L., Saltiel, A. and Rodriguez-Boulan, E. (
1988
). Polarized apical distribution of glycosyl phosphatidylinositol anchored proteins in a renal epithelial line.
Proc. Natl. Acad. Sci. USA
85
,
9557
-9561.
Lisanti, M., Caras, I. P., Davitz, M. A. and Rodriguez-Boulan, E. (
1989a
). A glycophospholipid membrane anchor acts as an apical targeting signal in polarized epithelial cells.
J. Cell Biol.
109
,
2145
-2156.
Lisanti, M. P., Le Bivic, A., Sargiacomo, M. and Rodriguez-Boulan, E. (
1989b
). Steady-state distribution and biogenesis of endogenous Madin-Darby canine kidney glycoproteins: evidence for intracellular sorting and polarized cell surface delivery.
J. Cell Biol.
109
,
2117
-2127.
Lisanti, M. P., Caras, I. W., Gilbert, T., Hanzel, D. and Rodriguez-Boulan, E. (
1990
). Vectorial apical delivery and slow endocytosis of a glycolipid-anchored fusion protein in transfected MDCK cells.
Proc. Natl. Acad. Sci. USA
87
,
7419
-7423.
Liu, Y. W., Surka, M. C., Schroeter, T., Lukiyanchuk, V. and Schmid, S. L. (
2008
). Isoform and splice-variant specific functions of dynamin-2 revealed by analysis of conditional knock-out cells.
Mol. Biol. Cell
19
,
5347
-5359.
Loktev, A. V., Zhang, Q., Beck, J. S., Searby, C. C., Scheetz, T. E., Bazan, J. F., Slusarski, D. C., Sheffield, V. C., Jackson, P. K. and Nachury, M. V. (
2008
). A BBSome subunit links ciliogenesis, microtubule stability, and acetylation.
Dev. Cell
15
,
854
-865.
Low, S. H., Chapin, S. J., Wimmer, C., Whiteheart, S. W., Komuves, L. G., Mostov, K. E. and Weimbs, T. (
1998
). The SNARE machinery is involved in apical plasma membrane trafficking in MDCK cells.
J. Cell Biol.
141
,
1503
-1513.
Low, S. H., Marmorstein, L. Y., Miura, M., Li, X., Kudo, N., Marmorstein, A. D. and Weimbs, T. (
2002
). Retinal pigment epithelial cells exhibit unique expression and localization of plasma membrane syntaxins which may contribute to their trafficking phenotype.
J. Cell Sci.
115
,
4545
-4553.
Luton, F., Hexham, M. J., Zhang, M. and Mostov, K. E. (
2009
). Identification of a cytoplasmic signal for apical transcytosis.
Traffic
10
,
1128
-1142.
Maeda, Y., Tashima, Y., Houjou, T., Fujita, M., Yoko-o, T., Jigami, Y., Taguchi, R. and Kinoshita, T. (
2007
). Fatty acid remodeling of GPI-anchored proteins is required for their raft association.
Mol. Biol. Cell
18
,
1497
-1506.
Marzolo, M. P., Bull, P. and Gonzalez, A. (
1997
). Apical sorting of hepatitus B surface antigen (HBsAg) is independent of N-glycosylation and glycosylphosphatidylinositol-anchored protein segregation.
Proc. Natl. Acad. Sci. USA
94
,
1834
-1839.
Marzolo, M. P., Yuseff, M. I., Retamal, C., Donoso, M., Ezquer, F., Farfan, P., Li, Y. and Bu, G. (
2003
). Differential distribution of low-density lipoprotein-receptor-related protein (LRP) and megalin in polarized epithelial cells is determined by their cytoplasmic domains.
Traffic
4
,
273
-288.
Matter, K. and Mellman, I. (
1994
). Mechanisms of cell polarity: sorting and transport in epithelial cells.
Curr. Opin. Cell Biol.
6
,
545
-554.
Matter, K., Brauchbar, M., Bucher, K. and Hauri, H. P. (
1990
). Sorting of endogenous plasma membrane proteins occurs from two sites in cultured human intestinal epithelial cells (Caco-2).
Cell
60
,
429
-437.
Maxfield, F. R. and McGraw, T. E. (
2004
). Endocytic recycling.
Nat. Rev. Mol. Cell. Biol.
5
,
121
-132.
Mazelova, J., Astuto-Gribble, L., Inoue, H., Tam, B. M., Schonteich, E., Prekeris, R., Moritz, O. L., Randazzo, P. A. and Deretic, D. (
2009
). Ciliary targeting motif VxPx directs assembly of a trafficking module through Arf4.
EMBO J.
28
,
183
-192.
Meiss, H. K., Green, R. F. and Rodriguez-Boulan, E. (
1982
). Lectin-resistant mutants of polarized epithelial cells.
Mol. Cell Biol.
2
,
1287
-1294.
Mellman, I. and Nelson, W. J. (
2008
). Coordinated protein sorting, targeting and distribution in polarized cells.
Nat. Rev. Mol. Cell. Biol.
9
,
833
-845.
Morelle, W., Stechly, L., Andre, S., Van Seuningen, I., Porchet, N., Gabius, H. J., Michalski, J. C. and Huet, G. (
2009
). Glycosylation pattern of brush border-associated glycoproteins in enterocyte-like cells: involvement of complex-type N-glycans in apical trafficking.
Biol. Chem.
390
,
529
-544.
Mostov, K., Apodaca, G., Aroeti, B. and Okamoto, C. (
1992
). Plasma membrane protein sorting in polarized epithelial cells.
J. Cell Biol.
116
,
577
-583.
Mostov, K. E. (
1994
). Transepithelial transport of immunoglobulins.
Annu. Rev. Immunol.
12
,
63
-84.
Muller, T., Hess, M. W., Schiefermeier, N., Pfaller, K., Ebner, H. L., Heinz-Erian, P., Ponstingl, H., Partsch, J., Rollinghoff, B., Kohler, H. et al. (
2008
). MYO5B mutations cause microvillus inclusion disease and disrupt epithelial cell polarity.
Nat. Genet.
40
,
1163
-1165.
Musch, A. (
2004
). Microtubule organization and function in epithelial cells.
Traffic
5
,
1
-9.
Musch, A., Cohen, D. and Rodriguez-Boulan, E. (
1997
). Myosin II is involved in the production of constitutive transport vesicles from the trans-Golgi network.
J. Cell Biol.
138
,
291
-306.
Musch, A., Cohen, D., Kreitzer, G. and Rodriguez-Boulan, E. (
2001
). cdc42 regulates the exit of apical and basolateral proteins from the trans-Golgi network.
EMBO J.
20
,
2171
-2179.
Muth, T. R. and Caplan, M. J. (
2003
). Transport protein trafficking in polarized cells.
Annu. Rev. Cell Dev. Biol.
19
,
333
-366.
Noda, Y., Okada, Y., Saito, N., Setou, M., Xu, Y., Zhang, Z. and Hirokawa, N. (
2001
). KIFC3, a microtubule minus end-directed motor for the apical transport of annexin XIIIb-associated Triton-insoluble membranes.
J. Cell Biol.
155
,
77
-88.
Nokes, R. L., Fields, I. C., Collins, R. N. and Folsch, H. (
2008
). Rab13 regulates membrane trafficking between TGN and recycling endosomes in polarized epithelial cells.
J. Cell Biol.
182
,
845
-853.
Ohno, H., Tomemori, T., Nakatsu, F., Okazaki, Y., Aguilar, R. C., Foelsch, H., Mellman, I., Saito, T., Shirasawa, T. and Bonifacino, J. S. (
1999
). Mu1B, a novel adaptor medium chain expressed in polarized epithelial cells.
FEBS Lett.
449
,
215
-220.
Omori, Y., Zhao, C., Saras, A., Mukhopadhyay, S., Kim, W., Furukawa, T., Sengupta, P., Veraksa, A. and Malicki, J. (
2008
). Elipsa is an early determinant of ciliogenesis that links the IFT particle to membrane-associated small GTPase Rab8.
Nat. Cell Biol.
10
,
437
-444.
Orzech, E., Cohen, S., Weiss, A. and Aroeti, B. (
2000
). Interactions between the exocytic and endocytic pathways in polarized Madin-Darby canine kidney cells.
J. Biol. Chem.
275
,
15207
-15219.
Oztan, A., Silvis, M., Weisz, O. A., Bradbury, N. A., Hsu, S. C., Goldenring, J. R., Yeaman, C. and Apodaca, G. (
2007
). Exocyst requirement for endocytic traffic directed toward the apical and basolateral poles of polarized MDCK cells.
Mol. Biol. Cell
18
,
3978
-3992.
Paladino, S., Sarnataro, D., Pillich, R., Tivodar, S., Nitsch, L. and Zurzolo, C. (
2004
). Protein oligomerization modulates raft partitioning and apical sorting of GPI-anchored proteins.
J. Cell Biol.
167
,
699
-709.
Paladino, S., Pocard, T., Catino, M. A. and Zurzolo, C. (
2006
). GPI-anchored proteins are directly targeted to the apical surface in fully polarized MDCK cells.
J. Cell Biol.
172
,
1023
-1034.
Paladino, S., Sarnataro, D., Tivodar, S. and Zurzolo, C. (
2007
). Oligomerization is a specific requirement for apical sorting of glycosyl-phosphatidylinositol-anchored proteins but not for non-raft-associated apical proteins.
Traffic
8
,
251
-258.
Paladino, S., Lebreton, S., Tivodar, S., Campana, V., Tempre, R. and Zurzolo, C. (
2008
). Different GPI-attachment signals affect the oligomerisation of GPI-anchored proteins and their apical sorting.
J. Cell Sci.
121
,
4001
-4007.
Parczyk, K. and Koch-Brandt, C. (
1991
). The role of carbohydrates in vectorial exocytosis. The secretion of the gp 80 glycoprotein complex in a ricin-resistant mutant of MDCK cells.
FEBS Lett.
278
,
267
-270.
Percival, J. M., Hughes, J. A., Brown, D. L., Schevzov, G., Heimann, K., Vrhovski, B., Bryce, N., Stow, J. L. and Gunning, P. W. (
2004
). Targeting of a tropomyosin isoform to short microfilaments associated with the Golgi complex.
Mol. Biol. Cell
15
,
268
-280.
Perret, E., Lakkaraju, A., Deborde, S., Schreiner, R. and Rodriguez-Boulan, E. (
2005
). Evolving endosomes: how many varieties and why?
Curr. Opin. Cell Biol.
17
,
423
-434.
Pfeffer, S. R. and Rothman, J. E. (
1987
). Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi.
Annu. Rev. Biochem.
56
,
829
-852.
Pocard, T., Le Bivic, A., Galli, T. and Zurzolo, C. (
2007
). Distinct v-SNAREs regulate direct and indirect apical delivery in polarized epithelial cells.
J. Cell Sci.
120
,
3309
-3320.
Potter, B. A., Ihrke, G., Bruns, J. R., Weixel, K. M. and Weisz, O. A. (
2004
). Specific N-glycans direct apical delivery of transmembrane, but not soluble or glycosylphosphatidylinositol-anchored forms of endolyn in Madin-Darby canine kidney cells.
Mol. Biol. Cell
15
,
1407
-1416.
Potter, B. A., Hughey, R. P. and Weisz, O. A. (
2006
). Role of N- and O-glycans in polarized biosynthetic sorting.
Am. J. Physiol. Cell Physiol.
290
,
C1
-C10.
Puertollano, R. and Alonso, M. A. (
1999
). MAL, an integral element of the apical sorting machinery, is an itinerant protein that cycles between the trans-Golgi network and the plasma membrane.
Mol. Biol. Cell
10
,
3435
-3447.
Puertollano, R., Martin-Belmonte, F., Millan, J., de Marco, M. C., Albar, J. P., Kremer, L. and Alonso, M. A. (
1999
). The MAL proteolipid is necessary for normal apical transport and accurate sorting of the influenza virus hemagglutinin in Madin-Darby canine kidney cells.
J. Cell Biol.
145
,
141
-151.
Ramnarayanan, S. P., Cheng, C. A., Bastaki, M. and Tuma, P. L. (
2007
). Exogenous MAL reroutes selected hepatic apical proteins into the direct pathway in WIF-B cells.
Mol. Biol. Cell
18
,
2707
-2715.
Remy, L. (
1986
). The intracellular lumen: origin, role and implications of a cytoplasmic neostructure.
Biol. Cell
56
,
97
-106.
Ridley, A. J. (
2006
). Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking.
Trends Cell Biol.
16
,
522
-529.
Rindler, M. J., Ivanov, I. E. and Sabatini, D. D. (
1987
). Microtubule-acting drugs lead to the nonpolarized delivery of the influenza hemagglutinin to the cell surface of polarized Madin-Darby canine kidney cells.
J. Cell Biol.
104
,
231
-241.
Rodriguez-Boulan, E. and Pendergast, M. (
1980
). Polarized distribution of viral envelope proteins in the plasma membrane of infected epithelial cells.
Cell
20
,
45
-54.
Rodriguez-Boulan, E. and Gonzalez, A. (
1999
). Glycans in post-Golgi apical targeting: sorting signals or structural props?
Trends Cell Biol.
9
,
291
-294.
Rodriguez-Boulan, E. and Musch, A. (
2005
). Protein sorting in the Golgi complex: shifting paradigms.
Biochim. Biophys. Acta
1744
,
455
-464.
Rodriguez-Boulan, E., Kreitzer, G. and Musch, A. (
2005
). Organization of vesicular trafficking in epithelia.
Nat. Rev. Mol. Cell. Biol.
6
,
233
-247.
Rojas, R., Ruiz, W. G., Leung, S. M., Jou, T. S. and Apodaca, G. (
2001
). Cdc42-dependent modulation of tight junctions and membrane protein traffic in polarized Madin-Darby canine kidney cells.
Mol. Biol. Cell
12
,
2257
-2274.
Roland, J. T., Kenworthy, A. K., Peranen, J., Caplan, S. and Goldenring, J. R. (
2007
). Myosin Vb interacts with Rab8a on a tubular network containing EHD1 and EHD3.
Mol. Biol. Cell
18
,
2828
-2837.
Roland, J. T., Lapierre, L. A. and Goldenring, J. R. (
2009
). Alternative splicing in class V myosins determines association with Rab10.
J. Biol. Chem.
284
,
1213
-1223.
Rollason, R., Korolchuk, V., Hamilton, C., Jepson, M. and Banting, G. (
2009
). A CD317/tetherin-RICH2 complex plays a critical role in the organization of the subapical actin cytoskeleton in polarized epithelial cells.
J. Cell Biol.
184
,
721
-736.
Salas, P. J., Misek, D. E., Vega Salas, D. E., Gundersen, D., Cereijido, M. and Rodriguez-Boulan, E. (
1986
). Microtubules and actin filaments are not critically involved in the biogenesis of epithelial cell surface polarity.
J. Cell Biol.
102
,
1853
-1867.
Salvarezza, S. B., Deborde, S., Schreiner, R., Campagne, F., Kessels, M. M., Qualmann, B., Caceres, A., Kreitzer, G. and Rodriguez-Boulan, E. (
2009
). LIM kinase 1 and cofilin regulate actin filament population required for dynamin-dependent apical carrier fission from the trans-Golgi network.
Mol. Biol. Cell
20
,
438
-451.
Sarnataro, D., Paladino, S., Campana, V., Grassi, J., Nitsch, L. and Zurzolo, C. (
2002
). PrPC is sorted to the basolateral membrane of epithelial cells independently of its association with rafts.
Traffic
3
,
810
-821.
Sato, T., Mushiake, S., Kato, Y., Sato, K., Sato, M., Takeda, N., Ozono, K., Miki, K., Kubo, Y., Tsuji, A. et al. (
2007
). The Rab8 GTPase regulates apical protein localization in intestinal cells.
Nature
448
,
366
-369.
Scheiffele, P., Peranen, J. and Simons, K. (
1995
). N-glycans as apical sorting signals in epithelial cells.
Nature
378
,
96
-98.
Scheiffele, P., Roth, M. G. and Simons, K. (
1997
). Interaction of influenza virus hemagglutinin with sphingolipid-cholesterol membrane rafts via its transmembrane domain.
EMBO J.
16
,
5501
-5508.
Schell, M. J., Maurice, M., Stieger, B. and Hubbard, A. L. (
1992
). 5′nucleotidase is sorted to the apical domain of hepatocytes via an indirect route.
J. Cell Biol.
119
,
1173
-1182.
Schuck, S., Gerl, M. J., Ang, A., Manninen, A., Keller, P., Mellman, I. and Simons, K. (
2007
). Rab10 is involved in basolateral transport in polarized Madin-Darby canine kidney cells.
Traffic
8
,
47
-60.
Schwartz, G. J. and Al-Awqati, Q. (
2005
). Role of hensin in mediating the adaptation of the cortical collecting duct to metabolic acidosis.
Curr. Opin. Nephrol. Hypertens
14
,
383
-388.
Sfakianos, J., Togawa, A., Maday, S., Hull, M., Pypaert, M., Cantley, L., Toomre, D. and Mellman, I. (
2007
). Par3 functions in the biogenesis of the primary cilium in polarized epithelial cells.
J. Cell Biol.
179
,
1133
-1140.
Sharma, N., Low, S. H., Misra, S., Pallavi, B. and Weimbs, T. (
2006
). Apical targeting of syntaxin 3 is essential for epithelial cell polarity.
J. Cell Biol.
173
,
937
-948.
Shtutman, M., Chausovsky, A., Prager-Khoutorsky, M., Schiefermeier, N., Boguslavsky, S., Kam, Z., Fuchs, E., Geiger, B., Borisy, G. G. and Bershadsky, A. D. (
2008
). Signaling function of alpha-catenin in microtubule regulation.
Cell Cycle
7
,
2377
-2383.
Simons, K. and Fuller, S. D. (
1985
). Cell surface polarity in epithelia.
Annu. Rev. Cell Biol.
1
,
243
-288.
Singla, V. and Reiter, J. F. (
2006
). The primary cilium as the cell's antenna: signaling at a sensory organelle.
Science
313
,
629
-633.
Slimane, T. A., Trugnan, G., Van, I. S. C. and Hoekstra, D. (
2003
). Raft-mediated trafficking of apical resident proteins occurs in both direct and transcytotic pathways in polarized hepatic cells: role of distinct lipid microdomains.
Mol. Biol. Cell
14
,
611
-624.
Spodsberg, N., Alfalah, M. and Naim, H. Y. (
2001
). Characteristics and structural requirements of apical sorting of the rat growth hormone through the O-glycosylated stalk region of intestinal sucrase-isomaltase.
J. Biol. Chem.
276
,
46597
-46604.
Stechly, L., Morelle, W., Dessein, A. F., Andre, S., Grard, G., Trinel, D., Dejonghe, M. J., Leteurtre, E., Drobecq, H., Trugnan, G. et al. (
2009
). Galectin-4-regulated delivery of glycoproteins to the brush border membrane of enterocyte-like cells.
Traffic
10
,
438
-450.
Steegmaier, M., Lee, K. C., Prekeris, R. and Scheller, R. H. (
2000
). SNARE protein trafficking in polarized MDCK cells.
Traffic
1
,
553
-560.
Sudhof, T. C. and Rothman, J. E. (
2009
). Membrane fusion: grappling with SNARE and SM proteins.
Science
323
,
474
-477.
Tai, A. W., Chuang, J. Z., Bode, C., Wolfrum, U. and Sung, C. H. (
1999
). Rhodopsin's carboxy-terminal cytoplasmic tail acts as a membrane receptor for cytoplasmic dynein by binding to the dynein light chain Tctex-1.
Cell
97
,
877
-887.
Tai, A. W., Chuang, J. Z. and Sung, C. H. (
2001
). Cytoplasmic dynein regulation by subunit heterogeneity and its role in apical transport.
J. Cell Biol.
153
,
1499
-1509.
Takeda, T., Yamazaki, H. and Farquhar, M. G. (
2003
). Identification of an apical sorting determinant in the cytoplasmic tail of megalin.
Am. J. Physiol. Cell Physiol.
284
,
C1105
-C1113.
Tzaban, S., Massol, R. H., Yen, E., Hamman, W., Frank, S. R., Lapierre, L. A., Hansen, S. H., Goldenring, J. R., Blumberg, R. S. and Lencer, W. I. (
2009
). The recycling and transcytotic pathways for IgG transport by FcRn are distinct and display an inherent polarity.
J. Cell Biol.
185
,
673
-684.
Tanos, B. and Rodriguez-Boulan, E. (
2008
). The epithelial polarity program: machineries involved and their hijacking by cancer.
Oncogene
27
,
6939
-6357.
ter Beest, M. B., Chapin, S. J., Avrahami, D. and Mostov, K. E. (
2005
). The role of syntaxins in the specificity of vesicle targeting in polarized epithelial cells.
Mol. Biol. Cell
16
,
5784
-5792.
Theard, D., Steiner, M., Kalicharan, D., Hoekstra, D. and van Ijzendoorn, S. C. (
2007
). Cell polarity development and protein trafficking in hepatocytes lacking E-cadherin/beta-catenin-based adherens junctions.
Mol. Biol. Cell
18
,
2313
-2321.
Tzaban, S., Massol, R. H., Yen, E., Hamman, W., Frank, S. R., Lapierre, L. A., Hansen, S. H., Goldenring, J. R., Blumberg, R. S. and Lencer, W. I. (
2009
). The recycling and transcytotic pathways for IgG transport by FcRn are distinct and display an inherent polarity.
J. Cell Biol.
185
,
673
-684.
van Meer, G., Gumbiner, B. and Simons, K. (
1986
). The tight junction does not allow lipid molecules to diffuse from one epithelial cell to the next.
Nature
322
,
639
-641.
van Meer, G. and Simons, K. (
1988
). Lipid polarity and sorting in epithelial cells.
J. Cell Biochem.
36
,
51
-58.
van Zeijl, M. J. and Matlin, K. S. (
1990
). Microtubule perturbation inhibits intracellular transport of an apical membrane glycoprotein in a substrate-dependent manner in polarized Madin-Darby canine kidney epithelial cells.
Cell Regul.
1
,
921
-936.
van't Hof, W., Rodriguez-Boulan, E. and Menon, A. K. (
1995
). Nonpolarized distribution of glycosylphosphatidylinositols in the plasma membrane of polarized Madin-Darby canine kidney cells.
J. Biol. Chem.
270
,
24150
-24155.
Vega-Salas, D. E., Salas, P. J. I. and Rodriguez-Boulan, E. (
1988
). Exocytosis of vacuolar apical compartment (VAC): a cell-cell contact controlled mechanism for the establishment of the apical plasma membrane domain in epithelial cells.
J. Cell Biol.
107
,
1717
-1728.
Verges, M., Sebastian, I. and Mostov, K. E. (
2007
). Phosphoinositide 3-kinase regulates the role of retromer in transcytosis of the polymeric immunoglobulin receptor.
Exp. Cell Res.
313
,
707
-718.
Vieira, O. V., Verkade, P., Manninen, A. and Simons, K. (
2005
). FAPP2 is involved in the transport of apical cargo in polarized MDCK cells.
J. Cell Biol.
170
,
521
-526.
Vieira, O. V., Gaus, K., Verkade, P., Fullekrug, J., Vaz, W. L. and Simons, K. (
2006
). FAPP2, cilium formation, and compartmentalization of the apical membrane in polarized Madin-Darby canine kidney (MDCK) cells.
Proc. Natl. Acad. Sci. USA
103
,
18556
-18561.
Wagner, M., Morgans, C. and Koch-Brandt, C. (
1995
). The oligosaccharides have an essential but indirect role in sorting gp80 (clusterin, TRPM-2) to the apical surface of MDCK cells.
Eur. J. Cell Biol.
67
,
84
-88.
Wakabayashi, Y., Kipp, H. and Arias, I. M. (
2006
). Transporters on demand: intracellular reservoirs and cycling of bile canalicular ABC transporters.
J. Biol. Chem.
281
,
27669
-27673.
Wang, E., Brown, P. S., Aroeti, B., Chapin, S. J., Mostov, K. E. and Dunn, K. W. (
2000
). Apical and basolateral endocytic pathways of MDCK cells meet in acidic common endosomes distinct from a nearly-neutral apical recycling endosome.
Traffic
1
,
480
-493.
Wang, Q. and Margolis, B. (
2007
). Apical junctional complexes and cell polarity.
Kidney Int.
72
,
1448
-1458.
Wojtal, K. A., Hoekstra, D. and van Ijzendoorn, S. C. (
2007
). Anchoring of protein kinase A-regulatory subunit IIalpha to subapically positioned centrosomes mediates apical bile canalicular lumen development in response to oncostatin M but not cAMP.
Mol. Biol. Cell
18
,
2745
-2754.
Woolner, S. and Bement, W. M. (
2009
). Unconventional myosins acting unconventionally.
Trends Cell Biol.
6
,
245
-252.
Yeaman, C., Le Gall, A. H., Baldwin, A. N., Monlauzeur, L., Le Bivic, A. and Rodriguez-Boulan, E. (
1997
). The O-glycosylated stalk domain is required for apical sorting of neurotrophin receptors in polarized MDCK cells.
J. Cell Biol.
139
,
929
-940.
Yoshimura, S., Egerer, J., Fuchs, E., Haas, A. K. and Barr, F. A. (
2007
). Functional dissection of Rab GTPases involved in primary cilium formation.
J. Cell Biol.
178
,
363
-369.
Zuo, X., Guo, W. and Lipschutz, J. H. (
2009
). The exocyst protein Sec10 is necessary for primary ciliogenesis and cystogenesis in vitro.
Mol. Biol. Cell
20
,
2522
-2529.
Zurzolo, C., Le Bivic, A., Quaroni, A., Nitsch, L. and Rodriguez-Boulan, E. (
1992
). Modulation of transcytotic and direct targeting pathways in a polarized thyroid cell line.
EMBO J.
11
,
2337
-2344.
Zurzolo, C., Lisanti, M. P., Caras, I. W., Nitsch, L. and Rodriguez-Boulan, E. (
1993
). Glycosylphosphatidylinositol-anchored proteins are preferentially targeted to the basolateral surface in Fischer Rat Thyroid epithelial cells.
J. Cell Biol.
121
,
1031
-1039.