regulation of actin-based apical structures on epithelial cells · 2018. 10. 16. · on intestinal...

REVIEW SUBJECT COLLECTION: CILIA AND FLAGELLA

Regulation of actin-based apical structures on epithelial cellsThaher Pelaseyed1,* and Anthony Bretscher2

ABSTRACTCells of transporting epithelia are characterized by the presence ofabundant F-actin-based microvilli on their apical surfaces. Likewise,auditory hair cells have highly reproducible rows of apical stereocilia(giant microvilli) that convert mechanical sound into an electricalsignal. Analysis of mutations in deaf patients has highlighted thecritical components of tip links between stereocilia, and relatedstructures that contribute to the organization of microvilli on epithelialcells have been found. Ezrin/radixin/moesin (ERM) proteins, whichare activated by phosphorylation, provide a critical link between theplasma membrane and underlying actin cytoskeleton in surfacestructures. Here, we outline recent insights into how microvilli andstereocilia are built, and the roles of tip links. Furthermore, wehighlight how ezrin is locally regulated by phosphorylation, and thatthis is necessary to maintain polarity. Localized phosphorylation isachieved through an intricate coincidence detection mechanism thatrequires the membrane lipid phosphatidylinositol 4,5-bisphosphate[PI(4,5)P2] and the apically localized ezrin kinase, lymphocyte-oriented kinase (LOK, also known as STK10) or Ste20-like kinase(SLK). We also discuss how ezrin-binding scaffolding proteinsregulate microvilli and how, despite these significant advances, itremains to be discovered how the cell polarity program ultimatelyinterfaces with these processes.

KEY WORDS: Actin, Cell polarity, Cytoskeleton, ERM proteins,Microvilli, Stereocilia

IntroductionCells throughout the vertebrate body are polarized. By regulating itsinternal organization and plasma membrane composition in apolarized manner, the cell ensures organism viability and theexecution of central biological functions, such as cell division,proliferation, signaling and migration. One critical aspect of cellpolarity is how cells build and restrict morphological features to onedomain of a polarized cell. The epithelial cell serves as thearchetypal example of a polarized cell. Its apical membrane domainis decorated with finger-like protrusions called microvilli (Grangerand Baker, 1950). Microvilli expand the interface between the celland extracellular milieu, and accommodate membrane proteins thattake part in signaling as well as ion and nutrient transport. Bycontrast, the basolateral membrane harbors a different compositionof membrane proteins and displays a membrane morphology that isvery distinct from the apical brush border membrane (Bryant andMostov, 2008). In this Review, we discuss recent insights into theregulation and assembly of the apical microvilli and stereocilia –

related finger-like protrusions that are found on the apical membraneof hair cells. We highlight the contribution of bundled actinfilaments to apical organization, and the contribution of tip-linkingto the structure and function of microvilli and stereocilia.Furthermore, we describe an emerging mechanism of howbundled actin filaments are connected to the plasma membrane ina way that restricts their presence to the apical membrane.

Morphological features and arrangement of actin-basedsurface structuresShaping the plasma membrane into a finger-like structure requiresa core of bundled cytoskeletal filaments with lateral attachmentsto the plasma membrane. In the case of cilia, the filaments aremicrotubules, whereas filaments of actin support filopodia,microvilli and stereocilia (DeRosier and Tilney, 2000; Lewisand Bridgman, 1992). Here, we restrict our discussion tomicrovilli and stereocilia, which are found on the apical domainof epithelial cells.

The plasma membrane of microvilli is supported by a bundle of∼30–40 parallel, inherently polarized actin filaments with theirbarbed ends at the microvillus tip (Mooseker and Tilney, 1975).Early morphological studies showed that the F-actin core is bundledand attached to the plasma membrane by lateral cross-bridges of∼30 nm length, indicating that the actin filaments are bothcrosslinked to one another and to the plasma membrane (Brunserand Luft, 1970; Matsudaira and Burgess, 1979; Mooseker andTilney, 1975). On intestinal epithelia and the epithelium of thekidney proximal tubule, the microvilli are of uniform length andgenerally hexagonally packed. Despite over 40 years of research, itis still not clear what drives the assembly of actin filaments inmicrovilli, but much has been learned about their morphogenesisby studying microvillar components. Today, we appreciate thatevents such as cytoskeletal remodeling, membrane–cytoskeletoncrosslinking and extracellular adhesion are parts of the cellularprogram that shapes the apical brush border domain (Crawley et al.,2014b). We discuss some of these cellular programs in this Review.

Stereocilia are found on the apical aspect of hair cells of thevertebrate cochlea and vestibular systems. Early studies of birdcochlea revealed that the length of hair cell stereocilia range from1.5 to 5 µm, increasing in length from the proximal to distal end(Tilney and Saunders, 1983). However, in contrast to what is seen inbirds, stereocilia on individual cells of the mammalian cochleaexhibit a staircase-like pattern with a predetermined graded increasein stereociliary length between rows (Fig. 1A, see Box 1). Themammalian cochlea contains two types of hair cells: a single row ofinner hair cells with a linear array of stereocilia measuring ∼5 µm inlength, and three rows of outer hair cells with stereocilia arranged ina V-shaped pattern and measuring 2–3 µm in length (Dallos, 1996;Kaltenbach et al., 1994). Vestibular hair bundles in the utricle ofadult mouse range from ∼2 µm for the shortest stereocilia in thestaircase to up to ∼15 µm (Li et al., 2008; Rzadzinska et al., 2004).Each mature stereocilium contains 400–3000 actin filaments withtheir barbed ends at the tip and pointed ends associated with the

1Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology,University of Gothenburg, SE-405 30 Gothenburg, Sweden. 2Weill Institute for Celland Molecular Biology, Department of Molecular Biology and Genetics,Cornell University, Ithaca, NY 14853, USA.

*Author for correspondence ([email protected])

T.P., 0000-0002-6434-3913; A.B., 0000-0002-1122-8970

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© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs221853. doi:10.1242/jcs.221853

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http://jcs.biologists.org/collection/cilia_flagellamailto:[email protected]://orcid.org/0000-0002-6434-3913http://orcid.org/0000-0002-1122-8970

base, where it tapers and inserts into the cuticular plate of the haircell (Tilney et al., 1980, 1989; Zheng et al., 2000). In an extendedseries of studies, Tilney et al. have documented the assemblyof stereocilia during development in ultrastructural detail(Tilney et al., 1992). Stereocilia on mature hair cells are linkedby tip links that activate mechano-electrical transduction channelswhen the stereocilia are bent at their base in response to sound.A prerequisite for proper mechano-electrical transduction is adirectional mechanosensitivity based on hair cells being arrangedso that their axis of sensitivity is aligned with the direction ofstimulation (Shotwell et al., 1981). Recently, a series of studies hasrevealed the molecular mechanisms that instruct the organization ofthe characteristic V-shaped staircase-like architecture of stereocilia incochlear hair cells (see Box 1).It is now clear that assembly of actin-based protrusions requires

the assembly of protein complexes, the timing and spatialdistribution of which are beginning to be elucidated. In the nextsection, we focus on how membrane traffic provides support forassembly of distinct membrane domains in epithelial cells.

Apical membrane morphology: the cell polarity program andmembrane trafficMicrovilli and stereocilia are restricted to the apical domain oftheir respective cells (Fig. 1A,B). This apical-basolateral polarityis directed by an epithelial polarity program that involves intrinsicpolarity proteins as well as signals from adjacent cells and theextracellular matrix (Rodriguez-Boulan and Macara, 2014). Thisprogram was elucidated mostly from studies in C. elegans andDrosophila (Campanale et al., 2017) and, in the fly, it consists ofBazooka, Partitioning defective-6 and Atypical protein kinase C(Baz–Par-6–aPKC), which function antagonistically throughaPKC kinase to exclude the basolateral complex Lethal giantlarvae, Discs large 1 and Scribble (Lgl–Dlg1–Scrib). Basolaterally, inturn, Par-1 kinase antagonizes the apical complex. aPKC and Par-1establish phosphorylation-dependent feedback loops in order toexclude trespassing of proteins and to maintain membranedomain identity (Benton and St Johnston, 2003). Crumbs (Crb),a large transmembrane protein that forms the so-called Crumbscomplex, participates in an essential epithelial polarity program

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StereociliaTip link

Microvilli

Myo7a

Myo7b

SANS

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MLPCDH

PCDH15

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Fimbrin Espin Fascin

Villin Fimbrin

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EspinKey

Fig. 1. Organization and regulationof stereocilia and microvilli.(A) Stereocilia of auditory hair cells arearranged in a staircase configuration.Actin bundles are assembled andstabilized through actin-binding proteinssuch as fimbrin, espin and fascin.Stereocilia of each row are connected tostereocilia of adjacent rows through tiplinks. Tip links require a ternary complexof Myo7a, USH1C (harmonin) and SANSthat binds to the cadherin CDH23.CDH23 forms a heteromericinterstereociliary link with protocadherinPCDH15 on the adjacent stereocilium.The cytoplasmic region of PCDH15interacts with the mechano-electricaltransduction (MET) channels. Auditorystimulus causes deflection towards tallerrows, which generates an influx of K+ andCa2+ ions through the MET channels. (B)Microvilli covering the apical aspects ofintestinal epithelial cells requireheterotypic extracellular linkages that aremediated by the protocadherin PCDH24and mucin-like protocadherin MLPCDH.By analogy with stereocilia, thecytoplasmic tail of PCDH24 binds theternary complex of USH1C (harmonin),the SANS homolog ANKS4B andMyo7b. The actin cytoskeleton isassembled and stabilized by villin, espinand fimbrin.

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(Bachmann et al., 2001). The Crumbs complex localizes to thesubapical compartment of the cell (Tepass, 1996), and its deletionresults in complete loss of the apical membrane domain, whereasCrb overexpression expands the size of apical domain (Wodarzet al., 1995). The regulatory machinery of membrane polarityoverlaps with trafficking of cargos to specific membrane domains.For example, the GTPases Rab11 and, downstream, Rab8 cooperatewith the apical Par3–aPKC complex to deliver vesicles with earlyapical identity markers, such as podocalyxin, to membranes forinitiation of lumenogenesis (Bryant et al., 2010). Misregulation ofthe intracellular trafficking machinery that underlies cell polarityleads to disease such as microvillous inclusion disease (MVID), asevere neonatal enteropathy that affects villus enterocytes (Cutzet al., 1989; Davidson et al., 1978). Lack of absorptive channels,exchangers and nutrient transporters in apical membranes ofenterocytes leads to diarrhea, life-threatening dehydration andmetabolic disorders (Ruemmele et al., 2006). Neonates with MVIDexhibit increased numbers of subapical vesicles, a sporadic presenceof microvilli on the basolateral surface, loss of microvilli andmicrovillous inclusions, characterized by intracellular vesicle-likestructures luminally lined by microvilli (Sherman et al., 2004).MVID has been attributed to mutations in the genes MYO5B and

STX3, encoding the motor protein Myo5b and the SNARE proteinsyntaxin 3, which are involved in regulated apical transport andmembrane fusion, respectively (Müller et al., 2008; Wiegerincket al., 2014). A prominent feature in patients with defects in Myo5bis the accumulation of apical tubules and vesicles that appear torepresent Rab8 and Rab11 double-positive recycling compartments(Vogel et al., 2017). Myo5b interacts with Rab8 and Rab11, whichare both required for transport of cargos from the trans-Golginetwork to the plasma membrane (Chen et al., 1998; Roland et al.,2007). Rab8a-deficient mice exhibit a marked decrease in apicalproteins in intestinal cells, a phenotype that leads to nutrientmalabsorption and death (Sato et al., 2007). These mice also displaysubapical microvillus inclusion bodies produced by endocytosisfrom the apical membrane. Myo5b–Rab11 interaction is requiredfor delivery of cargo to plasma membranes in order to establish anapical membrane domain (Wakabayashi et al., 2005). Mice that aredeficient in both Rab8a and Rab11a show a severe enteropathy andextensive formation of basolateral microvilli (Feng et al., 2017).Syntaxin 3 is also known to localize apically in epithelial cellswhere it facilitates fusion of vesicles to the plasma membrane(Delgrossi et al., 1997; Sharma et al., 2006). In summary,appropriate apical morphology and physiology requires anintimate relationship between cargo trafficking, recycling andlocal assembly of delivered cargo into a functional membranedomain. Defects in any of these systems result in disease.

Tip-links in the structure and function of stereociliaand microvilliAnalyses of the molecular basis of deafness in patients withhereditary Usher Syndrome 1 have been instrumental in defining aprotein complex that links the tip of one stereocilium to the side ofthe adjacent one (Fig. 1A) (Pickles et al., 1984). Collectively, thiswork has shown that the MyTH4-FERM tail domain of the motorprotein Myo7a is essential to form a stable ternary complex with thePDZ-domain-containing scaffolding proteins USH1C (harmonin)and scaffold protein containing ankyrin repeats and SAM domain(SANS, also called USH1G) to bind the cytoplasmic tail of thecadherin CDH23 (Caberlotto et al., 2011; Grati and Kachar, 2011;Li et al., 2017). In addition, the extracellular region of the cadherindimer interacts with the extracellular region of the protocadherinPCDH15 dimer on a neighboring stereocilium (Siemens et al.,2004). There, the cytoplasmic region of PCDH15 interacts with themechano-electrical transduction (MET) channels located on the tipof the adjacent stereocilium. When sound induces the stereocilia tobend, the tip-link transmits tension that allows for the influx of K+

and Ca2+ (Beurg et al., 2006, 2009). The identity of MET channelsthat are linked to PCDH15 has not been firmly established.However, sensory transduction signals require the transmembranechannel-like proteins (TMC) 1 and 2, which form heteromericcomplexes at stereociliary tips and allow Ca2+ permeability (Kurimaet al., 2015; Pan et al., 2013). In addition, studies in frogs haveshown that Pcdh15a interacts with Tmc1 and Tmc2a, orthologs ofmammalian TMC1 and TMC2 (Maeda et al., 2014).

Remarkably, a similar type of linking system has been discoveredbetween microvilli on epithelial cells (Fig. 1B). Here, heterotypicextracellular linkages occur near the tips of microvilli betweenprotocadherin PCDH24 (encoded by CDHR2) and mucin-likeprotocadherin MLPCDH (encoded by CDHR5) to form links of50 nm width (Crawley et al., 2014a, 2016; Li et al., 2016; Yu et al.,2017). The cytoplasmic tail of PCDH24 binds USH1C (harmonin)and forms a ternary linkage with the SANS homolog ANKS4B tolink to the MyTH4-FERM domains of Myo7b. Since USH1C is a

Box 1. Emerging mechanism for the staircase-likearchitecture of stereociliaHair cells display two integrated levels of polarity. The cells aresystematically and uniformly oriented in the cochlea, thus they displayorgan-wide planar cell polarity (PCP) (Goodrich and Strutt, 2011).Additionally, hair cells have an intrinsic planar axis, outlined by thedirection of the staircase-like gradient of stereocilia height towards thetallest hair cell, the kinocilium, at the edge of the V-shaped bundle ofstereocilia (Pickles et al., 1984). Initially, epithelial cells of sensoryepithelium are covered by microvillar precursors. During apicalmorphogenesis, a lateral ‘bare zone’ emerges, uniquely devoid ofmicrovilli. The bare zone forms through apical recruitment of a complex ofthe mammalian adaptor protein inscuteable (mInsc), Leu-Gly-Asnrepeat-enriched protein (LGN, also known as GPSM2) and theinhibitory heteromeric G protein Gαi (Tarchini et al., 2013). Thecomplementary medial side is enriched in atypical PKC, which isexcluded from the bare zone. The mInsc–LGN–Gαi complex expandsthe bare zone, thus relocalizing the kinocilium from the lateral junctiontowards the cell center and shaping the stereocilia bundle (Tarchini et al.,2013). Mice lacking mInsc display impaired lateromedial extension of thebare zone and exhibit abnormal stereociliary sub-bundles. Deletion ofLGN results in disorganized or even missing bundles, displacement ofkinocilium and a generally disrupted apical morphology, indicating thatLGN is responsible for the inward relocalization of the kinocilium alongthe lateromedial axis and asymmetric positioning of stereocilia (Tarchiniet al., 2013). Deletion of Gαi causes delocalization of LGN and results inmisorientation of the kinocilia, suggesting that Gαi regulates the lateralhair cell orientation through the movement of the kinocilium. LGN andGαi, but not mInsc, are also enriched in the tip of stereocilia in the tallestrow at the medial boundary of the bare zone, and LGN mutants displayshorter stereocilia and an increased number of rows in the architecture ofthe staircase (Tarchini et al., 2016). Consequently, it has been suggestedthat the mInsc–LGN–Gαi first orchestrates the formation of the bare zoneand the V-shaped stereocilia, before LGN and Gαi are localized tostereociliary tips in order to define the height of the tallest first row(Tarchini et al., 2016). A fourth component, dishevelled-associatingprotein with a high frequency of leucine residues (Daple, encoded byCCDC88C), couples the PCP pathway to the formation of the cell-intrinsic planar axis by connecting the PCP protein dishevelled to thecell-instrinsic signaling component Gαi. Mice lacking Daple exhibitmisoriented and misshapen staircases of stereocilia, caused byuncoupling of Gαi from the kinocilium (Siletti et al., 2017).

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common component of tip-linkages between stereocilia andmicrovilli, and is abundant in the intestine (Crawley et al., 2014a),this explains why a fraction of patients with mutations in theUSH1Cgene, besides suffering from hearing defects, are also affected byintestinal disease (Bitner-Glindzicz et al., 2000), includingprotracted diarrhea (Kobayashi et al., 1998, 1999; Powell et al.,1982). The membrane proteome of intestinal epithelial cellsincludes proteins that are involved in ion transport, nutrientuptake and bacterial sensing (van der Post and Hansson, 2014)and Ush1c-knockout mice display loss of microvilli in a fraction ofintestinal epithelial cells (Crawley et al., 2014a), which explains theobserved enteropathy. Loss of other components of the inter-microvillar linkage also results in irregular microvillar lengths andloss of the hexagonal packing that is typical of fully differentiatedcells in culture or in the mouse intestine (Crawley et al., 2014a).Interestingly, the Drosophila ortholog of PCDH15, Cad99C, isfound on the microvilli of follicle cells, and its loss results inshortened and disordered microvilli (D’Alterio et al., 2005).Additionally, the adhesion molecule Fasciclin 2 has been found tolocalize to the brush border of the renal tubules in Drosophila, andinterference studies indicate that it is necessary for the regulation ofmicrovilli length and organization (Halberg et al., 2016). Thus,these data suggest an emerging principle showing that correct lengthregulation and organizational packing of microvilli requiresappropriate inter-microvillar links.It is attractive to think that tip links might be a simple mechanism

to automatically give rise to the beautiful hexagonal packingcharacteristic of microvilli on intestinal epithelial cells. As shownin both the vertebrate and fly models, tethering microvilli to eachother could also be an effective physical mechanism for regulationof microvilli length on individual cells. Whereas the length ofmicrovilli between adjacent cells is usually quite uniform,occasionally this can be variable, but remains strikingly uniformon individual cells, which is consistent with this model (Crawleyet al., 2014a). However, how hair cells generate rows of stereociliawith the same length adjacent to rows with longer lengths is not yetfully understood. For a discussion of a potential shared evolutionaryorigin of microvilli and stereocilia, see Box 2.

Actin filament crosslinkersThe length ofmicrovilli varies depending on cell type. In enterocytes,each microvillus measures ∼1–2 µm and contains a core of ∼30–40unipolar actin filaments (Mooseker, 1985). Stereocilia can beconsidered an exaggerated form of microvilli with variable lengthsbased on hair cell type and localization in the cochlea or vestibularsystem, as outlined above (Tilney and Saunders, 1983; Tilney et al.,1988). The length of microvilli and stereocilia is, at least in part,regulated by the growth rate of the core actin bundle, where longeractin protrusions have faster rates of actin polymerization (Narayananet al., 2015; Rzadzinska et al., 2004).The actin-crosslinking proteins espin, fimbrin and villin

each play a part in regulation of microvillar length (Fig. 1B)(Grimm-Günter et al., 2009; Loomis et al., 2003; Ubelmann et al.,2013). Mice carrying mutations in the Espn gene, so-called jerkermice (Grüneberg et al., 1941; Zheng et al., 2000), which exhibitcharacteristic head-jerking movements and rapid circling, showgradual shortening and thinning of stereocilia followed by hair celldegeneration and vestibular dysfunction (Anniko et al., 1989;Sekerková et al., 2011). The role of espin in length regulation has alsobeen highlighted in epithelial cells in which espin overexpressionresults in lengthening of microvilli (Loomis et al., 2003). However,whether espin is directly involved in length regulation or whether

shortened actin protrusions are an indirect effect of diminishedcrosslinking and smaller actin bundle diameter remains to bedetermined. Fimbrin acts as an F-actin bundler in stereocilia as wellas in microvilli (Hanein et al., 1998; Tilney et al., 1989), whereasvillin is a multifunctional protein that acts as an F-actin-severing and-capping protein, as well as an actin bundler and nucleator thatregulates the distribution of actin filaments (Bretscher and Weber,1980; Khurana and George, 2008).

Remarkably, mice that lack espin and villin display noparticular microvillar phenotype, whereas fimbrin-knockout miceexhibit shorter microvilli that lack rootlets (Ferrary et al., 1999;Grimm-Günter et al., 2009; Zheng et al., 2000). Nevertheless, tripleknockout mice lacking espin, fimbrin and villin are not only viable,but still develop microvilli. However, microvilli are sparse,disorganized and shorter in these animals (Revenu et al., 2012).Consequently, it has been proposed that one or more additionalbundling proteins participate in regulating actin dynamics andmicrovillus length; however, none have so far been identified(Revenu et al., 2012). A protein that has recently been implicated inregulation of microvillar length is cordon bleu, a WH2 domain-containing protein localized at the base of microvilli (Wayt andBretscher, 2014). Overexpression of cordon bleu results in shortermicrovilli in human syncytiotrophoblasts in a WH2 domain-dependent manner (Wayt and Bretscher, 2014), thus suggesting arole for the reported F-actin-severing features of these WH2 domains(Jiao et al., 2014). Other studies have shown that cordon bleu usuallylocalizes to the base of microvilli, and that overexpression of cordonbleu in intestinal epithelial cells generates more and longermicrovillaractin bundles in a WH2-dependent manner (Grega-Larson et al.,2016). It is likely that the inconsistent data on the function of cordonbleu reflects cell-specific effects. In fact, cordon bleu interacts withPACSIN-2 (also known as Syndapin-2) in both syncytiotrophoblastsand intestinal epithelial cells (Grega-Larson et al., 2016; Wayt andBretscher, 2014), but whereas this interaction is required fortargeting PACSIN-2 to microvilli in syncytiotrophoblasts, inintestinal epithelial cells, PACSIN-2 acts upstream of cordon bleufor its localization at the base of microvilli.

Box 2. Evolutionary origins of microvilli and stereociliaThe closely related structural and tip-link components of microvilli andstereocilia suggest they could share a common evolutionary origin. Asthe gut is an evolutionary older organ than the auditory system, it isintriguing to think of microvilli as the primordial actin-based structure anda precursor to stereocilia that appeared later in evolution. In support ofthis concept, stereocilia typically first emerge as small, short microvilli ofuniform length, before growing into the classic pattern of rows withincreasing heights (Tilney et al., 1992). The specialized tip links mostlikely arose through gene duplication followed by genetic divergence toencode tissue-specific complexes (Peña et al., 2016). The conservationof tip linkages in microvilli and stereocilia might be associated withmembrane remodeling and mechanosensing in each respective organ.Fluid shear stress has been shown to trigger microvilli formation throughCa2+ influx through mechanosensitive activation of the Ca2+-selectivetransient receptor potential cation channel vanilloid 6 (TRPV6), which isexpressed in intestinal epithelial cells (Bianco et al., 2006; Miura et al.,2015). Likewise, mechanical stimuli to deflect stereocilia result inopening of MET channels, giving rise to an electrical output (Ohmori,1985). Finally, both microvilli and stereocilia have to maintain tensiongenerated by the formation of actin-based protrusions on the apical cellmembrane. The closely related ERM protein family that crosslink theactin core to the plasma membrane appear to contribute to this functionin hair cell stereocilia and intestinal microvilli (Rouven Brückneret al., 2015).

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Less is known about the actin-crosslinking proteins of stereocilia.However, stereocilia lack villin, but utilize at least fimbrin, espinand fascin as F-actin crosslinkers (Fig. 1A). Fimbrin was the firstcrosslinking protein described (Flock et al., 1982; Tilney et al.,1989) and recent work indicates it is the most abundant one (Kreyet al., 2016). Espin was initially identified by analysis of the deafjerker mouse (Zheng et al., 2000), and fascin was the most recentlydescribed (Chou et al., 2011). A recent study points at the importantrole of barbed-end-capping proteins in stabilizing actin filaments bypreventing depolymerization. Here, the heterodimeric cappingprotein CAPZ is recruited to growing actin filaments in order toprevent depolymerization, especially when the concentration of freeactin monomers is low (Avenarius et al., 2017). Deletion of CAPZin mouse utricles results in both shorter and narrower stereocilia,and diminished auditory and vestibular function.In conclusion, microvilli and stereocilia are dynamic structures that

require a diverse set of actin-regulating proteins to provide accuratecontrol over actin filament assembly, disassembly and stability. In thenext section, we discuss another aspect of stereociliary andmicrovillar dynamics, namely the physical coupling of the actinbundle of microvilli and stereocilia by phosphoregulated crosslinkers.

Linking the actin core to the plasma membraneThe ERM proteins belong to the 4.1 superfamily of proteins thatshare a 4.1 protein, ezrin, radixin and moesin (FERM) domain as acommon feature. The ERM proteins, each encoded by a single genein mammals, act as tissue-specific crosslinkers between the plasmamembrane and the underlying cortical F-actin cytoskeleton in manydifferent cellular contexts (Fehon et al., 2010). Most epithelial cellsonly express ezrin; stereocilia on hair cells of the cochlea containsome ezrin, but predominantly express radixin (Kikuchi et al., 2002;Kitajiri et al., 2004). Consequently, ezrin-knockout mice diesoon after birth with short microvilli and disorganized rootlets(Saotome et al., 2004) and the intestinal defects are even moresevere upon tissue-specific depletion of ezrin in adult animals(Casaletto et al., 2011). The mouse radixin knockout is viable, butstereocilia degenerate after formation, resulting in deafness(Kitajiri et al., 2004). Thus, ERM proteins have central roles inthe structure and function of microvilli and stereocilia.ERM proteins consist of an N-terminal FERM domain, a central

α-helical domain and a C-terminal domain (CTD) in which theF-actin-binding site lies (Turunen et al., 1994). In their inactive(closed) form, the FERM domain binds with high affinity to theCTD with the α-helical region forming an anti-parallel coiled coil(Gary and Bretscher, 1995; Li et al., 2007). In this closed state, boththe sites on the FERM domain for binding membrane proteins andthe F-actin-binding site in the CTD are masked (Li et al., 2007).Phosphorylation of a conserved threonine residue (T567 in ezrin) inthe CTD lowers the affinity between the FERM domain and CTD,resulting in ezrin adopting a more open configuration (Nakamuraet al., 1995). Open ezrin can now act as a crosslinker by engagingplasma membrane proteins through its N-terminal FERM domain,whereas the CTD binds F-actin in the bundled filaments (Gary andBretscher, 1995) (Fig. 2B). Oscillation of ezrin between the openand closed states is regulated by rapid cycles of phosphorylationand dephosphorylation (phosphocycling), exhibiting a turnovertime of∼1 min in cultured cells (Viswanatha et al., 2012). If the cycleof ezrin phosphorylation and dephosphorylation is decoupled –either by inhibiting ezrin dephosphorylation or by expressing aconstitutively active phosphomimetic ezrin mutant (T567E) – thepolarized distribution of ezrin in the apical membrane is lost(Viswanatha et al., 2012). Consequently, restricting ezrin function

to the apical domain requires local phosphorylation at the apicalmembrane, followed by dephosphorylation in the cytoplasm(Fig. 2A). Remarkably, the basal rate of ezrin phosphocyclingcorrelates with the turnover rate of fluorescently tagged ezrin in theapical membrane (∼1–2 min) and is significantly faster than theturnover of the microvillus itself (∼12 min) (Garbett and Bretscher,2012; Gorelik et al., 2003). The fast cycling of ezrin activation reflectsits role as a crosslinker between the continuously treadmilling actinfilaments and the numerous membrane proteins in the overlyingplasma membrane.

A coincidence detection mechanism restricts activeezrin apicallyTwo closely related kinases, lymphocyte-oriented kinase (LOK,also known as STK10) and Ste20-like kinase (SLK), have beenidentified as the major ezrin kinases in epithelial cells (Viswanathaet al., 2012). Phosphorylation of ERM proteins by LOK and SLK isfunctionally conserved: in Drosophila, Slik is the only kinase thatphosphorylates the single ERM protein present in flies, Moesin,on the regulatory threonine residue that corresponds to residueT567 in human ezrin (Carreno et al., 2008; Hipfner et al., 2004;Kunda et al., 2008). LOK-mediated phosphorylation of ezrin is anintricate multistep process that culminates in T567 phosphorylation(Pelaseyed et al., 2017). The FERM domain of ezrin containsa binding pocket for phosphatidylinositol 4,5-bisphosphate[PI(4,5)P2] (Niggli et al., 1995) located adjacent to the coiled-coilα-helix connecting the FERM domain to the CTD. It is likely thatbinding of PI(4,5)P2 to this particular binding site triggers aconformational change that lowers the affinity between the FERMdomain and CTD. The lower-affinity FERM–CTD complex allowsfor the CTD of LOK to wedge in between the FERM and CTD, thusprying ezrin open and allowing LOK kinase domain to gain accessto the phosphorylatable T567 residue (Fig. 2B). Several importantfeatures of this mechanism are highlighted below: the transition ofthe FERM–CTD complex from the high-affinity closedconformation to a lower-affinity conformation requires that theFERM and CTD domains are connected through the coiled-coilα-helix. In fact, a FERM–CTD complex that lacks this coiled-coilregion cannot be phosphorylated by LOK, even in the presence ofPI(4,5)P2 (Pelaseyed et al., 2017). Thus, binding of PI(4,5)P2 to full-length ezrin – a process we call ‘priming’ – does not simply changethe conformation of the FERM domain, but requires activeinvolvement of the α-helical region to reduce the affinity betweenthe FERM and CTD domains (Pelaseyed et al., 2017). In this sense,the central helical region can function as a spring to reduce theaffinity between the domains. Ezrin is explicitly primed byPI(4,5)P2 and not by inositol trisphosphate (IP3),phosphatidylinositol 3-phosphate [PI(3)P], phosphatidylinositol4-phosphate [PI(4)P] or phosphatidylinositol (3,4,5)-trisphosphate[PI(3,4,5)P3] (Pelaseyed et al., 2017). The specificity of PI(4,5)P2 forezrin is in agreement with reports showing that PI(4,5)P2 constitutes∼1% of the phospholipids in the apical membrane, where it recruitsproteins at the interface between the plasma membrane and thecytoplasm. By contrast, PI(3,4,5)P3 is found in the inner membraneleaflet on the basolateral surface (Blazer-Yost et al., 2004; van denBogaart et al., 2011). As recruited proteins engage, throughelectrostatic interactions, with negatively charged phosphates inthe inositol ring of phosphoinositides (Balla, 2005), our findingssuggest that the specific location of negative charges in PI(4,5)P2dictates the specificity for the ezrin FERM domain. LOK is the onlyknown kinase to be specifically localized to the apical membrane ofepithelial cells, and this is mediated through its CTD (Viswanatha

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et al., 2012). Since LOK and PI(4,5)P2 are both enriched on theapical membrane, and both are required for ezrin phosphorylation,this provides a powerful coincidence detection mechanism to ensurethat ezrin activation only occurs on the apical domain (Pelaseyedet al., 2017).Importantly, two additional features ensure LOK kinase specificity

for ezrin. First, the LOK kinase domain requires a distal docking site,situated N-terminal to T567, in order to efficiently phosphorylateT567 (Pelaseyed et al., 2017). Second, LOK and SLK are highlyspecific kinases for ERM proteins, because they exhibit a strongpreference for substrates with a tyrosine at position −2 from T567, aresidue that is conserved in all ERM proteins (Belkina et al., 2009).Replacing the tyrosine residue with a positively charged lysineresidue greatly diminishes the ability of LOK to phosphorylate ezrin(T.P., unpublished data). LOK and its relative SLK belong to thegerminal center-like kinase (GCK)-V subfamily of kinases (Danet al., 2001; Delpire, 2009). They consist of a conserved N-terminalkinase domain, a less-conserved intermediate region and amoderately conserved CTD (Sabourin et al., 2000). The LOK CTDinhibits the kinase activity of LOK, most likely through a cisinteraction (Pelaseyed et al., 2017). In fact, even small amounts of theLOKCTDprevent phosphorylation of ezrin in vitro as well as in cells,where it inhibits microvilli assembly (Viswanatha et al., 2012).Remarkably, the CTD of LOK itself localizes to the distal region ofmicrovilli (Viswanatha et al., 2012), suggesting that the CTD targetsLOK to microvilli through an unidentified mechanism. Interestingly,cells expressing delocalized LOK lacking the CTD still phosphorylate

ezrin, but ezrin is mislocalized to basolateral membranes.Furthermore, intentional delocalization of LOK to basolateralsubdomains caused mislocalized ezrin enrichment at thesebasolateral sites (Viswanatha et al., 2012).

In summary, a coincidence detection mechanism controls thespecificity of ezrin phosphorylation by a combination of twodifferent identity codes – PI(4,5)P2 and LOK – both of which arespatially restricted to apical membranes (Pelaseyed et al., 2017).This high specificity, coupled with dynamic ezrin phosphocyclinginvolving local phosphorylation and delocalized dephosphorylation(Viswanatha et al., 2012) (Fig. 2A,B), reveals that morphologicalpolarity is not a static, but rather a dynamic and spatiallycontrolled process.

Regulation of membrane protein compositionThe microvilli of epithelial cells increase the interface between thecell and the extracellular environment∼10-fold, whereas the proteincontent of the microvillar membrane controls the physiologicalprocesses within the cell. Microvillar proteins include theintermicrovillar adhesion molecules discussed previously, as wellas specialized receptors that elicit intracellular signaling upon ligandrecognition, channels, exchangers and transporter of ions andnutrients, and enzymes such as hydrolases that process digestednutrients (McConnell et al., 2011; van der Post and Hansson,2014). Large, extended glycoproteins, such as transmembranemucins, constitute a glycocalyx that protects the cell againstextracellular insults (Pelaseyed et al., 2013).

Ezrin

LOK

PI(4,5)P2

F-actin

(+)

(–)

FERM

CTD

1

2

3

4

P

T567

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Tip of microvilli(localized ezrinphosphorylation)

Base of microvilli(global ezrin

dephosphorylation)

A

Ezri

nPh

osph

oryl

ated

ezri

n

LOK

CN

P

Fig. 2. Model for spatial control of ezrinphosphocycling required for microvilli formation.(A) Ezrin undergoes continuous cycles of localphosphorylation in the tip region of microvilli where LOKresides; this is followed by global dephosphorylation,most likely at the base of microvilli where the LOKconcentration is lower, before ezrin cycles back to itsactive, phosphorylated form. (B) Model of thecoincidence detection mechanism behind LOK-mediated phosphorylation of ezrin. (1) Inactivecytoplasmic ezrin is recruited to the first apical identitycode PI(4,5)P2. Priming by PI(4,5)P2 results in loweraffinity between the FERM domain and the CTD.(2) The CTD of the second apical identity code, LOK,binds in a a wedge-like manner between the FERMdomain and the CTD of ezrin. (3) LOK kinasedomain can now phosphorylate ezrin on T567.(4) Phosphorylated active ezrin adopts an openconformation that allows crosslinking of the plasmamembrane to the underlying actin cytoskeleton.Adapted from Pelaseyed et al. (2017), where it waspublished under a CC BY 4.0 license (https://creativecommons.org/licenses/by/4.0/).

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https://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/

The protein composition of the microvillar membrane isprimarily determined by sorting and delivery pathways duringmembrane trafficking (Apodaca et al., 2012; Garcia-Castillo et al.,2017). It is believed that the unmasked FERM domain of ezrinengages the cytoplasmic tails of a number of membrane proteins andthis might occur directly or through additional scaffolding proteins(Viswanatha et al., 2013). Ezrin oscillates between open/active andclosed/inactive conformations (Jayaraman and Nicholson, 2007).Consequently, the interaction between ezrin and specific partnersthat bind the FERM domain and CTD, respectively, depends on the‘openness’ of ezrin, a feature that is controlled in part by thephosphorylation status of T567 in the CTD (Nakamura et al., 1999;Pearson et al., 2000). Studies on ezrin indicate that phosphorylationof T567 in CTD creates a partially open ezrin (Chambers andBretscher, 2005). A fully open ezrin with an unmasked FERMdomain requires a synthetic truncation in the CTD that completelyabolishes the interaction between ezrin FERM and CTD (Reczek andBretscher, 1998). A global analysis of ezrin-binding partners hasidentified several membrane and cytosolic proteins (Viswanathaet al., 2013) that can be classified into three groups: (1) proteinsthat preferentially associate with phosphorylated and/or activeezrin [e.g. LOK, SLK and dystroglycan (DAG1)], (2) proteins thatassociatemost strongly with a synthetic hyperactive ezrin variant witha fully unmasked FERM domain [e.g. ezrin-radixin-moesin-bindingphosphoprotein 50 (EBP50)] and, (3) proteins that selectivelyinteract with non-phosphorylated and/or inactive ezrin [e.g. theF-actin-nucleating formin FHOD-1, the Ras GTPase-activatingprotein RASA1, and protein-associating with the carboxyl-terminaldomain of ezrin (SCYL3, also known as PACE-1) (Viswanathaet al., 2013)]. The comprehensive interactome of ezrin supports a‘conformation activation model’ in which some, mostly cytoplasmic,proteins associate with inactive ezrin, and some microvillar proteinsassociate with partially active ezrin and others with fully open ezrin.Fully active ezrin binds the PDZ domain-containing scaffolding

proteins of the NHE3-regulatory factor (NHERF) family, which caninteract with a host of other proteins. The family consists of fourmembers: EBP50 (also known as NHERF1 and SLC9A3R1),NHE3 kinase A regulatory protein (E3KARP, also known asNHERF2 and SLC9A3R2), PDZ domain-containing protein 1(PDZK1, also known as NHERF3) and intestinal and kidney-enriched PDZ protein (IKEPP, also known as NHERF4 andPDZD3). EBP50 and the paralog E3KARP contain two PDZdomains, whereas PDZK1 and IKEPP have four PDZ domains intandem. EBP50 and E3KARP both have an ERM-binding domainin their C-terminal tails (Reczek and Bretscher, 1998; Yun et al.,1998), whereas PDZK1 and IKEPP do not. The four PDZ proteinsalso exhibit differences in terms of tissue distribution andsubcellular localization. EBP50 and PDZK1 are expressedapically in polarized cells of the kidney, small intestine and theliver, whereas E3KARP resides apically in lungs and kidney(Donowitz et al., 2005; Ingraffea et al., 2002; Yun et al., 1998). Bycontrast IKEPP is a primarily cytoplasmic protein found in theintestine and kidneys (Scott et al., 2002).EBP50 associates with the FERM domain of ezrin at a site that is

fully masked by the CTD in the closed inactive conformation(Reczek and Bretscher, 1998; Reczek et al., 1997). EBP50colocalizes with ezrin in microvilli, but is much more dynamicthan ezrin, exhibiting a remarkable in vivo exchange rate of ∼10 s,compared with ∼1 min for ezrin (Garbett and Bretscher, 2012).Knockdown of EBP50 in human syncytiotrophoblasts reduces, butdoes not fully eliminate, the number of cells with microvilli(Garbett et al., 2010; Hanono et al., 2006). The PDZ1 domain and

ezrin-binding site of EBP50 are crucial elements for restoration ofmicrovilli in EBP50-deficient cells. Surprisingly, mutations thatinactivate the PDZ1 and PDZ2 of EBP50, thus precluding ligandbinding, greatly diminish its in vivo exchange rate, suggesting thatproteins interacting with the PDZ domains in EBP50 influence itsin vivo dynamics (Garbett and Bretscher, 2012). EBP50 undergoesphosphorylation by Cdc2 (He et al., 2001) and PKC (Fouassieret al., 2005; Li et al., 2009; Raghuram et al., 2003). Inphosphorylated EBP50, ligand binding to PDZ2 renders PDZ1inaccessible to its ligand(s), resulting in inactive EBP50. OncePDZ2 is unoccupied, phosphorylated EBP50 can function inmicrovilli assembly (Garbett et al., 2010). EBP50 is a scaffoldingprotein for numerous membrane proteins, such as cystic fibrosistransmembrane conductance regulator (CFTR) and β2-adrenergicreceptor (β2AR) (Cao et al., 1999; Hall et al., 1998a,b; Viswanathaet al., 2014), which in turn are under tight regulation (Guggino andStanton, 2006; Yang et al., 2015). Therefore, it is plausible that ahighly dynamic EBP50 reflects the need for a tunable proteinscaffold that can be controlled through phosphorylation as well asPDZ interactions in order to meet the demands from rapidlychanging physiological cues. As noted above, EBP50 contributesin an unknown manner to the regulation of microvilli assembly, ascultured cells with reduced EBP50 expression have greatlyreduced apical microvilli number (Garbett et al., 2010).

In summary, phosphorylation-dependent regulation of theinteractions between ERM proteins and their binding partnersplays a central role in instructing the dynamic morphological andfunctional changes that take place in the apical aspects of polarizedepithelial cells.

Conclusions and perspectivesEver since microvilli were first seen on intestinal epithelial cells byelectron microscopy more than 60 years ago (Granger and Baker,1950), their structure and function have fascinated cell biologists. Inparallel, over the past 40 years, our understanding of the programthat specifies epithelial cell polarity has advanced immensely;however, how it restricts microvilli, or stereocilia, to the apicalmembrane has not been fully elucidated. Furthermore, we do notknow how epithelial cells regulate the precise number of microvillior sterocilia on their surface – in one case with structures of uniformlength, and in the other with precisely defined different lengths.Whereas the role of protocadherins in distal tip complexes ofstereocilia and microvilli has been established, it remains to beproven whether other apical membrane proteins, such as theextended transmembrane mucins of the glycocalyx, play importantroles in membrane bending and curvature when actin protrusionspush through the apical plasma membrane to form tight bundles ofstereocilia and microvilli (Stachowiak et al., 2012).

The proposed mechanism of ezrin phosphorylation, whichinvolves two apical identity codes – PI(4,5)P2 and the ERM-specific kinases LOK and SLK – represents one aspect of therequirements for localized microvilli assembly at apical membranes.However, we still have little knowledge about how microvillarassembly is initiated at this specific membrane domain.Furthermore, how do LOK and SLK localize to microvilli andhow does the CTD of LOK contribute to its localization? How doesthe presence of microvilli impact on the steady-state apicalmembrane proteome, and what role does the dynamics of ezrinand scaffolding proteins play in retention or regulation of membraneproteins? Addressing both general questions regarding cell polarityas well as highly specific questions regarding microvilli andstereocilia will be greatly facilitated by combining recent advances

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in organoid engineering with modern techniques, such asquantitative proteomics, genome editing with CRISPR/Cas9 andhigh-resolution in vivo imaging.

AcknowledgementsWe are grateful to Dr Andrew Lombardo for valuable comments on the manuscript.

Competing interestsThe authors declare no competing or financial interests.

FundingOur research is support by the Wenner-Gren Foundation (T.P.), Swedish CysticFibrosis Foundation (Riksförbundet Cystisk Fibros; T.P.), Birgit and Hellmuth HertzFoundation (T.P.) andNational Institutes of Health (A.B., NIHGM036552). Depositedin PMC for release after 12 months.

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