Cilia and Polycystic Kidney Disease, Kith and KinLiwei Huang* and Joshua H. Lipschutz

In the past decade, cilia have been found to play important roles in renalcystogenesis. Many genes, such as PKD1 and PKD2 which, when mutated,cause autosomal dominant polycystic kidney disease (ADPKD), have beenfound to localize to primary cilia. The cilium functions as a sensor to transmitextracellular signals into the cell. Abnormal cilia structure and function areassociated with the development of polyscystic kidney disease (PKD). Ciliaassembly includes centriole migration to the apical surface of the cell, ciliaryvesicle docking and fusion with the cell membrane at the intended site ofcilium outgrowth, and microtubule growth from the basal body. This review

summarizes the most recent advances in cilia and PKD research, with specialemphasis on the mechanisms of cytoplasmic and intraciliary protein transportduring ciliogenesis. Birth Defects Research (Part C) 00:000–000, 2014.

VC 2014 Wiley Periodicals, Inc.

Key words: polycystic kidney disease; cilia; planar cell polarity; exocyst

IntroductionCilia are thin rod-like organelles found on the surface ofhuman eukaryotic cells. First described by Anthony vanLeeuwenhoek in 1675 (Dobell, 1932), they were originallydefined by their motility, being structurally and functionallysimilar to eukaryotic flagella. In 1876 and 1898 (Langer-hans, 1876; Zimmermann, 1898), another class of cilia wasdescribed, the solitary (or nonmotile) cilia, which wererenamed primary cilia in 1968 (Sorokin, 1968). Motile cilia/flagella have been studied for many years in single-celledorganisms, such as protozoa, and important insights intothe physiology and biochemistry of these organelles haveresulted. However, despite the established anatomical pres-ence of primary cilia in eukaryotic cells, until recently, littlehas been known about their specific function, and they hadeven been considered vestigial organelles (Webber and Lee,1975). Over the past decade, the primary cilium began toreceive increasing attention, after it was discovered thatproteins mutated in different forms of polycystic kidney dis-ease (PKD) were tightly associated with primary cilia.

PKD is a group of genetic disorders characterized bythe growth of numerous cysts in the kidney. Cysts are nor-mal “building blocks” for epithelial organs, but abnormalregulation of cystogenesis in the kidney results in cystickidney disease. Autosomal dominant PKD (ADPKD), whichaffects approximately 500,000 Americans and 12,000,000people world wide, is the most common potentially lethal

genetic disorder in humans (Grantham, 2001). Mutationsin PKD1, the gene encoding polycystin-1, and PKD2, thegene encoding polycystin-2, have been identified as thecause of ADPKD (The International Polycystic Kidney Dis-ease Consortium, 1995; Mochizuki et al., 1996). Autosomalrecessive PKD (ARPKD), a severe form of PKD thatpresents primarily in infancy and childhood, is caused by amutation in the polycystic kidney and hepatic disease1(Pkhd1) gene (Zerres et al., 1994). Nephronophthisis, aform of PKD that is the most common genetic cause ofrenal failure through age 30, is caused by mutations inmultiple (16 and counting) different nephronophthisis(NPHP) genes (Hildebrandt et al., 2009).

More and more evidence has accumulated showingthat gene mutations resulting in structural or functionaldefects of the primary cilium cause PKD. For example,ADP-ribosylation factor–like protein 13b (Arl13b), a smallGTPase of the Arf family, is highly enriched in cilia and isrequired for cilia formation. Knockdown of the Arl13bgene results in disorganized cilia structure and kidneycyst formation in zebrafish (Duldulao et al., 2009). Muta-tions in Arl13b in patients lead to the nephronophthisisform of PKD (Cantagrel et al., 2008). Another example iskinesin-like protein 3A (KIF3A), one of the subunits of thecilia motor protein, kinesin-2, which is essential for ciliaformation. Inactivation of KIF3A in renal epithelia inducedkidney cyst formation and severe PKD (Lin et al., 2003).Virtually all forms of PKD in human patients and animalmodels are associated with perturbations in renal primarycilia structure and/or function. Although maintenance ofprimary cilia structure and function appears to be key topreventing/treating PKD, it is not entirely clear how aber-rant ciliogenesis promotes the disease.

The purpose of this review is to summarize the mostrecent advances in cilia and PKD research, with an empha-sis on ciliary protein trafficking. Proteins located on theprimary cilium play important roles in cilia structure andassembly, and are also critical for the pathogenesis of PKD.Primary cilia function as sensors that transmit extracellu-lar signals into the cell and regulate downstream signal

1Department of Medicine, Eastern Virginia Medical School, Norfolk, Virginia2Department of Medicine, Philadelphia Veterans Affairs Medical Center,Philadelphia, Pennsylvania; Department of Medicine, University ofPennsylvania, Philadelphia, Pennsylvania

Supported by a grant from the Veterans Affairs (Merit Award to J.H.L.), andNIH (DK093625 to L.H., and 5P30 DK047757-18 pilot award to J.H.L.).

*Correspondence to: Liwei Huang, Lewis Hall Rm 2124, 740 W. Olney Rd,Norfolk, VA 23501. Email: huangle@evms.edu

Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). Doi: 10.1002/bdrc.21066

VC 2014 Wiley Periodicals, Inc.

pathways, such as the planar cell polarity (PCP) pathway,cell proliferation, and differentiation. The mechanisms ofprotein transport to, and inside, the primary cilia, andtheir role in ciliary function and PKD, are critical forproper ciliogenesis and function.

Ciliary Structure and AssemblyCilia are organelles protruding from the surface of humaneukaryotic cells, which extend outward from the basalbody, a cellular organelle related to the centriole. Figure 1shows the structure of the primary cilium. The membraneof the cilium is an extension of the cell membrane,although it is separated from the apical membrane by aseptin band (Hu et al., 2010). Cilia contain a central axo-neme composed of microtubules. The primary ciliary axo-neme, a cytoskeletal component that extends from thebasal body, consists of nine microtubule doublets arrangedtangentially around the center in a configuration known as910, whereas the motile ciliary axoneme is characterizedby a 912 architecture with nine outer microtubule dou-blets plus a central pair of microtubules.

Cilia are rapidly assembled and disassembled at differ-ent stages of the cell cycle, indicating that ciliary dynamicsare precisely coordinated with the cell cycle (Sorokin,1962; Gilula and Satir, 1972). Most cells in the humanbody will enter the G0 phase, or quiescent stage, after celldivision. Primary cilia present in G0 cells are regarded aspostmitotic structures of quiescent cells. Cilia assemblyincludes several different stages, as observed by electronmicroscopy (Sorokin, 1962; Pedersen, 2008). To assembleprimary cilia, the centrosomes, which are comprised oftwo centrioles (mother and daughter centrioles), migrateto the apical membrane of the cell where the mother cen-triole forms the basal body. This migration involvesvesicles, which bind at the distal end of the mother cen-triole, the actin cytoskeleton, and the exocyst complex,which assists with basal body migration, membrane dock-ing, and fusion of the ciliary vesicles at the intended siteof cilium outgrowth (Dawe et al., 2007; Zuo et al., 2009;Garcia-Gonzalo and Reiter, 2012). The detailed mecha-nisms of how centrioles migrate during primary ciliogene-sis, however, remain unclear. The Meckel–GruberSyndrome proteins, MKS1 and MKS3, were found to berequired for migration of the centrosomes to the plasmamembrane (Dawe et al., 2007). After migration, basalbodies dock with the cell surface through vesicular fusion.Before docking, basal bodies acquire accessory structures,including basal feet and striated rootlets. The basal bodyalso has other accessories, including centriolar satellitesand transitional fibers, but the timing of acquisition ofthese structures is also unclear (Dawe et al., 2007). ThePCP protein, Dishevelled (Dvl), is required to establish theapical actin network necessary for vesicle targeting, trans-port, and docking of basal bodies (Vladar and Axelrod,

2008). After basal body docking, post-translationally modi-fied tubulin begins to polymerize from the basal body andgrows away from the surface of the membrane. Tubulinthen is transported from the cytoplasm to the ciliary tipby intraflagellar transport (IFT) to construct the cilium. Atthe same time, a disassembly process occurs, and the bal-ance between these two processes determines the lengthof the cilia (Avasthi and Marshall, 2012). In cystic kidneydisease, excessively long cilia and the absence of cilia haveboth been associated with cyst development (Mokrzanet al., 2007; Besschetnova et al., 2010; Hildebrandt et al.,2011).

Primary cilia do not contain the machinery for proteinsynthesis, so the cell must transport proteins that arerequired for cilia assembly, sensory perception, and signal-ing from the site of synthesis into the cilium. Trafficking ofproteins from the cytosol and Golgi to the primary cilium,and the movement of proteins along the ciliary axonemeare critical for normal cilia structure and function, and areregulated by polarized vesicle trafficking and IFT,respectively.

FIGURE 1. Structure of primary cilia. The cilium protrudes from the apical sur-

face of the cell. The axoneme, or cilium core, grows out from the basal body

and is covered by ciliary membrane. The axoneme is composed of nine dou-

blet microtubules nucleated directly from the basal body. Intraflagellar trans-

port (IFT) particles transport proteins to the distal end of the cilium. Kinesin-II

is the microtubule motor responsible for anterograde IFT. Retrograde transport

utilizes dynein. Polycystin-2 is a calcium channel located on the primary cil-

ium, where it interacts with polycystin-1.

2 CILIA AND PKD

IFT refers to the process of transporting vital proteinswithin the cilium. Because of the similarity to the proc-esses required to build flagella, it is named “intraflagellar”transport. IFT describes the bidirectional movement ofnonmembrane-bound particles along the doublet microtu-bules of the flagellar or ciliary axoneme, and between theaxoneme and plasma membrane. Movement of IFT par-ticles along the microtubule is carried out by two differentmicrotubule-based motors: movement towards the tip ofthe cilium is termed anterograde IFT and depends uponkinesin-2 microtubule motors, while the reverse retro-grade transport requires dynein (Goetz and Anderson,2010). The IFT particle consists of two subcomplexes,each made up of several individual IFT proteins. ComplexA has five IFT subunits and complex B is composed of 12IFT subunits. Collingridge et al. found that compartmental-ized intraciliary Ca21 signaling can regulate the movementof IFT particles and is therefore likely to play a centralrole in directing the movement and distribution of manyciliary proteins (Collingridge et al., 2013). Bhogaraju et al.,recently identified the transport mechanism of the keyprotein tubulin, the most abundant protein and the mainbackbone of the cilium, and found that two proteins,IFT74 and IFT81, work together to form a tubulin-bindingmolecule (Bhogaraju et al., 2013).

Normal Ciliary Function in Embryonic KidneyDevelopment and in the Adult KidneyThe mammalian kidney originates from intermediate meso-derm, and three kidneys form in succession during embryo-genesis: the pronephros, mesonephros, and metanephros(Lipschutz, 1998; Vize et al., 2003). The metanephrosbecomes the permanent kidney in mammals. It forms whenthe epithelial ureteric bud grows out of the mesonephric(aka Wolffian) duct and contacts the loose metanephricmesenchyme in the caudal region of the embryo. The ure-teric bud then elongates and branches to form a tree-likestructure, which becomes the highly branched collectingduct system of the mature kidney (Saxen and Sariola, 1987).The metanephric mesenchyme cells condense around thetip of the ureteric bud, aggregate, epithelialize, and undergomorphogenetic movement and differentiation to form thepodocytes, and proximal and distal renal tubules. Stromalmesenchymal cells surround the condensed mesenchymeand differentiate into the pericytes of the mature kidney(Cullen-McEwen et al., 2005).

It has been known for over a hundred years that epi-thelial cells along the length of the nephron possess pri-mary cilia (Zimmermann, 1898). In the mammalian kidney,cilia have been observed on cells in the parietal layer ofthe Bowman’s capsule, the proximal tubule, the distaltubule, and in the principal, but not intercalated, cells ofthe collecting duct (Webber and Lee, 1975). Their specificfunction, however, has only recently begun to beunderstood.

Primary cilia play key roles in the development ofmany organs by coordinating extracellular signaling withcellular physiology (Corbit et al., 2005; Haycraft et al.,2005; Simons et al., 2005). The ciliary membrane is richin receptors, ion channels, and signaling proteins, whichcan be activated by mechanical and/or chemical stimuli(Rosenbaum, 2002). In adult kidney, cilia function asmechanosensors for luminal flow [as covered in a recentreview by Kotsis et al. (2013)]. Aside from their importantsensory functions, primary cilia also play important rolesin the control of cell proliferation, and are involved in sev-eral developmental signaling pathways, including Hedge-hog, Wnt, and mammalian target of rapamycin (mTOR)(Morgan et al., 2002; Cano et al., 2004; Haycraft et al.,2005).

Ciliary Dysfunction in ADPKDWork with C. elegans provided early clues that the gene prod-ucts for PKD1 and PKD2 might also be involved with ciliastructure and/or function. During the examination of muta-tions that affect mating behavior in C. elegans, Barr et al.(1999, 2001) identified worm homologs of PKD1 as lov-1 (forlocation of vulva), and PKD2 as pkd2. The protein productsfor lov-1 and pkd2 localized to the cilia of sensory neurons inC. elegans (Barr and Sternberg, 1999; Barr et al., 2001).

The relationship between primary renal cilia and thepathogenesis of PKD was further suggested by the OakRidge polycystic kidney (orpk) mouse. In these animals,insertional mutation of the Tg737 gene product resulted ina hypomorphic allele, and mice carrying this mutationdeveloped complex phenotypes, including polycystic kid-ney and pancreas, and portal fibrosis. Kidney cysts andhepatic fibrosis are also seen in humans with ARPKD, andthis mouse became one of the models for this disease(Moyer et al., 1994). The Tg737 gene is a homolog of Chla-mydomonas IFT88, a subunit of the intraflagellar trans-porter. IFT proteins are required for cilia formation andmaintenance, and IFT88 mutants showed a completeabsence of flagella in Chlamydomonas (Pazour et al.,2000). Subsequently, localization of Tg737 to the cilia ofrenal tubule epithelial cells was demonstrated using theMadin-Darby canine kidney (MDCK) epithelial cell line(Taulman et al., 2001). The primary cilia in the kidney ofTg737 mutant mice are short and stunted, indicating thatprimary cilia have an important function in the kidney,and that defects in their assembly lead to PKD (Pazouret al., 2000). The co-localization of polycystins-1 and 22,cystin, and Tg737 in mouse cortical collecting-ductal cells,further indicated that primary cilia play a key role in nor-mal physiological functions of renal tubule epithelia, andthat defects in ciliary function contribute to the pathoge-nesis of PKD (Yoder et al., 2002).

Although these studies suggested the important con-nection between cilia and normal kidney architecture, the

BIRTH DEFECTS RESEARCH (PART C) 00:00–00 (2014) 3

role of the primary cilium in renal cyst formation remainspoorly defined. While mice with insertional mutation ofthe Tg737 gene developed renal cysts, Tg737 null muta-tions are embryonic lethal, and are noted to have situsinversus. In this animal model, it was found that theembryonic node cells lack cilia on their apical surface. Theloss of cilia on these cells can cause abnormal nodal flowof morphogens and leads to defects in right-left patterning(nodal flow hypothesis). Polycystin-2, the protein encodedby PKD2, localizes to the primary cilia. PKD2-deficientmice also showed associated defects in left-right asymme-try, further indicating a functional link between primarycilia and polycystin-2 (Pennekamp et al., 2002). Researchinto pkd2 function in zebrafish has better elucidated thefunction of polycystin-2 in cilia. Knockdown of pkd2 bymorpholinos (MO) (Bisgrove et al., 2005), or in mutants(Sun et al., 2004; Schottenfeld et al., 2007), produced phe-notypes consistent with a role in cilia function, such ascurved tails, pronephric cysts, and edema. Further studiesshowed that polycystin-2 functions as a calcium-activatedintracellular calcium release channel in vivo, and that PKDresults from the loss of a regulated intracellular calciumrelease signaling mechanism (Koulen et al., 2002).

Several groups have shown that primary cilia functionas mechanosensors, which transmit extracellular mechani-cal signals into the cell through calcium influx. For exam-ple, MDCK cells respond to bending of the cilium by amicropipette or fluid flow with influx of calcium (Praetor-ius and Spring, 2001). This calcium influx in response tofluid flow was abolished after removal of the MDCK cellprimary cilium by chemical means, indicating that the pri-mary cilium is the flow sensor in MDCK cells (Praetoriusand Spring, 2003). Isolated renal collecting ducts fromcilia-deficient orpk mice also showed blunted flow-sensing(Liu et al., 2005). The identification of polycystin-2 as acalcium channel localized on the primary cilia indicated itsimportant role in the mechanosensory function of the pri-mary cilia. Blocking antibodies against polycystin-2 alsoabrogated the calcium influx in response to fluid flow(Nauli et al., 2003). Mechanical stimulation of the primarycilia, such as by fluid flow, initiates an intracellular calciumrise, as well as nitric oxide release, and proteinmodifications.

The cilium has also been shown to be a chemosensor.In addition to being involved in hedgehog signaling as dis-cussed previously, Masyuk et al. (2008) reported that chol-angiocyte cilia are chemosensors that detect biliarynucleotides through ciliary P2Y12 receptors and transducecorresponding signals into a cAMP response. Abdul-Majeedet al. (2011) found that dopamine receptor—type 5 (DR5),located on the primary cilia of mouse vascular endothelialcells, can sense systemic dopamine and plays an importantrole in cilia length and function. Silencing DR5 completelyabolished mechano-ciliary function through changes in sen-sitivity to fluid-shear stress (Abdul-Majeed et al., 2011).

Cilia and Planar Cell PolarityWe have discussed above the role of cilia as mechano/che-mosensors that transmit extracellular flow signal into thecells. Downstream effects of this transmitted signalingpathway include changes in cell differentiation and polar-ity. Planar cell polarity (PCP), also called tissue polarity,describes the coordinated orientation of cells and cellularstructures along an axis within the plane of an epithelialsurface (Simons and Mlodzik, 2008). Polarized cellular ori-entation and migration controlled by PCP are critical formultiple developmental processes. Park et al. (2006)showed that two planar polarity effectors, inturned (int)and fuzzy (fy), play a role in cilia formation. Heydeck et al.(2009) further demonstrated that fy plays an importantrole in cilia formation, hedgehog signal transduction, andembryonic development in mice. Strong evidence implicat-ing cilia in PCP came from mice missing cilia throughtissue-specific targeting of Ift88 and Kif3a. For example,these animals displayed highly disordered tissue architec-ture in the inner ear (Jones et al., 2008). However, IFT88mutant zebrafish, which do not form cilia, had a normalPCP phenotype, indicating that IFT88 plays cilia- and PCP-independent roles in controlling oriented cell division(Borovina and Ciruna, 2013).

The relationship of renal primary cilia, PCP, and PKDwas supported by the inversin (Inv) mouse. Inv is encodedby the inversion of embryo turning (Inv) gene (Mochizukiet al., 1998, Otto et al., 2003), and was discovered due toits role, during mammalian embryonic development, inestablishment of left–right asymmetry. Inv is a ciliary pro-tein that regulates developmental processes and tissuehomeostasis partly through the degradation of Dishevelled(Dvl) proteins to coordinate Wnt signaling in planar cellpolarity. The Inv mutation in mice resulted in both a rever-sal of left–right axis polarity (situs inversus) and cyst for-mation in the kidneys and pancreas (Yokoyama et al.,1993; Mochizuki et al., 1998). The human homolog of Invwas later found to cause nephronophthisis type 2(NPHP2) (Otto et al., 2003). Inv localizes to the base ofprimary cilia and is required for the localization of NPHP3and Nek8/NPHP9 to that region (Shiba et al., 2010).

Another PCP-associated gene, fat4, is required for outermedullary collecting duct (OCD) and tubule elongationduring kidney development, and its loss results in cystickidneys in mice (Saburi et al., 2008). Fat4 localizes to pri-mary cilia and is hypothesized to act in a partially redun-dant fashion with Vangl2 during cyst formation. Severalother genes that are known to give rise to cystic kidneyshave also been shown to cause defects in cilia and PCPsignaling, including Pkd1, Bbs4, Bbs6, and Ofd1.

Another important polarized event is oriented cell divi-sion, in which mitotic cells are oriented along the tubularaxis during tubule elongation. Orientated cell division dic-tates the maintenance of constant tubule diameter duringtubule lengthening, and defects in this process trigger

4 CILIA AND PKD

renal tubule enlargement and cyst formation (Fischeret al., 2006). Cilia also play a role in oriented cell division.Inactivation of KIF3A, the ciliary transport gene, in renalepithelial cells resulted in kidney cyst formation anddefects in oriented cell division (Patel et al., 2008). IFT20mutant mice also lacked cilia and developed renal cyststhat were associated with a disoriented axis of cell divi-sion (Jonassen et al., 2008). Taken together the cilia, PCP,and oriented cell division data suggest that primary ciliaplay an important role in transmitting extracellular infor-mation, such as urine flow, into the cell, and work throughthe PCP pathway. The hypothesis, therefore, is that genetic

disorders that result in abnormal ciliary sensing of fluidmovement lead to loss of PCP, which causes randomizedcell division. This, in turn, results in an increased tubulediameter, rather than tubule elongation, leading to cystformation.

Cilia, Exocyst, and Cdc42The cilium and the rest of the apical surface are both cov-ered by plasma membrane; however, there is regulatedmovement of both proteins and lipids into the cilium, andthe membrane composition is different in these two areas

FIGURE 2. Sec10 knockdown inhibits, while Sec10 overexpression enhances, primary ciliogenesis. We grew control, Sec10 overexpressing (Sec10 OE), and

Sec10 knockdown (KD) cells on Transwell filters for 14 days. The cells were fixed in glutaraldehyde and electron microscopy (EM) was performed (Phillips XL20

SEM). Confirming the results by IF and scanning EM (SEM) (data not shown here), by transmission EM (TEM) primary cilia were longer in Sec10 OE cells com-

pared to control cells and were rarely identified in Sec10 KD cells. Bar 50.5 mm. (Zuo et al., 2009) (Reproduced with reproduced with permission from Molecu-

lar Biology of the Cell).

FIGURE 3. Knockdown of sec10 phenocopies loss of pkd2. (A–F) Gross phenotypes of zebrafish embryos at 3 dpf, lateral view, 43 magnification. Uninjected

embryo (A), 4ng pkd2MO embryo with a severe curly tail up, small eyes, and glomerular edema (B), 15ng sec10MO embryo with a moderate curly tail up, small

eyes and glomerular edema (C). A synergistic genetic interaction was observed upon co-injection of suboptimal doses of 0.25/2ng pkd2 MO 1 7.5ng sec10MO

(D)—which do not result in curly tail up when injected alone (E, F). This suggests that exocyst sec10 and pkd2, one of two genes which are mutated in ADPKD,

act in the same pathway. (Reproduced with permission from PLoS Genetics).

BIRTH DEFECTS RESEARCH (PART C) 00:00–00 (2014) 5

FIGURE 4..

6 CILIA AND PKD

(Rohatgi and Snell, 2010). There is a diffusion barrier atthe base of the cilia membrane. This diffusion barrier2involves Septin 2 (SEPT2), a member of the septin familyof guanosine triphosphatases, and is essential for retainingciliary proteins (Hu et al., 2010.). Since protein synthesisdoes not occur in the cilium, the �700 proteins which areknown to reside there (Li et al., 2004) must be trans-ported to and integrated into the cilium. Although theroles that ciliary proteins play in diverse biologic proc-esses are being elucidated (Emmer et al., 2010), we knowvery little about how these proteins are transported fromthe sites of synthesis in the endoplasmic reticulum to thecilium.

A significant number of proteins destined for the cili-ary compartment are likely transported in Golgi-derivedvesicles to the base of the cilium, where the ciliary pro-teins are delivered and associate with IFT particles. How-ever, of the IFT proteins located in the cilia, only IFT20has been shown, to date, to also be located in the Golgicomplex and function in the delivery of ciliary proteinsfrom the Golgi to the cilium (Follit et al., 2006). At the cili-ary base, proteins containing specific ciliary targetingmotifs are allowed access through the zone defined by thetransition fibers (Follit et al., 2010). Lateral movement andrecycling of proteins from the apical membrane to the cil-ium involving the BBSome (Nachury et al., 2007) are alsopossible routes of entry into the cilium. Following entryinto the ciliary compartment, these proteins, along withinactive cytoplasmic dynein 2, are transported in ananterograde fashion along the axoneme by kinesin II-mediated IFT. At the ciliary tip, IFT particles and ciliaryturnover products (e.g., inactive receptors) are remodeled,kinesin-II is inactivated, and cytoplasmic dynein 2 is acti-vated. Ciliary turnover products are, in turn, transportedin a retrograde fashion along the ciliary axonemes by cyto-plasmic dynein 2 for recycling or degradation in thecytoplasm.

Trafficking of proteins from the cytosol and Golgi to theprimary cilium is regulated by polarized vesicle trafficking.The primary cilium, the Golgi, and the nucleus are consis-

tently found in proximity to each other (Poole et al., 1997),but the functional links between these organelles are notknown. The exocyst, originally identified in the yeast, Sac-charomyces cerevisiae (Novick et al., 1980), is a highly con-served 750-kD eight-protein complex known for thetargeting and docking of vesicles carrying membrane pro-teins to the plasma membrane (Lipschutz and Mostov,2002), and is composed of Sec3, Sec5, Sec6, Sec8, Sec10,Sec15, Exo70, and Exo84 (also known as EXOC1–8) (Hsuet al., 1996). Notably, in addition to being found near thetight junction, exocyst proteins were localized to the pri-mary cilium in kidney cells (Rogers et al., 2004; Zuo et al.,2009). Of the exocyst components, Sec10 and Sec15 aremost vesicle-proximal, and Sec15 directly binds Sec4 (Rab8in mammals), a Rab GTPase found on the surface of trans-port vesicles. Sec10 then acts as a “linker” by bindingSec15 (which is attached to the vesicle) to the other exocystcomponents at the membrane (Guo et al., 1999). It wasshown that knockdown of exocyst Sec10 in MDCK cellsabrogated ciliogenesis, while Sec10 overexpressionenhanced ciliogenesis (Zuo et al., 2009) (Fig. 2). Further-more, Sec10 knockdown decreased the levels of other exo-cyst components (and IFT88) and caused abnormalcystogenesis when the cells were grown in a collagenmatrix (Zuo et al., 2009). In vivo, knockdown in zebrafishusing antisense morpholinos of sec10 and pkd2 phenocop-ied each other, with a curly tail up, small eyes, and glomer-ular edema. Furthermore, small amounts of sec10 and pkd2morpholinos, which alone had no effect, together had asevere effect. This is called genetic synergy, and suggeststhat sec10 and pkd2 act in the same pathway (Fig. 3)(Fogelgren et al., 2011). From these findings, together withtheir known role in trafficking proteins to the plasma mem-brane (Grindstaff et al., 1998; Lipschutz et al., 2000, 2003;Moskalenko et al., 2002), Sec10 and the exocyst are thoughtto be required to build primary cilia by targeting and dock-ing vesicles carrying ciliary proteins.

Small guanosine triphophatases (GTPases) are versatiletemporal and spatial regulators of virtually all cellular proc-esses including signal transduction, cytoskeleton dynamics,and membrane trafficking. Rab GTPases are the largest

FIGURE 4. Lack of Cdc42 in kidney tubule cells leads to an early postnatal death due to lack of cilia and PKD. KspCre;Cdc42fl/1 mice (KspCadherin is expressed

in distal tubules and collecting ducts) were mated to Cdc42fl/fl mice, and all four possible genotypes were readily identified by PCR (data not shown here). (A) Of

67 pups tested from P11-P33, there were 22 Cdc42fl/fl, 19 Cdc42fl/1, 24 KspCre;Cdc42fl/1 mice, with one dead (mostly eaten) homozygous Cdc42 knockout

(KspCre;Cdc42fl/fl) mouse found at P15, and one long-surviving homozygous Cdc42 knockout (KspCre;Cdc42fl/fl) mouse that was euthanized at P33 due to failure

to thrive, growth restriction, and renal failure. (B) By linear regression modeling, BUN levels in Cdc42 kidney-specific knockout mice were significantly higher

than in littermate control KspCre;Cdc42fl/1 and Cdc42fl/fl mice at P10 (p 5 0.035). (C) By immunofluorescence, Cdc42 is not seen in the cysts (cy), but is seen

in the normal-appearing tubules of Cdc42 kidney-specific knockout mice (arrow). Bar 5 10 mm. (D–F) Immunostaining of acetylated alpha tubulin (red) in kidneys

from control (D) and Cdc42 kidney-specific knockout mice (E) at P4, shows lack of cilia in cells surrounding cysts. (F) Quantification from kidney tubule cell-

specific Cdc42 knockout mice shows a significant decrease in the number of cilia seen per cell in cysts (which presumably are deficient in Cdc42, as no cysts

were seen in control mice) compared to normal-appearing tubules, which most likely express Cdc42. Bar 5 20 mm. (G, H) Hematoxylin/eosin stained sections of

kidneys from control (G) and Cdc42 kidney-specific knockout mice (H) at P4 (left) and P6 (right). Bar for kidneys on left in G, H 5 200 mm. Bar for kidney sec-

tions on right in G, H 5 20 mm. (Reproduced with permission from the Journal of American Society of Nephrology).

BIRTH DEFECTS RESEARCH (PART C) 00:00–00 (2014) 7

group of the Ras superfamily of small GTPases. The exocystinteracts with Rab GTPases as described above (Zhanget al., 2004; Wu et al., 2005). The Cdc42 protein (Johnsonet al., 1987) is a member of the Rho GTPase subfamily ofthe Ras superfamily, and acts as a molecular switch to regu-late many cellular processes (Johnson and Pringle, 1990).Like all GTPases, Cdc42 can exist in two states, a GTP-bound, active state, and a GDP-bound, inactive state. Thecycling of Cdc42 between these states is controlled by twosets of proteins: guanine nucleotide exchange factors (GEFs)and GTPase-activating proteins (GAPs). Cdc42 is an impor-tant regulator of exocytosis in yeast (Wu et al., 2010). Incell culture, Cdc42 biochemically interacts with Sec10, andthe two co-localize at the primary cilium. Expression ofdominant negative Cdc42 and shRNA-mediated knockdownof both Cdc42 and Tuba, a Cdc42 GEF, inhibit ciliogenesisand result in MAPK pathway activation (Zuo et al., 2011).

In zebrafish, Cdc42 knockdown was recently shown to phe-nocopy many aspects of sec10 (and pkd2) knockdown. Inmice, kidney-specific knockout of Cdc42 resulted in renalfailure within weeks of birth. Histology revealed cystogene-sis in distal tubules and collecting ducts, decreased ciliogen-esis in cyst cells, increased tubular cell proliferation,increased apoptosis, increased fibrosis, and MAPK activa-tion, consistent with a nephronophthisis phenotype (Choiet al., 2013) (Fig. 4). In fact, a family with Joubert syn-drome, a nephronophthisis form of PKD, had a mutation inthe exocyst (Dixon-Salazar et al., 2012). Indeed, of the 1969proteins found in the mouse photoreceptor sensor cilium,there are 96 small GTPases, GEFs, and GAPs (Liu et al.,2007). Taken together, these results suggest that Cdc42localizes the exocyst to primary cilia, whereupon the exo-cyst targets and docks vesicles carrying ciliary proteins.Abnormalities in this pathway result in deranged ciliogene-sis and PKD (Fig. 5).

FIGURE 5. Model for the role of Cdc42 and the exocyst in delivery of ciliary proteins. (A) Data support a model in which the exocyst complex is localized to the pri-

mary cilium by Cdc42, which is located all along the apical surface, but is activated at the primary cilium by Tuba, a Cdc42 GEF. The exocyst is then stabilized

by Sec10 binding directly to the Par complex via Par6. Once the exocyst complex is stabilized at the nascent primary cilium, it then targets and docks vesicles

carrying ciliary proteins, by interacting with a small GTPase, Rab8, found on vesicles. (B) When Cdc42 (or the exocyst or the PAR complex) is lacking in a cell,

the exocyst no longer localizes to primary cilia and increased cell proliferation, apoptosis, and fibrosis occur, with the result being ciliary phenotypes (hydrocepha-

lus, small eyes, tail curvature, and edema in zebrafish; and PKD in mice). (Reproduced with permission from the Journal of American Society of Nephrology).

8 CILIA AND PKD

CONCLUSIONSAfter having been discovered more than 300 years agoand having been “ignored” for the vast majority of thattime, primary cilia have caught the attention of manyinvestigators in recent years because of research connect-ing primary cilia to PKD. Cilia extend from the surface ofmost eukaryotic cells, so structural or functional defectscan cause multisystemic disorders, and this apparently dis-parate group of human diseases was recently given a com-mon name, “ciliopathies.” Among this group, PKD is by farthe most common. Cilia are present on the apical surfaceof renal tubule epithelial cells, and function as mechano-and chemosensors that sense and transmit extracellularsignals, such as urine flow, into the cell, and maintain nor-mal renal epithelial planar cell polarity and oriented celldivision. Defects in cilia structure and/or function causePKD. Protein trafficking and IFT play very important rolesin normal ciliogenesis, and the exocyst complex and itsregulatory GTPases appear to be key players in this pro-cess. Although we now have considerable insight into themolecular and cellular basis of ciliopathies such as PKD,many questions remain. For instance, the mechanisms con-necting mechanosensing components at the cilium and thedownstream signal cascades are poorly understood. Fur-ther studies are clearly needed to better understand howcilia-related effects cause cyst formation to devise rationaltreatments for human PKD, diseases for which noapproved treatments currently exist.

AcknowledgmentAndrea Wecker is gratefully acknowledged for editing thepaper and offering helpful comments and suggestions.

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