ORGANIZATION OF VESICULAR TRAFFICKING IN EPITHELIA

Epithelial cells carry out key vectorial functions forhost multicellular organisms that depend on thepolarized distribution of plasma-membrane proteinsinto apical and basolateral domains. The first observa-tions of the vectorial properties of epithelial cells dateback to the 1940s: physiologists, using radioactivetracers in combination with electrophysiological pro-cedures, showed that epithelial cells could transportnutrients vectorially against steep electrical and chem-ical gradients (reviewed in REF. 1). In the followingthree decades (see the TIMELINE), electron micro-scopists described the complex asymmetric structureof epithelial cells, including the polarized organelles inthe secretory pathway2 and the junctional complex(TIGHT JUNCTIONS, ADHERENS JUNCTIONS and DESMOSOMES) atthe border of the apical and lateral regions of the cell(the apical–lateral border)3. In the late 1970s, the fun-damental question of how epithelial cells establish andmaintain their polarized phenotype became experi-mentally approachable when Cereijido, Misfeld andco-workers4,5 made the groundbreaking discovery thatMadin–Darby canine kidney (MDCK) cells developan electrically tight epithelial monolayer when platedon a permeable substratum4,5 (BOX 1). The MDCKmodel was refined to study polarized protein targetingafter it was shown that influenza virus assembles fromthe APICAL SURFACE and vesicular stomatitis virus (VSV)assembles from the BASOLATERAL SURFACE guided by thepolarized distribution of their envelope glycoproteins

— influenza haemagglutinin (HA) and VSV G protein(VSVG)6,7 (BOX 1). The impact that was generated by theMDCK model (see REF. 1 for its history) can be estimatedfrom the steep increase in the number of publications —on epithelial polarity, epithelial morphogenesis andthe epithelial junctional complex — that followed itsintroduction.

The experimental advantages that were provided byvirus-infected MDCK cells (such as high biosyntheticlevels of envelope glycoproteins and no interfering poolof endogenous plasma-membrane proteins) led to theidentification of the TRANS-GOLGI NETWORK (TGN; the distalregion of the Golgi complex)8 as the sorting compart-ment in the biosynthetic route for apical influenza HAand basolateral VSVG9–11. In the late 1980s, epithelialcell biologists characterized the biosynthetic, endo-cytic, recycling and transcytotic routes of MDCK cellsand other epithelial cell lines using sensitive protein-targeting assays12,13 (BOX 1). This knowledge led to theconcept of the flexible epithelial phenotype — that is,that different epithelial cell types can change the finallocalization and transport routes of apical and basolat-eral proteins to carry out tissue-specific vectorial func-tions14 (BOX 2). Insights into the mechanisms that areresponsible for this variation first emerged in the late1980s and early 1990s with the discovery of apical andbasolateral sorting signals15,16, and in the 1990s withthe identification of cellular components that regulatepolarized vesicular trafficking17,18. The molecular

ORGANIZATION OF VESICULARTRAFFICKING IN EPITHELIAEnrique Rodriguez-Boulan*, Geri Kreitzer ‡ and Anne Müsch*

Abstract | Experiments using mammalian epithelial cell lines have elucidated biosynthetic andrecycling pathways for apical and basolateral plasma-membrane proteins, and have identifiedcomponents that guide apical and basolateral proteins along these pathways. Thesecomponents include apical and basolateral sorting signals, adaptors for basolateral signals, anddocking and fusion proteins for vesicular trafficking. Recent live-cell-imaging studies provide areal-time view of sorting processes in epithelial cells, including key roles for actin, microtubulesand motors in the organization of post-Golgi trafficking.

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 6 | MARCH 2005 | 233

*Margaret Dyson VisionResearch Institute, WeillMedical College of CornellUniversity, 1300 YorkAvenue, New York, New York10021, USA.‡Department of Cell andDevelopmental Biology,Weill Medical College ofCornell University, 1300York Avenue, New York,New York 10021, USA.e-mails:[email protected];[email protected];[email protected]:10.1038/nrm1593

TIGHT JUNCTION

The most apical intercellularjunctions in mammalianepithelial cells, which functionas selective (semi-permeable)diffusion barriers betweenindividual cells. They areidentified as a belt-like region inwhich two lipid-apposingmembranes lie close together.

ADHERENS JUNCTION

A cell–cell adhesion complexthat contains cadherins andcatenins that are attached tocytoplasmic actin filaments.

R E V I E W S

DESMOSOME

A patch-like adhesiveintercellular junction found invertebrate tissues that is linkedto intermediate filaments.

APICAL SURFACE

The surface of an epithelial orendothelial cell that faces thelumen of a cavity or tube or theoutside of the organism.

BASOLATERAL SURFACE

The surface of an epithelial cellthat adjoins underlying tissue.

TRANS-GOLGI NETWORK

(TGN). Membranouscompartment from whichvesicles bud to deliver proteinsand other materials to the cellsurface or to the late endosomesfor delivery to lysosomes.

DOCKING/FUSION SITE

Vesicles and tubules that aretargeted to the plasma membranefrom the Golgi complex or fromendosomes possess mechanismsby which to dock and fuse at thecell surface. These includetethering factors, Rab proteinsand SNARE proteins.

GPI-ANCHOR

The function of this post-translational modification is toattach proteins to the exoplasmicleaflet of membranes, possiblyto specific domains therein. Theanchor is made of one moleculeof phosphatidylinositol towhich a carbohydrate chain islinked through the C-6hydroxyl of the inositol. Thisanchor is linked to the proteinthrough an ethanolaminephosphate moiety.

234 | MARCH 2005 | VOLUME 6 www.nature.com/reviews/molcellbio

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in the biosynthetic route of MDCK cells was carriedout in the TGN (routes 1 and 2 in FIGS 1,2), recent workindicates that some apical–basolateral sorting in thebiosynthetic route could occur in common recyclingendosomes, immediately at the exit of the TGN (routes3, 4 and 5 in FIGS 1,2)21. Common recycling endosomesare also the main sorting site for membrane proteinsthat are internalized from the apical and basolateralsurfaces22. Different epithelial cells vary widely in theuse of direct and transcytotic routes for apical proteins(BOX 2). Until recently, it was assumed that most apicalproteins are transferred to the apical surface of MDCKcells directly from the TGN, but a recent paper23 hasintroduced lively controversy on this issue. The authorsfound that addition of tannic acid, a mild fixative, tothe basolateral medium of live MDCK cells preventedthe delivery of newly synthesized glycosylphosphatidyli-nositol (GPI)-ANCHORED proteins (GPI-APs) to the apicalsurface, but did not interfere with the transport ofanother apical protein, the p75 neurotrophin receptor(p75NTR). Apical GFP-tagged GPI-APs and basolateralVSVG were present in common tubular transporters,which indicates that some apical proteins might besorted after arrival at the basolateral surface. These latestresults indicate that the transcytotic mode of apicaltransport might be more widespread among epithelialcells than was previously thought, a proposition thatneeds to be further tested by the field.

Apical sorting mechanismsApical sorting signals. Apical sorting signals (FIG. 1) arelocalized in EXOPLASMIC, membrane or cytoplasmicdomains of the sorted protein. An initial clue on thenature of apical sorting signals was the demonstra-tion that endogenous GPI-APs were localized at theapical surface of MDCK cells and other epithelial celllines24. The first demonstration of an epithelial sort-ing signal came from experiments showing thatrecombinant addition of GPI to a secretory protein25,26

resulted in the apical localization of the chimericplasma-membrane proteins.

analysis of genetically tractable model organisms —Saccharomyces cerevisiae, Caenorhabditis elegans andDrosophila melanogaster — led to the discovery of agrowing list of ‘polarity genes’, the products of which areclustered around the junctional complex. Recent workhas provided fascinating details of their complex inter-actions and roles in epithelial morphogenesis19,20, buttheir role in polarized vesicular trafficking remainslargely unknown. In the late 1990s and the new millen-nium, the advent of green fluorescent protein (GFP)and its derivatives, combined with high-resolutionoptical-imaging techniques (BOX 1), started to provide areal-time view of the sorting processes that occur in theGolgi complex and recycling endosomes of polarizedcells. This provided an increased understanding of thefundamental role that is carried out by the cytoskeleton inthe organization of the delivery and fusion of transportvesicles to the plasma membrane.

Here, the mechanisms that have a role in vesiculartrafficking to apical and basolateral membranes arereviewed, with a predominant focus on MDCK cells, inwhich most of this work has been carried out. A histori-cal perspective, which highlights the main discoveries inthe field, the current status, and the remaining openquestions, is provided. We focus sequentially on themechanisms that: first, carry out the initial segregation ofapical and basolateral proteins into different transportintermediates; second, mediate the formation and move-ment of these transport intermediates to the cell surface;and, third, determine DOCKING and FUSION at specific sitesin the plasma membrane. Because of the restrictions inreference numbers and length, the review focuses onvesicular trafficking and omits exciting parallel discov-eries on cell–cell junctions or epithelial morphogenesisand the role of cell–cell adhesion1,17,18, unless the findingsare directly relevant to vesicular-trafficking mechanisms.

Trafficking routes in epithelial cellsThe biosynthetic, endocytic, recycling and transcytoticroutes of MDCK cells are summarized in FIG. 1.Althoughit was initially believed that most of the protein sorting

Radioisotopesfirst used(early 1940s).

Ion transportacross epithelia.

Cell fractionationdiscovered.

Electron microscopydeveloped.

Junctional complexesin epithelia.

Endocytosis andendocytic signals.

Secretory pathway inexocrine pancreas.

Polarized viral buddingfrom MDCK cells.

Ca2+-switchprotocol developed.

Sorting of apical andbasolateral proteinsin Golgi complex.

Transcytosis routesin MDCK, intestinaland liver cells.

Assays to studypolarized proteindelivery developed.

Zona occludens-1identified as first tight-junction component.

Polarized MDCK cells grownon monolayers and culturedon permeable substrates.

1940 1946 1949 1952 1963 1964–1966 1974 1976, 1978 1978 1984 1985 1985–1987 1985–1990 1986

Timeline | Discoveries in the field of epithelial polarity: the impact of the MDCK model.

Important discoveries in epithelial polarity are represented by a red outline; important methodologies are outlined in blue boxes. Even once the polarized function and structure of epithelial cells had beenknown for several decades, it was only after the introduction of the Madin–Darby canine kidney (MDCK) model in the late 1970s that the fields of epithelial polarity, epithelial morphogenesis and epithelialjunctions developed, as evidenced by the large increase in the number of publications in these areas, many of them involving MDCK cells or other epithelial cell lines. Publications in all areas of epithelialcell biology totalled about 100 in 1970 but, from 1980, rapidly increased to the present level of several thousand.

Laser scanningconfocal microscopydeveloped.


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implied by experiments showing that the apical secre-tion of some soluble non-glycosylated proteins becamesaturated at high expression levels and could be outcom-peted by the overexpression of a second apical protein39.However, these receptors have not yet been identified.N-glycans have been proposed to sort apically by inter-acting with a LECTIN sorting receptor32. A candidatelectin, VIP36, localizes to the TGN when it is overex-pressed. However, endogenous VIP36 is present mainlyin the cis-Golgi, and its possible role in apical proteinsorting is now considered doubtful40. Finally, there aremany exceptions to the ability of N-glycans to functionas conventional apical sorting signals, which impliesthat they might have a different, unconventional role inapical protein sorting.

The lipid-raft hypothesis. An alternative to the conven-tional sorting-receptor model for apical sorting ispresented by the lipid-raft hypothesis, which was putforward by van Meer and Simons in 1988 (REFS 41,42). Thelipid-raft hypothesis postulates that many proteins aresorted apically because they have an affinity for micro-domains of glycosphingolipids and cholesterol (lipidrafts) that are assembled in the Golgi complex.Accordingto this hypothesis, lipid rafts and their associated proteinsform sorting platforms that, on recognition by special-ized machinery in the TGN, recycling endosomes orplasma membrane, become incorporated into apicaltransport intermediates that deliver them to the apicalmembrane (FIG. 1). The experimental evidence support-ing the lipid-raft hypothesis is substantial and is discussedin BOX 3. Examples of proteins that show affinity for lipid-rafts are GPI-APs and influenza HA; the association ofthese proteins with lipid rafts is mediated by the GPIanchor and by the transmembrane domain, respectively,and occurs at the level of the Golgi complex (BOX 3).

However, full acceptance of the lipid-raft hypothesisfor apical sorting has been compromised by inconsis-tencies that have, so far, been difficult to explain. Forexample, several GPI-APs are found on the basolateralsurface of MDCK cells, even when they are associated

A second group of apical signals comprises N-GLYCANS27

and O-glycans28,29 — the latter are usually present injuxtamembrane regions of transmembrane proteins.Removal of N- or O-glycans results in the non-polarizedrelease of luminal secretory proteins in MDCK cells27,30.Removal of N-glycans blocks the exit from the Golgicomplex of two apically targeted plasma-membraneproteins31. N- and O-glycans usually function as reces-sive signals relative to basolateral sorting signals(described below); their wide distribution in plasma-membrane and secretory proteins explains the frequentfinding that removal of basolateral signals from abasolateral protein results in its apical localization16,28,32.

A third group of apical sorting signals encompassesproteinaceous motifs in the exoplasmic, transmem-brane or cytoplasmic domains of the protein. Completedeletion of N-glycans does not alter the apical secretionof some soluble glycoproteins33, endogenous plasma-membrane proteins34 or exogenous glycoproteins30,which implies that there are also proteinaceous apicaltargeting motifs. The transmembrane domain ofinfluenza HA contains apical sorting information, whichmediates incorporation of the protein into LIPID RAFTS

(discussed below). The cytoplasmic domain of the light-sensitive protein rhodopsin35 has binding sites for themicrotubule motor protein cytoplasmic dynein, whichmediates translocation of rhodopsin to the apical mem-brane in MDCK cells. The cytoplasmic domains of mega-lin36,37 and other apical proteins contain apical sortingsignals that have not yet been fully characterized38.

Apical sorting receptors? Apical sorting signals thatmediate association with dynein are thought to functionby promoting microtubule-mediated directional trans-port to the apical surface (discussed later in this review).By contrast, the sorting mechanisms that are used byother apical signals are still poorly understood. Theycould function as conventional sorting signals — forexample, by interacting with an apical sorting receptorthat promotes incorporation into an apical transportvesicle. The existence of apical sorting receptors was

EXOPLASMIC

Facing the outside of the cell orthe topologically equivalentlumen of organelles in thesecretory pathway; the oppositeof cytoplasmic.

GLYCAN

A polymer that consists ofseveral monosaccharide residues(polysaccharide). In the case ofGPI-anchored proteins, the basicunit is composed of glucosamineand three mannose residues.

LIPID RAFTS

Membrane microdomains thatare enriched in cholesterol,sphingolipids and lipid-modified proteins such as GPI-linked proteins andpalmitoylated proteins. Thesemicrodomains often function asplatforms for signalling events.

LECTIN

A protein that can bind tocarbohydrates with highselectivity. For example,concanavalin A is a lectin withaffinity for mannose residues inglycoproteins.

MDCK cellsmechanically andbiochemically perforated.

GPI identified asapical sorting signal.

Crumbs identified asapical polarity gene.

Advent of greenfluorescent protein.

AP1B, anepithelial-specificadaptor, identified.

Lipid-raft hypothesisproposed.

E-cadherinidentified asepithelial organizer.

Apical and basolateralendosomes observedin MDCK cells.

Basolateralsorting signalsidentified.

N-Glycans and O-glycansidentified as apical sorting signals.

Polarized exocytictrafficking viewedin live MDCK cells.

Molecules thatregulate polarizedtrafficking discovered.

Polarity genesidentified injunctional complex.

1987 1987–1990 1988 1988–1989 1989 1990 1991 1995 1995, 1997 1995–2000 1998–2002 1999 2003

promote additional association with lipid rafts. Finally,lipid rafts are difficult to visualize by optical microscopy;indeed, recent studies45 indicate that lipid rafts are smalland highly dynamic structures with a capacity toaccommodate a maximum of 3–5 GPI-APs.

with lipid rafts according to the usual criterion of insol-ubility in non-ionic detergents at 4oC (discussed in REF. 43;see BOX 3). Some GPI-anchored proteins also require thepresence of N-glycans before they can localize apically44,even though the presence of N-glycans does not always

Influenza virus Vesicular stomatitis virus

Polarized MDCK cells

GFP–apical marker GFP–basolateral marker

Total-internal-reflectionfluorescence microscopy

GolgiTubulo–vesicularstructuresNucleus

Golgi

Nucleus

12

11

10

8

6

4

µmLive confocal microscopy

IS

a b

c d


R E V I E W S

Box 1 | The Madin–Darby canine kidney (MDCK) cell model

MDCK monolayers MDCK cells were the first epithelial cells that were used to form electrically tight monolayers on permeable filters in vitro4,5 (see figure, part a). MDCK cells that were grown on filters were also used to develop all of the techniques that are available to study polarized molecular targeting. These include:• Biotinylation and protease assays to study steady-state polarity and domain-specific biosynthetic protein delivery9,132,133.

• Assays that use mechanically or biochemically (streptolysin-O-) permeabilized cells to experimentally manipulate post-Golgi exocytosis65,134–136.

• Assays to study the establishment of polarity, such as the Ca2+ switch117, collagen embedding or the collagen overlay ofMDCK cells18,118,121.

Polarized viral assembly The envelope glycoproteins of viruses that bud from either the apical (influenza haemagglutinin) or basolateral (vesicularstomatitis virus G protein) domains of MDCK cells are still widely used apical and basolateral markers6 . They provided thefirst evidence of polarized biosynthetic routes and sorting at the Golgi complex (see figure, part b). IS, intercellular space.

Live-cell-imaging of polarized post-Golgi exocytosisApical or basolateral cargo proteins that are tagged with green fluorescent protein (GFP) accumulate in the trans-Golginetwork of polarized MDCK cells at 20oC (REFS 23,88). Their exit from the Golgi complex and delivery to the cell surface isvisualized using time-lapse fluorescence microscopy, total-internal-reflection microscopy (see figure, part c) or confocalmicroscopy (see figure, part d).


R E V I E W S

lipid-raft-associated apical proteins. In the case ofinfluenza HA, the evidence supports a role for theproteolipid MAL (myelin and lymphocyte protein)52 inapical targeting, but whether this protein participatesin lipid-raft clustering is not known52.

Basolateral sorting mechanismsBasolateral signals. Basolateral signals are usually locatedin cytoplasmically exposed regions of the protein; theirparticipation in biosynthetic and recycling trafficking inMDCK cells is shown in FIG. 1. Mostov and co-workersshowed in 1986 that deletion of the cytoplasmic domainof polymeric immunoglobulin-A receptor preventedits basolateral localization53 and in 1991 that the signalwas transplantable to other proteins54. Mellman andco-workers reported in 1991 (REF. 55) the existence ofbasolateral signals in the cytoplasmic domain of the low-density lipoprotein (LDL) receptor and subsequentlyshowed that these signals were also transplantable56. Twoother groups showed in 1991 that basolateral signalscould be created by introducing mutations that gener-ated endocytic motifs in the cytoplasmic domains of theapical proteins influenza HA57 and p75NTR58.

The finding that basolateral proteins had sortingsignals was surprising, as it was then thought that trans-port to the basolateral plasma membrane occurred bydefault13 — an extension of an earlier hypothesis onbiosynthetic transport to the cell surface in non-polar-ized cells. Many studies have since confirmed that baso-lateral signals are localized in the cytoplasmic domain ofbasolateral proteins and consist of tyrosine55,57 ordileucine59 motifs, which are often in the vicinity ofpatches of acidic amino acids, as well as other uncharac-terized motifs (recently reviewed in REF. 60). More recentwork has uncovered a new type of basolateral signal thatconsists of a single leucine motif 61. Mutations in baso-lateral signals might result in disease — for example, aform of familial hypercholesterolaemia is caused by a mutated signal in the LDL receptor62.

Relative to single-span basolateral proteins, such asthose discussed above, less information is available onthe sorting signals of hetero-oligomeric membraneproteins. Many ion or nutrient transporters are non-glycosylated multispan proteins that require chaperon-ing by a glycosylated single-span protein for transportto the cell surface. Among the most well-studied exam-ples are the Na+/K+-ATPase, which is found basolater-ally in all mammalian epithelia (with the exception ofneuroepithelia, the retinal pigment epithelium andCHOROID PLEXUS) and the H+-ATPase, which is found apically in the parietal (hydrochloric-acid-secreting)cells of the stomach and in other cells60. In these twoproteins, the apical and basolateral exocytic-traffickingsignals are contained in the multispan non-glycosylatedα-subunit. The glycosylated β-subunit in the H+-ATPasecontains a tyrosine-based endocytic motif that mediatesre-incorporation into a regulated secretory compartmentthat, when the cell is stimulated, is exocytosed to the api-cal plasmalemma. However, trafficking signals do notsolely account for the basolateral-surface localization ofthe Na+/K+-ATPase. The α-subunit also has binding sites

A recent report, together with older data, providessome clues to the mystery of how lipid-rafts might con-tribute to apical sorting: Zurzolo and co-workers43

showed that GPI-anchored GFP is targeted to the apicalmembrane of MDCK cells, but is missorted to the baso-lateral membrane when mutations are introduced intoGFP that prevent its natural tendency to oligomerize.These experiments agree with decade-old data byHannan et al.46 that show that newly synthesized GPI-APs arrive at the apical surface of MDCK cells in largeimmobile clusters (as determined by FRAP), which pro-gressively disperse into small mobile clusters (as deter-mined by FRET). That the formation of large clusters ofGPI-APs is required for their apical sorting is shown bystudies with lectin (concanavalin A)-resistant MDCKcells that prevent the ability of GPI-APs to formlarger clusters in the apical route. These GPI-APs aremissorted to the basolateral membrane46. Parallelexperiments imply that clustering is important in thegeneral endocytic sorting of GPI-APs. Crosslinking of GPI-APs by antibodies that are directed againsttheir ectodomains promotes their incorporation intopre-existing CAVEOLAE in fibroblasts47 and into newlyformed caveolae at the apical surface of MDCK cells48.(Like most epithelial cells, MDCK cells have only baso-lateral caveolae, but they develop apical caveolae aftercrosslinking of GPI-APs.)

The experiments that are described above indicatethat the lipid-raft hypothesis might be refined as follows:apical proteins are sorted by association with small lipidrafts that are converted into functional apical sortingplatforms by a ‘clustering event’ (see FIG. 1 and insets;also discussed in REF. 43). This modified lipid-rafthypothesis gives rise to secondary hypotheses, whichcould be tested using available experimental data orfuture experiments. The first secondary hypothesis isthat any feature of a lipid-raft-associated protein thatpromotes oligomerization could promote apical tar-geting. This could be tested using approaches that aresimilar to those used in REF. 43. The next secondaryhypothesis is that the formation of clustered lipid raftsmight be promoted by a luminal lectin (FIG. 1, insets). Onthe basis of available data with lectin-resistant MDCKcells, a luminal lectin with affinity for mannose-rich N-glycan cores could participate in such clustering49,50.The third secondary hypothesis is that the formation offunctional lipid rafts could be promoted by proteinsthat promote the coalescence of small lipid rafts intolarger rafts. Indeed, there is evidence that caveolin-1,a cholesterol-binding protein that resides on the cyto-plasmic side of lipid rafts, forms large homo-oligomers inthe apical route51 (FIG. 1, insets). Luminal and cytoplasmicclustering of lipid rafts could facilitate apical sorting ofGPI-APs not only at the TGN but also at other sortingstations in the biosynthetic or recycling routes (FIG. 1).

So, a luminal lectin with an affinity for the mannosecores of GPI-APs might promote their clustering andsorting into the apical route, in cooperation witholigomerized caveolin on the cytoplasmic side of themembrane (FIG. 2, insets). There is no evidence yetthat a similar mechanism could operate for other

FRAP

(fluorescence recovery afterphotobleaching). A live-cell-imaging technique used to studythe mobility of fluorescentmolecules. A pulse of high-intensity light is used toirreversibly photobleach apopulation of fluorophores in atarget region. Recovery offluorescence in the bleachedregion represents movement offluorophores into that region.

FRET

(fluorescence resonance energytransfer). The non-radiativetransfer of energy from a donorfluorophore to an acceptorfluorophore that is typically <80 Å away. FRET will onlyoccur between fluorophores inwhich the emission spectrum of the donor has a significantoverlap with the excitation ofthe acceptor.

CAVEOLAE

Flask-shaped invaginations ofthe plasma membrane that arecoated with the protein caveolin.Caveolae are endocytosed in aclathrin-independent manner.

CHOROID PLEXUS

A capillary bed that is coveredby transporting epithelial cells,and that protrudes on thecerebral ventricles. The cells areresponsible for producingcerebral spinal fluid.


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recombinant-DNA techniques causes their retention atthe Golgi complex31. Adding peptides that contain thetail of VSVG to the cytoplasm of MDCK cells blocks thevesicular release of the VSVG protein from both theGolgi complex and the endoplasmic reticulum64–66 butdoes not affect the exit of the epidermal growth factorreceptor (which has a different basolateral signal) fromthese organelles66. This indicates that basolateral-likesignals operate at various levels of the secretory pathway.Similar peptide-blocking experiments in fibroblasts,which lack apical–basal polarity, showed that basolat-eral signal peptides selectively abrogated the exit ofexogenous basolateral proteins from the TGN, butdid not affect the exit of exogenous apical proteins65.

for ankyrin — a spectrin-binding protein that is linkedto the basolateral actin cytoskeleton — which mediatesits selective retention at the basolateral membrane63.Finally, the Na+/K+-ATPase might be retained at the lateral membrane through its β-subunits, which haveadhesive properties1. Alternative mechanisms for baso-lateral- (and apical-) domain-selective retention includeinteraction with scaffold proteins, such as proteins thatcontain PDZ DOMAINS (reviewed in REFS 38,60).

A recognized role of both apical and basolateraltrafficking signals is to promote the sorting of cargoproteins into different vesicles at the Golgi complex,plasma membrane or recycling endosomes. Removingsorting signals from certain basolateral proteins by

PDZ DOMAIN

A protein-interaction domainthat often occurs in scaffoldingproteins, and is named after thefounding members of thisprotein family (Post-synapticdensity protein of 95 kDa, Discslarge and Zona occludens-1).

Caveolin?AP2Clathrin

AP1BClathrin

Caveolin?MAL?

5 4

4

1

86

6

7

7

3c

3b

3a

2b

2a

8

AP4?

AP3?

AP4?

AP1AGGA

AP2Clathrin

AP1A?Clathrin

Lipidrafts

Tightjunction

BSE

LE LYS

CREpH 5.8

AREpH 6.5

Caveolin?

Caveolin?MAL?

Apical sorting signals• GPI, N-glycans, O-glycans• Proteinaceous motifs

ASE

TGN

TGN

Clusteredlipid rafts Non-clathrin

AP4 coat?

Smalllipid raft

Adaptor?

Clathrin

Clustering factoror lectin

Caveolin-1 oligomers

CRE

Clusteredlipid rafts

Clathrin

Smalllipid raft

AP1B

Basolateral sorting signals• YXXΦ• NPXY• LL• L

Figure 1 | Trafficking routes and sorting mechanisms in epithelial cells. Apical exocytic routes (1 and 4):glycosylphosphatidylinositol (GPI) anchors, N-glycans and O-glycans sort apical proteins at the trans-Golgi network (TGN), commonrecycling endosomes (CREs) and apical recycling endosomes (AREs). This sorting might involve clustering small lipid rafts into largerfunctional lipid rafts, a process that might be promoted by a luminal lectin, caveolin oligomers and MAL (inset). Basolateral exocyticroutes (2 and 5): basolateral signals interact with adaptors of the clathrin (adaptor protein-1 (AP1), AP3, GGA (Golgi-localized, γ-ear-containing, Arf-binding protein)) or non-clathrin (AP4) type at the TGN or CREs. AP1B operates at the level of CREs (route 5; inset);the participation of other adaptors in basolateral sorting is hypothetical. AP4 might mediate basolateral sorting through microtubulemotors. Newly synthesized lysosomal membrane proteins (LAMPs) seem to be transported to the lysosome via the basolateralmembrane151 (route 2 followed by route 7 to basal sorting endosomes (BSEs), late endosomes (LEs) and lysosomes (LYS)), althoughsome authors believe they follow a direct intracellular route (3c)152. Endocytic routes (6 and 7): endocytosed apical (route 6) orbasolateral (route 7) membrane proteins are internalized into apical sorting endosomes (ASEs) or BSEs by AP2, mix in CREs18,22,153

and are sorted into apical and basolateral exocytic routes by sorting signals that are similar to those used in the biosynthetic route69,70

(routes 4 and 5). Soluble proteins are sorted from membrane proteins in ASEs and BSEs, mixed in LEs and degraded in LYS.Biosynthetic route through endosomes (routes 3a–c): some newly synthesized basolateral proteins reach CREs directly from theTGN21,76–78 (routes 3a and 3b; via unknown adaptors) from where they are sorted to the basolateral membrane via AP1B (route 5).Mannose-6-phosphate receptors and their ligands (lysosomal hydrolases) move through clathrin-coated vesicles, possibly into LEs(route 3c) from where they are transported back to the TGN. Recycling and transcytosing apical membrane proteins transfer fromCREs to AREs (route 4). Some GPI-anchored proteins seem to use a transcytotic route (route 8) in Madin–Darby canine kidney cells23.


R E V I E W S

signals that interact with a family of organelle-specificadaptor protein (AP) complexes71. The interaction ofbasolateral signals with organelle-specific APs (whichcan be thought of as sorting receptors) explains the find-ing that the basolateral sorting of the LDL receptor canbe saturated by overexpression of the protein, whichresults in apical transport of the excess protein56.

Available evidence indicates that two members of afamily of heterotetrameric APs — AP1B72 and AP4(REF. 73) — are involved in the sorting of basolateralmembrane proteins (FIG. 2). AP1B shares subunits β, γand σ with AP1A, but has an epithelial-specific mediumsubunit (µ1B)74. This subunit is absent in LLC-PK1 cells(a porcine epithelial cell line), which explains the apicalmissorting of transferrin receptors and LDL receptorsin these cells72. Transfection of the µ1B subunit intoLLC-PK1 cells enables the AP1B complex to assembleand reverses the targeting defect72. AP1B promotes the

Accordingly, some biochemical properties that are asso-ciated with polarized pathways (for example, lipid-raftassociation) are preserved in fibroblastic cell lines67,68,which implies that vesicular-trafficking pathways with‘apical’ and ‘basolateral’ features might be present innon-epithelial cells that are normally thought to be‘non-polarized’.

Adaptins. A striking realization that came from the dis-covery of basolateral sorting signals was their remarkableresemblance to the signals used by receptors that areendocytosed through coated pits from the plasma mem-brane, or to signals that are used for endocytosis andtransport to lysosomes56,57 (FIG. 1). Early experiments hadindicated that similar basolateral sorting signals operatein the recycling and biosynthetic routes69,70. These find-ings are now understood, as endocytic, recycling andbasolateral sorting signals belong to a family of peptide

RETROMER COMPLEX

A protein complex that consistsof Vps35,Vps26,Vps29,Vps17and Vps5, which was discoveredthrough genetic screens inSaccharomyces cerevisiae. Itfunctions in the retrieval ofproteins from the prevacuolarcompartment and transport tothe Golgi.

Rab5

Cdc42Rab8RalAExocystPKDMyo1Actin

5 4

4

1

86

6

7

7

3c

3b

3a

2b

2a

8

Cdc42Rab8RalAExocystPKD

Rab4,5Dynamin 2

Lipidrafts

Tightjunction

BSE

LE LYS

CRE

AREASE

TGN

CREDynamin

Clustering factoror lectin

Caveolin-1 oligomers

Myo6DAB2ARF6/ARNODynamin 1

Rab17

MyoVbRab11aRab11-FIP2Rab25Rab3bRetromer

KinesinDyneinCdc42Dynamin

Hip1R

PKD

RalARab8Exocyst

TGN

DynaminKinesin?

PKD

Kinesin

Adaptor AP1B

Figure 2 | Machinery that controls polarized vesicular trafficking in epithelial cells. The vesicular-trafficking routes correspond tothose in FIG. 1. Sorting signals promote the incorporation of apical proteins at the trans-Golgi network (TGN) or in common recyclingendosomes (CREs) into clustered lipid rafts that recruit microtubule motors (such as kinesin) directly or through unknown adaptors; insome cases (rhodopsin) the motor (dynein) is recruited through direct interactions. The motors generate tubular elements that movealong microtubules (inset). Basolateral proteins are incorporated into clathrin-coated or into uncoated tubules (which are pulled byunidentified microtubule motors that are presumably different from those used by apical proteins). Cdc42 stimulates the exit of apicalproteins and inhibits the exit of basolateral proteins from the TGN92 through unknown downstream effectors and might have similarfunctions at CREs. Fission of apical transporters is mediated by the GTPase dynamin83,86; protein kinase D (PKD) promotes vesiclerelease for the basolateral route110 (see inset). Dynamins 1 and 2, the adaptor DAB2 and the GTPase ARNO regulate specific endocyticsteps38. Other regulators of protein trafficking include phosphatidylinositol 4-kinases154, heterotrimeric G-proteins65,135 and RETROMER155.Microtubule and actin motors (myosins I, II, Vb and VI) probably participate at several stages of endocytic and biosynthetictrafficking38,93. Hip1R, a linker between actin and clathrin, promotes the release of clathrin-coated vesicles that contain mannose-6-phosphate receptors from the TGN156. The production of a vesicle from a donor compartment is coordinated with delivery to theacceptor compartment by various compartment-specific Rab proteins18,38. The exocyst, together with RalA and Rab8, coordinatesbasolateral exocytic routes, whereas Rab25 and Rab11 coordinate apical recycling157. ARE, apical recycling endosome; ASE, apicalsorting endosome; BSE, basal sorting endosome; LE, late endosome; LYS, lysosome.


R E V I E W S

LDL, transferrin and mannose-6-phosphate, but theirstudy did not fully clarify the organelle location of thissorting event. CLATHRIN is expected to be involved inbasolateral protein sorting (FIG. 2), as AP1, AP2 and AP3(but not AP4) have binding sites for clathrin71. Andbiochemical and immunoelectron-microscopy experi-ments show that clathrin is associated with the trans-ferrin receptor in endosomes81 and with VSVG proteinin µ1B-expressing LLC-PK1 cells, but not in wild-typecells76. However, no functional evidence is available yetto support a role for clathrin in polarized transport.

Transport to the plasma membraneThe sorting of apical and basolateral proteins at theTGN and common recycling endosomes must be coor-dinated with their incorporation into vesicular andtubular transport intermediates and the transport ofthese intermediates across the cytosol to specific areas of the cell surface. These different processes are coordi-nated by various factors including organelle-specific RAB proteins, elements of the actin and microtubulecytoskeletons and a particle known as the exocyst (FIG. 2).

The microtubule cytoskeleton. Extensive experimenta-tion with microtubule-disrupting agents showed a selec-tive requirement for microtubules in the transport ofapical membrane proteins (reviewed in REF. 82) but didnot determine at what step microtubules were required.Recent live-cell-imaging experiments indicate impor-tant roles for microtubules and microtubule motors inthe exit of apical proteins (and possibly some basolat-eral proteins) from the TGN and endosomes (FIG. 2,inset) and in their transport across the cytosol83. Innon-polarized cells84–89 and in fully polarized MDCKcells23,88, GFP-tagged apical and basolateral markerswere shown to leave the Golgi complex in long, tubulartransport intermediates and small vesicles that movealong microtubule tracks.

Three lines of evidence imply that specific micro-tubule motors participate in the sorting/transport ofapical proteins. First, antibodies that were directedagainst the motor protein kinesin blocked the produc-tion of tubules that contained the apical markerp75NTR–GFP; they also blocked transport from theGolgi area to the cell surface of small vesicles contain-ing p75NTR–GFP, which were presumably releasedfrom the Golgi by an independent mechanism86. Theseantibodies cause basolateral missorting of apical mark-ers in polarized MDCK cells (G.K., unpublished data).Second, cytoplasmic dynein, a minus-end-directedmicrotubule motor, interacts with the C-terminal endof rhodopsin through the dynein light chain TCTEX,and is crucially involved in the apical transport ofrhodopsin in MDCK cells35. Third, the kinesin-familymember KIFC3, another minus-end-directed micro-tubule motor, has been associated with the apical deliv-ery of influenza HA and annexin-13b (REF. 90); however,the motor function of KIFC3 in this process has stillnot been shown. These experiments are consistentwith the observation that microtubules orientate theirnegative ends towards the apical pole of polarized

basolateral sorting of the LDL and transferrin receptorsat recycling endosomes rather than at the TGN, as hasbeen established by recycling assays75 and colocalizationwith endosomal, rather than TGN, markers75,76. Recentwork21,76 indicates that VSVG protein might transitthrough recycling endosomes en route to the plasmamembrane (see routes 3a and 3b in FIGS. 1, 2), in agree-ment with previous data for other basolateral pro-teins77,78. Experiments with antibodies that block thefunction of AP1B indicate that AP1B might sort baso-lateral proteins in a post-TGN compartment at thecrossroads of both the recycling and the biosyntheticroutes (J. Cancino, A. Soza, G. Mardonez, E.R.-B. andA. Gonzalez, manuscript in preparation).

AP1A and GGAs (Golgi-localized, γ-ear-containing,Arf-binding proteins) are ubiquitous ADAPTINS that,unlike AP1B, localize to the TGN and cooperate insorting the mannose-6-phosphate receptor from theTGN to endosomes71 (see route 3c in FIGS 1,2). It isunknown whether they participate in basolateral sort-ing at the TGN or endosomes. An interesting outcomeof the studies on AP1A and AP1B is the realizationthat their different µ-subunits, which are more than80% identical, mediate the different organelle localiza-tions of these adaptors. This could depend on the µ-subunits binding to TGN- or endosome-specificphosphatidylinositol (PtdIns) phosphate lipids, as itwas recently shown that AP1A binds through its µ1Asubunit to PtdIns 4-phosphate that is synthesized by aGolgi-localized PtdIns 4-kinase79.

The AP3 adaptor protein regulates the exit of exoge-nous VSVG from the Golgi complex in non-polarizedcells80, but its role in basolateral sorting in polarized cellshas not been studied yet. Simmen et al.73 have proposeda role for AP4 in basolateral sorting of receptors for

ADAPTIN

A tetrameric (for example, AP1,AP2, AP3 and AP4) ormonomeric (such as GGAs)protein that promotes theformation of coated vesicles.Adaptins might interact withclathrin, small GTPases (such asArf1) and microtubule-basedmotor proteins.

CLATHRIN

The main component of the coatthat is associated with clathrin-coated vesicles, which areinvolved in membrane transportboth in the endocytic andbiosynthetic pathways.

RAB

A small protein with GTPaseactivity that is involved in theformation and delivery ofvesicles.

Box 2 | The flexible epithelial phenotype

Different epithelial cell types show ‘flexibility’ in theirpolarized trafficking, which is defined as the ability tolocalize a given protein to different regions of the cellor to use a different targeting route to reach the same(usually apical) region15. Examples of the firstscenario occur in the retinal pigment epithelium137

and the LLC-PK1 kidney-tubule cell line138, in whichseveral proteins that are basolateral in Madin–Darbycanine kidney (MDCK) cells are concentrated on theapical surface. Examples of the second scenario areprovided by apical proteins such as dipeptidylpeptidaseIV or influenza haemagglutinin, which aretransported by a direct route from the trans-Golginetwork in MDCK cells, but follow a transcytoticroute in the liver, intestinal epithelium or retinalpigment epithelium139,140, or even change from atranscytotic to a direct route (for dipeptidylpeptidaseIV) as the epithelial monolayer matures in vitro141.Liver cells represent an extreme case in which a largemajority of apical proteins use the transcytoticroute142; however, some multispan membrane proteinsuse a direct delivery route from the Golgi complex tothe bile canalicular surface of hepatocytes143.


R E V I E W S

TGN of MDCK cells86 and the release of VSVG–GFPin non-polarized cells97. Finally, actin could negativelyregulate the release of some proteins from the TGN byforming a physical barrier that must be removed forvesicular budding to occur.

In summary, the microtubule and the actincytoskeletons have important regulatory roles in sortingapical and basolateral proteins in the TGN, recyclingendosomes and at the plasma membrane. The mecha-nisms underlying this regulation and the roles of spe-cific actin and microtubule motors in the generation oftransport intermediates are still largely unknown. Themicrotubule and actin cytoskeletons are also involved atspecific steps in the transport of vesicular and tubulartransport intermediates across the cytosol (FIG. 2). Futurework needs to elucidate in detail the participation ofthese motors in the transport of specific cargos andtheir regulation.

Polarized exocytosis of transport intermediatesThe release of transport intermediates that carry apicaland basolateral proteins from the TGN and endosomesmust be coordinated with their transport across thecytoplasm and their insertion in the plasma membraneat specific sites. Work over the past 10 years has identi-fied various components of the exocytic machinerythat transport apical and basolateral proteins and hasstarted to identify the mechanisms that regulate theirlocalization and function (FIG. 3).

Apical exocytosis is mediated by a tetanus-toxin-insensitive vesicle membrane (v)-SNARE (ti-VAMP)98 andby the target membrane (t)-SNAREs syntaxin-3 andSNAP23 (REFS 99–101). Epithelial-specific Munc18-2 reg-ulates the assembly of the apical SNARE complex102.Annexin-2 and annexin-13B might promote docking of

epithelial cells (discussed below). Microtubules alsoform arrays of mixed polarity underneath the apicalmembrane, which implies that plus-end-directedmotors might also participate in apical delivery.

The actin cytoskeleton. The actin cytoskeleton has anincreasingly recognized role in the assembly of vesiculartransport intermediates at the Golgi complex and onrecycling endosomes, and in their translocation acrossthe cytoplasm83. Actin, actin-associated proteins (suchas spectrin, ankyrin and myosins I, II and VI) andactin-regulatory proteins (Cdc42 and several of itsdownstream effectors, and dynamin) are present in theGolgi complex (reviewed in REF. 91). Interfering with thefunction of Cdc42 using constitutively active or inactivemutants slows the exit of the basolateral protein N-CAM (neuronal cell-adhesion molecule) but accel-erates exit of the apical protein p75NTR from theTGN92. Inhibiting actin polymerization causes missort-ing of basolateral proteins from recycling endosomesand promotes their TRANSCYTOSIS to the apical surface93,94.

How do actin filaments work in apical–basolateralsorting? They could function conventionally as tracks forthe myosin-driven movement of vesicles by Rabs — ashas been suggested for myosin VI and Rab11 in apicalrecycling95. In vitro assays indicate that a myosin activitypromotes the release of basolateral, but not apical,vesicles from the TGN of MDCK cells96, but the iden-tity of this myosin activity remains controversial83.Alternatively, by analogy with its role at the plasmamembrane, actin might participate in a less conventionalrole — that is, in the fission of vesicles from the Golgicomplex in response to dynamin or downstream effec-tors of Cdc42 (REF. 97) (FIG. 3, inset). Dominant-negativedynamin inhibits the release of p75NTR from the

TRANSCYTOSIS

Transport of macromoleculesacross a cell, which consists ofthe endocytosis of amacromolecule at one side of amonolayer and its exocytosis atthe other side.

PRENYLATION

The enzymatic addition ofprenyl moieties (geranyl,farnesyl or geranylgeranylgroups) to a protein as a post-translational modification.

Box 3 | Sorting by lipid rafts

The influential lipid-raft hypothesis41 postulates that many apical proteins are sorted through their affinity formicrodomains of glycosphingolipids and cholesterol (lipid rafts) that are initially assembled in the Golgi complex. Thisproposal is supported by strong experimental evidence:• First, many apical proteins, such as influenza haemagglutinin (HA)67 and glycosylphosphatidylinositol (GPI)-anchoredproteins (GPI-APs)144, become insoluble in non-ionic detergents at 4oC as they reach the Golgi complex. Detergentinsolubility reflects lipid-raft-association, as raft lipid components are insoluble in non-ionic detergents at 4oC.Biophysical techniques such as fluorescence recovery after photobleaching (FRAP), which measure lipid-raft associationmore accurately than detergent insolubility does, show a good correlation between apical targeting for HAs andmutations in the transmembrane anchor that disrupt association with lipid rafts145.

• Second, epithelial cell types that fail to incorporate GPI-APs into detergent-insoluble complexes missort these proteinsto the basolateral membrane146.

• Third, depletion of glycosphingolipids or cholesterol results in the missorting of GPI-APs and influenza HA on theirway to the apical plasma membrane of Madin–Darby canine kidney (MDCK) cells147,148. Additional work is necessary toshow that missorting results from the disruption of lipid rafts in the sorting compartment rather than indirectly fromdefective PRENYLATION of key apical sorting factors or from increased signalling by ceramide precursors in cells that havebeen exposed to inhibitors of glycosphingolipid synthesis.

• Finally, lipid rafts are enriched in the apical membrane41,42. There is strong evidence for the presence and sorting of lipidrafts in the endocytic and recycling pathways but there is no definitive evidence that raft lipids have the intrinsic ability tobe sorted apically in the trans-Golgi network (TGN). Early results in MDCK cells149 have been contested by laterevidence150. Furthermore, it remains unclear whether lipid sorting drives protein sorting or vice versa. Progress in thisarea has been slowed by technical difficulties with available raft lipids tagged with fluorescent dyes; these lipids frequentlylose their association with lipid rafts, which makes the interpretation of live-cell-imaging studies difficult.


R E V I E W S

The exocyst. Further factors are required, in addition to the SNARE machinery, to promote translocation oftransport intermediates to the apical or basal poles of cells. Basolateral vesicle delivery requires tethering ofthe vesicles by a highly conserved complex of eight pro-teins that is known as the exocyst107 (FIG. 3). A tetheringfactor for vesicles in the apical route has not been identi-fied yet. Exocyst antibodies selectively block the transportof basolateral proteins in MDCK cells but do not inhibitthe delivery of apical proteins108. Interestingly, the exocysthas a second role at sorting organelles — it probablyparticipates in the formation of basolateral transportvesicles from a perinuclear compartment109 — and a thirdrole at the endoplasmic reticulum, where it selectivelymodulates the synthesis of basolateral proteins107.

Consistent with these different functions, the exocystlocalizes not only at the junctional complex108, but alsoat a perinuclear compartment109, which is more likely to comprise recycling endosomes than the TGN76. Bothof these localizations might be regulated by AP1B76,whereas the junctional localization of the exocyst is promoted by E-cadherin and nectin110. The smallGTPases RalA111 and Rab8 (REF. 112; the yeast orthologueof which (Sec4) interacts genetically with the exocyst)have activities that are consistent with the regulation ofthe vesicular-trafficking roles of the exocyst. In MDCKcells, dominant-negative Rab8 inhibited basolateralexocytosis113, and constitutively active Rab8 promotedmissorting of basolateral proteins112. The dual regulationof both production and tethering of basolateral vesiclesby the exocyst resembles the dual trafficking role of Rabproteins and probably contributes to the homeostasis ofthe basolateral membrane.

The cytoskeleton and polarized traffickingGrowing evidence indicates that exit from the sortingorganelles (TGN and recycling endosomes) and translo-cation to the plasma membrane are also coordinated bythe actin and microtubule cytoskeletons, which show adivision of function in this regard. Whereas micro-tubules seem to be important in the organization ofapical exocytosis, the actin cytoskeleton seems to be anorganizer for basolateral exocytosis.

Microtubules and apical delivery. The organizing roleof the microtubule cytoskeleton in apical exocytosis is shown by live-cell-imaging experiments in non-polarized and polarized MDCK cells (FIG. 4). TOTAL-

INTERNAL-REFLECTION FLUORESCENCE MICROSCOPY, which allowshigh-resolution analysis of cell–substrate contact areas,shows extensive fusion of post-Golgi transport inter-mediates that contain p75NTR–GFP with the basalplasma membrane of non-polarized MDCK cells88

(and also, presumably, with the rest of the plasmamembrane, which cannot be imaged with this tech-nique). By contrast, post-Golgi transport intermediatesthat contain p75NTR–GFP fuse with neither the basalnor the lateral membrane in polarized MDCK cells;therefore, they must fuse with the apical surface, althoughthis event has been difficult to image so far88 (FIG. 4).The restriction of fusion sites to the apical membrane

apical transport intermediates by remodelling corticalactin at the apical pole and/or chaperoning the interme-diates to specific attachment sites103,104.

Basolateral post-Golgi transporters dock at the cellsurface using a tetanus-sensitive v-SNARE (ts-VAMP)and the basolateral v-SNAREs syntaxin-4 and SNAP23(REFS 98,100). The assembly of the basolateral SNAREcomplex is regulated by Munc18c (REF. 105). It wasrecently shown that Mlgl, the mammalian homologueof Lethal giant larvae (Lgl, a D. melanogaster proteinthat is essential for epithelial polarity) interacts withsyntaxin-4 at the lateral membrane of MDCK cells106.Although functional data are still lacking, the availableevidence indicates that Mlgl regulates the syntaxin system that is involved in basolateral exocytosis (FIG. 3).

SNARE

(soluble N-ethylmaleimide-sensitive fusion proteinattachment protein (SNAP)receptor). SNARE proteins are afamily of membrane-tetheredcoiled-coil proteins that regulatefusion reactions and targetspecificity in vesicle trafficking.They can be divided into v-SNAREs and t-SNAREs on thebasis of their localization.

Tethering particlesand receptors(unknown)

Non-centrosomalmicrotubules

UnknownSNAREs

Syntaxin-3SNAP23

Zonulaoccludens

Zonulaadherens

CrumbsPals1PatjPAR3

PAR6aPKC

PAR1

PAR4

Transcytoticand recyclingapical vesicles

Direct andrecyclingbasolateralvesicles

Syntaxin-4SNAP23

Syntaxin-4SNAP23

Nectin

Directapicalvesicle

ti-VAMP

Munc18-2

ts-VAMP

Munc18c

Junctionalcomplex

Syntaxin-4SNAP23

ScribbleDiscs large

Lethal giant larva

Cdc42RalARab8

Exocyst

Figure 3 | The exocytic machinery of MDCK cells. At the apical surface: apical vesicles in thedirect route contain a tetanus-insensitive v-SNARE (ti-VAMP) that heterotrimerizes with the apicalsyntaxins SNAP23 and syntaxin-3 (REF. 98), a process that is regulated by Munc18-2. Apicalsyntaxins are restricted apically by microtubules, using unknown mechanisms88. PAR1, which islocated near the junctional complex, organizes microtubules non-centrosomally into vertical andsubapical networks121. Unknown SNAREs mediate the fusion of transcytotic and recycling apicalvesicles with the apical membrane. At the basolateral surface: basolateral vesicles carry atetanus-sensitive v-SNARE (ts-VAMP) that interacts with syntaxin-4 and SNAP23, which arecomponents of the basolateral docking/fusion machinery. The syntaxins localize to the lateralmembrane independently of microtubules. Many proteins that are involved in regulatingbasolateral exocytosis (indicated by a green background) are orthologues of regulators of thesecretory pathway in Saccharomyces cerevisiae. These include Cdc42, exocyst, RalA, Rab8(Sec4 in yeast) and Lethal giant larvae (Lgl; Sro7/Sro77 in yeast). Lgl interacts with syntaxin-4 buta vesicular-trafficking regulatory role has not been shown. The recruitment of exocyst to thejunctional area is promoted by nectin158 and is a key determinant of the fusion of vesicles in thisarea. The products of three sets of physically and/or genetically interacting polarity genes(Crumbs–Pals1–Patj; atypical protein kinase C (aPKC)–PAR3–PAR6; scribble–discs-large–Lgl)19

along with PAR1 and PAR4 are also likely to have important vesicular-trafficking regulatory roles.These gene products are restricted to either the apical or lateral domain, and are often concentratedjust above or below the apical junctional complex. Their crosstalk ensures the maintenance ofdistinct epithelial surface domains. SNARE, soluble NSF (N-ethylmaleimide-sensitive fusion protein)accessory protein (SNAP) receptor; VAMP, vesicle-associated membrane protein.


R E V I E W S

cells, syntaxin-4 shifts from a random localization to alateral membrane distribution. That syntaxin-4 has abroader localization than just basolateral exocyticsites indicates that additional factors (such as the exo-cyst) contribute to the localized fusion of basolateraltransport intermediates near junctional areas of theplasma membrane.

The basolateral delivery routes to the lateral mem-brane and the lateral localization of syntaxin-4 are notantagonized by microtubule disruption; instead, evidenceis accumulating that the actin cytoskeleton and its regula-tors are involved in this process (FIG. 3). Several ortho-logues of gene products that control the docking andfusion of post-Golgi vesicles in S. cerevisiae regulate baso-lateral exocytosis. Paramount in both yeast and MDCKsystems is Cdc42. Expression of dominant-negative oractivated Cdc42 disrupts both post-Golgi targeting andendosomal recycling of basolateral proteins in MDCKcells115,116. Consistent with this observation, depolymeriz-ing the actin cytoskeleton disrupts basolateral targeting ofrecycling basolateral proteins93,94.

Epithelial lumen formationThe role of PAR proteins. Simple (non-stratified) epithe-lial cells position their lumen at the apex in columnarepithelia (which is therefore known as the apical domain,for example, in the kidney and intestine) or at the lateral

parallels the redistribution of syntaxin-3 to the apicalsurface88. Importantly, disrupting microtubules resultsin the depolarization of syntaxin-3 and the fusion ofpost-Golgi transport intermediates that containp75NTR–GFP with the basal membrane in a syntaxin-3-dependent manner88 (FIG. 4). These experiments stronglyindicate that microtubules can organize apical traffickingroutes, at least in part through their capacity to positionsyntaxin-3 at the apical membrane. How they accom-plish this is not yet understood. Also unclear is the exactsite in the apical membrane at which apical transportintermediates dock and fuse. Early experiments byLouvard indicate that delivery could occur in the vicinityof the junctional area114.

Actin and basolateral delivery. In contrast to the key roleof microtubules in organizing the apical exocyticmachinery, the leading role in the organization of vesicu-lar exocytosis to the basolateral pole seems to be held bythe actin cytoskeleton. In subconfluent, non-polarizedMDCK cells, post-Golgi vesicles carrying basolateralproteins fuse randomly with the plasma membrane;this correlates with the random distribution of thebasolateral t-SNARE syntaxin-4 (REF. 88). By contrast, inpolarized MDCK cells, post-Golgi vesicles with basolat-eral proteins fuse exclusively with the top third of thelateral membrane (FIG. 4). During polarization of MDCK

TOTAL-INTERNAL-REFLECTION

MICROSCOPY

Fluorescence-microscopytechnique with significant depthdiscrimination that canselectively excite only thosefluorescent molecules within 100 nm of the interface betweena cell and a coverslip.

Non-polarized MDCK cell Polarized MDCK cell

Nucleus

Golgi

Fusion withbasal membrane

Apical vesicle-fusion site

Basolateralvesicle-fusion site

Polarized MDCK cells+ microtubule depolymerization

Tightjunction

Basolateralvesicle-fusion site

Tightjunction

a b c

Apical vesicle-fusion site

Basolateral carrier Apical carrier

Figure 4 | Microtubules organize vesicular trafficking to the apical pole. a | Total-internal-reflection fluorescence microscopyshows fusion of apical and basolateral post-Golgi transport intermediates with the basal surface in subconfluent Madin–Darbycanine kidney (MDCK) cells. b | These fusions are not observed in polarized MDCK cells88. In polarized MDCK cells, basolateralpost-Golgi transport intermediates (which contain low density lipoprotein (LDL) receptors) fuse with the upper third of the lateralmembrane as determined by spinning disc confocal microscopy88 (see also BOX 1). The restriction of basolateral fusion sites to thelateral membrane correlates with a restricted localization of fusion (syntaxin-4) and tethering (exocyst) machinery (see FIG. 3). Inpolarized MDCK cells, post-Golgi transport intermediates that contain the apical marker p75 neurotrophin receptor (p75NTR)tagged with green fluorescent protein fuse directly with the apical surface. c | Microtubule depolymerization results in a dispersedGolgi complex and the fusion of apical, but not basolateral, post-Golgi transport intermediates with the basal membrane; thesefusions are inhibited by antibodies to syntaxin-3. Microtubule depolymerization does not disrupt the fusion of basolateral post-Golgitransport intermediates with the lateral membrane.


R E V I E W S

protein that, when prevented from binding 14-3-3 pro-teins by mutation of a potential PAR1 phosphorylationsite127, interferes with MDCK lumen formation in a sim-ilar manner to PAR1 (REF. 128). In some systems, PAR4(another serine/threonine kinase, also known as LKB1)activates PAR1, which implies that there are possiblecommon polarizing signalling routes that are mediatedby PAR1, PAR3 and PAR4 (REF. 129). Recent work130 hasshown that PAR4, under the influence of the adaptorprotein STRAD (STE20-related adaptor protein), pro-motes polarization of intestinal epithelial cells withpolarized apical MICROVILLI and luminal proteins, in theabsence of cell–cell contacts — that is, in single cells.

PAR1, however, seems to be unique in that it promotesnot only lumen formation per se, but it also regulateslumen position. Overexpression of PAR1 locks MDCKcells in a ‘liver polarity phenotype’, with lateral luminathat resemble bile canaliculi121 (FIG. 5) and, interestingly,promotes a transcytotic route for apical proteins,which is also a characteristic of liver epithelial cells131.These experiments imply that levels of PAR1 activitymight control the developmental decision between theestablishment of columnar or hepatic epithelia, a hypoth-esis that needs to be tested experimentally. The lumenpolarity and traffic-organizing functions of PAR1 mightbe mediated by its known ability to regulate micro-tubules. Inhibition of PAR1 function prevents thechange in the organization of microtubules from theradial centrosomal array that is found in non-polarizedMDCK cells (and in other non-polarized cells such asfibroblasts) to the non-centrosomal, vertical microtubulearrays that are characteristic of polarized epithelia (FIG. 5).Overexpression of PAR1 in MDCK cells organizesmicrotubules in horizontal arrays with their minus endsfacing lateral lumina, which is similar to the organizationthat is found in liver cells121.

Concluding remarksThe study of the generation and maintenance ofepithelial polarity has been, and will continue to be, achallenging endeavour, as polarization involves thecoordination of all of the main subcellular systems in

membrane in liver cells (which are known as BILE CANALI-

CULI)12,14. The mechanisms that are involved in the for-mation and positioning of epithelial lumina are stillpoorly understood. The process of lumen formationand tubulogenesis in MDCK cells has been studiedusing the Ca2+ switch117 and using MDCK cells embed-ded in collagen18,118. In the Ca2+-switch experiment (FIG. 5), MDCK cells that are cultured in low (~5 µM)concentrations of Ca2+ accumulate luminal (apical)markers intracellularly in large vacuoles that are knownas vacuolar apical compartments (VACs)119; equivalentstructures accumulate basolateral markers under theseconditions120.VAC-like structures have been observed inpathological conditions — for example, in transformedepithelial cells (reviewed in REF. 121) — but are difficultto observe in subconfluent MDCK cells. After switchingto normal concentrations of Ca2+ (2 mM), the VACsmove towards the lateral membrane, where they formtransient intercellular lumina that are reminiscent ofliver bile canaliculi; these lateral lumina progressivelyrelocate to the apical domain121,122. Similar transientlateral lumina are observed in MDCK cells that arepolarizing within collagen gels118,121 or under standardconditions after dissociation by trypsin (D. Cohen andA.M., unpublished data). Importantly, transient laterallumina are also observed in vivo, in developing ratintestinal epithelial cells123. These data indicate that tran-sient lateral lumina represent an intermediate morpho-genetic step in the generation of polarity in columnarepithelial cells.

Important regulators of the processes of lumen for-mation and polarization are the partitioning (PAR)genes (FIG. 3). PAR genes were discovered as cell-fatedeterminants in the C. elegans embryo; many studieshave characterized their participation in the formationof the tight-junction complex, which is a key aspect ofcell polarization124. Inhibition of PAR1 prevents the for-mation of MDCK-cell lumina in three-dimensional collagen-gel assays121. PAR1 is a serine/threonine kinasewith the unique ability to bind and transfer 14-3-3 PROTEINS

to its substrates125,126. A candidate PAR1 substrate in thelumen-assembly pathway is PAR3, a PDZ-domain

BILE CANALICULI

Tiny channels on the surface ofliver cells that collect the bile thatthey produce.

14-3-3 PROTEIN

A scaffolding protein thatregulates the localization ofother proteins by binding toconserved phosphotyrosine-containing motifs in aphosphorylation-dependentmanner.

MICROVILLI

Small, finger-like projections(1–2-µm long and 100-nmwide) that occur on the exposedsurfaces of epithelial cells tomaximize the surface area.

MICROTUBULE-ORGANIZING

CENTRE

(MTOC). Also called thecentrosome or spindle-polebody, this structure nucleatesand organizes microtubules.

Non-polarized MDCK cell Liver polarity stage Columnar MDCK cell

VAC

MTOC

Microtubules

PAR1(low levels)

PAR1

PAR1(high levels)

Apical

Basal

+

–

TJ

TJ

+ –

a b c

Figure 5 | PAR1 controls epithelial microtubule organization and lumen morphogenesis. a | Madin–Darby canine kidney(MDCK) cells cultured at 5 µM Ca2+ lack cell–cell contacts117 show a centrosomal microtubule array with the negative endsemanating from a juxtanuclear MICROTUBULE-ORGANIZING CENTRE (MTOC)121,159 and accumulate luminal (apical) markers (green) inlarge intracellular vacuoles that are known as vacuolar apical compartments (VACs)119. b | After addition of Ca2+, MDCK cellsestablish E-cadherin-mediated cell–cell contacts. VACs are exocytosed, forming intercellular lumina (green) that are surrounded bytight junctions (TJs; red) — these are reminiscent of liver bile canaliculi122. Microtubules rearrange to a horizontal position, with theMTOC immediately under the lumen121 — such an arrangement is typical of hepatocytes82. c | Several hours after the addition ofCa2+, the lateral lumen is displaced to the apical surface122 and the microtubules acquire a non-centrosomal vertical arrangementthat is typical of columnar epithelial cells121,159. PAR1 promotes the formation of lateral lumina and inhibits the progression tocolumnar morphology.


R E V I E W S

pathway from the mother cell to the budding daughtercell in S. cerevisiae.

Future research will uncover important details ofthe machinery that is involved in Golgi and endoso-mal sorting, including the roles of lipid rafts, sortingsignals, adaptins and the cytoskeleton. Individualmicrotubule- and actin-based motors that participatein the exit of various classes of proteins from the TGNneed to be identified. The molecular interactions thatunderlie the control by the microtubule cytoskeletonand the actin cytoskeleton of the establishment ofluminal and basolateral docking sites for post-Golgiroutes will be elucidated.

MDCK cells will continue to be an important tool inthis task, as they are the most well-studied mammalianepithelial cell and have provided the context for thedevelopment of the most incisive cell-biological tech-nology with which to test hypotheses and candidategenes in polarized vesicular trafficking.

response to cues that are provided by cell–cell andcell–substrate contacts. The establishment of polarizedtrafficking requires precise coordination betweenmechanisms that are involved in the sorting of plasma-membrane proteins into post-Golgi and post-endosomaltransport vesicles, and the delivery of these vesicles torestricted sites at the cell surface. Several apical andbasolateral routes exit the Golgi complex and fuse ran-domly with the cell surface in non-polarized cells. Thereorganization of the microtubule cytoskeleton thatoccurs during epithelial polarization under the controlof signalling proteins such as PAR1 and PAR4 leads tothe apical delivery of luminal markers, probably byrestricting specialized docking and fusion machineryto the apical surface. The organization of basolateraldelivery to the apical third of the lateral membrane is,instead, under the control of Cdc42, some of its down-stream effectors, and the actin cytoskeleton — thesame protagonists that are involved in the secretory

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AcknowledgementsWe gratefully acknowledge the support of the National Institutes ofHealth (E.R.-B.), a Jules and Doris Stein Professorship from theResearch to Prevent Blindness Foundation (E.R.-B.) and a CareerDevelopment Award from the American Heart Association (A.M.).We are grateful to A. Gonzalez, T. McGraw, J. Nelson and V. Malhotra for useful comments on the manuscript. Our goal tocite mostly primary references is not free of arbitrariness given thelimited number of references allowed by the format of the review andthe rapid growth of the field. We apologize that several excellentpapers could not be cited individually and could only be discussedindirectly or through reviews.

Competing interests statementThe authors declare no competing financial interests.

Online links

DATABASESThe following terms in this article are linked online to:Swiss-Prot: http://us.expasy.org/sprot/annexin 13b | µ1B | caveolin-1 | Cdc42 | GFP | HA | KIFC3 | LDLreceptor | p75NTR | PAR1 | PAR3 | Rab8 | Rab11 | RalA |rhodopsin | SNAP23 | STRAD | TCTEX | ti-VAMP | VIP-36 | VSVGAccess to this links box is available online.

ORGANIZATION OF VESICULAR TRAFFICKING IN EPITHELIA

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