Regulation of Actin-Based Apical Structures on Epithelial Cells Thaher Pelaseyed1,* and Anthony Bretscher2

REVIEW SUBJECT COLLECTION: CILIA AND FLAGELLA Regulation of actin-based apical structures on epithelial cells Thaher Pelaseyed1,* and Anthony Bretscher2

ABSTRACT related finger-like protrusions that are found on the apical membrane Cells of transporting epithelia are characterized by the presence of of hair cells. We highlight the contribution of bundled actin abundant F-actin-based microvilli on their apical surfaces. Likewise, filaments to apical organization, and the contribution of tip-linking auditory hair cells have highly reproducible rows of apical stereocilia to the structure and function of microvilli and stereocilia. (giant microvilli) that convert mechanical sound into an electrical Furthermore, we describe an emerging mechanism of how signal. Analysis of mutations in deaf patients has highlighted the bundled actin filaments are connected to the plasma membrane in critical components of tip links between stereocilia, and related a way that restricts their presence to the apical membrane. structures that contribute to the organization of microvilli on epithelial cells have been found. Ezrin/radixin/moesin (ERM) proteins, which Morphological features and arrangement of actin-based are activated by phosphorylation, provide a critical link between the surface structures plasma membrane and underlying actin cytoskeleton in surface Shaping the plasma membrane into a finger-like structure requires structures. Here, we outline recent insights into how microvilli and a core of bundled cytoskeletal filaments with lateral attachments stereocilia are built, and the roles of tip links. Furthermore, we to the plasma membrane. In the case of cilia, the filaments are highlight how ezrin is locally regulated by phosphorylation, and that microtubules, whereas filaments of actin support filopodia, this is necessary to maintain polarity. Localized phosphorylation is microvilli and stereocilia (DeRosier and Tilney, 2000; Lewis achieved through an intricate coincidence detection mechanism that and Bridgman, 1992). Here, we restrict our discussion to requires the membrane lipid phosphatidylinositol 4,5-bisphosphate microvilli and stereocilia, which are found on the apical domain of epithelial cells. [PI(4,5)P2] and the apically localized ezrin kinase, lymphocyte- oriented kinase (LOK, also known as STK10) or Ste20-like kinase The plasma membrane of microvilli is supported by a bundle of ∼ – (SLK). We also discuss how ezrin-binding scaffolding proteins 30 40 parallel, inherently polarized actin filaments with their regulate microvilli and how, despite these significant advances, it barbed ends at the microvillus tip (Mooseker and Tilney, 1975). remains to be discovered how the cell polarity program ultimately Early morphological studies showed that the F-actin core is bundled interfaces with these processes. and attached to the plasma membrane by lateral cross-bridges of ∼30 nm length, indicating that the actin filaments are both KEY WORDS: Actin, Cell polarity, Cytoskeleton, ERM proteins, crosslinked to one another and to the plasma membrane (Brunser Microvilli, Stereocilia and Luft, 1970; Matsudaira and Burgess, 1979; Mooseker and Tilney, 1975). On intestinal epithelia and the epithelium of the Introduction kidney proximal tubule, the microvilli are of uniform length and Cells throughout the vertebrate body are polarized. By regulating its generally hexagonally packed. Despite over 40 years of research, it internal organization and plasma membrane composition in a is still not clear what drives the assembly of actin filaments in polarized manner, the cell ensures organism viability and the microvilli, but much has been learned about their morphogenesis execution of central biological functions, such as cell division, by studying microvillar components. Today, we appreciate that proliferation, signaling and migration. One critical aspect of cell events such as cytoskeletal remodeling, membrane–cytoskeleton polarity is how cells build and restrict morphological features to one crosslinking and extracellular adhesion are parts of the cellular domain of a polarized cell. The epithelial cell serves as the program that shapes the apical brush border domain (Crawley et al., archetypal example of a polarized cell. Its apical membrane domain 2014b). We discuss some of these cellular programs in this Review. is decorated with finger-like protrusions called microvilli (Granger Stereocilia are found on the apical aspect of hair cells of the and Baker, 1950). Microvilli expand the interface between the cell vertebrate cochlea and vestibular systems. Early studies of bird and extracellular milieu, and accommodate membrane proteins that cochlea revealed that the length of hair cell stereocilia range from take part in signaling as well as ion and nutrient transport. By 1.5 to 5 µm, increasing in length from the proximal to distal end contrast, the basolateral membrane harbors a different composition (Tilney and Saunders, 1983). However, in contrast to what is seen in of membrane proteins and displays a membrane morphology that is birds, stereocilia on individual cells of the mammalian cochlea very distinct from the apical brush border membrane (Bryant and exhibit a staircase-like pattern with a predetermined graded increase Mostov, 2008). In this Review, we discuss recent insights into the in stereociliary length between rows (Fig. 1A, see Box 1). The regulation and assembly of the apical microvilli and stereocilia – mammalian cochlea contains two types of hair cells: a single row of inner hair cells with a linear array of stereocilia measuring ∼5µmin length, and three rows of outer hair cells with stereocilia arranged in 1Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology, a V-shaped pattern and measuring 2–3 µm in length (Dallos, 1996; University of Gothenburg, SE-405 30 Gothenburg, Sweden. 2Weill Institute for Cell and Molecular Biology, Department of Molecular Biology and Genetics, Kaltenbach et al., 1994). Vestibular hair bundles in the utricle of Cornell University, Ithaca, NY 14853, USA. adult mouse range from ∼2 µm for the shortest stereocilia in the staircase to up to ∼15 µm (Li et al., 2008; Rzadzinska et al., 2004). *Author for correspondence ([email protected]) Each mature stereocilium contains 400–3000 actin filaments with

T.P., 0000-0002-6434-3913; A.B., 0000-0002-1122-8970 their barbed ends at the tip and pointed ends associated with the Journal of Cell Science

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A Fig. 1. Organization and regulation of stereocilia and microvilli. Stereocilia (A) Stereocilia of auditory hair cells are arranged in a staircase configuration. Tip link Stimulus Harmonin Actin bundles are assembled and stabilized through actin-binding proteins such as fimbrin, espin and fascin. SANS Stereocilia of each row are connected to stereocilia of adjacent rows through tip CDH23 links. Tip links require a ternary complex of Myo7a, USH1C (harmonin) and SANS K+ that binds to the cadherin CDH23. Ca2+ MET channel CDH23 forms a heteromeric interstereociliary link with protocadherin Myo7a PCDH15 PCDH15 on the adjacent stereocilium. The cytoplasmic region of PCDH15 interacts with the mechano-electrical transduction (MET) channels. Auditory stimulus causes deflection towards taller rows, which generates an influx of K+ and F-actin F-actin Ca2+ ions through the MET channels. (B) Key Microvilli covering the apical aspects of Fimbrin Espin Fascin intestinal epithelial cells require heterotypic extracellular linkages that are mediated by the protocadherin PCDH24 B and mucin-like protocadherin MLPCDH. By analogy with stereocilia, the Microvilli cytoplasmic tail of PCDH24 binds the ternary complex of USH1C (harmonin), Harmonin the SANS homolog ANKS4B and Myo7b. The actin cytoskeleton is ANKS4B assembled and stabilized by villin, espin PCDH24 and fimbrin.

MLPCDH

Myo7b

F-actin F-actin Key Villin Espin Fimbrin

base, where it tapers and inserts into the cuticular plate of the hair Apical membrane morphology: the cell polarity program and cell (Tilney et al., 1980, 1989; Zheng et al., 2000). In an extended membrane traffic series of studies, Tilney et al. have documented the assembly Microvilli and stereocilia are restricted to the apical domain of of stereocilia during development in ultrastructural detail their respective cells (Fig. 1A,B). This apical-basolateral polarity (Tilney et al., 1992). Stereocilia on mature hair cells are linked is directed by an epithelial polarity program that involves intrinsic by tip links that activate mechano-electrical transduction channels polarity proteins as well as signals from adjacent cells and the when the stereocilia are bent at their base in response to sound. extracellular matrix (Rodriguez-Boulan and Macara, 2014). This A prerequisite for proper mechano-electrical transduction is a program was elucidated mostly from studies in C. elegans and directional mechanosensitivity based on hair cells being arranged Drosophila (Campanale et al., 2017) and, in the fly, it consists of so that their axis of sensitivity is aligned with the direction of Bazooka, Partitioning defective-6 and Atypical protein kinase C stimulation (Shotwell et al., 1981). Recently, a series of studies has (Baz–Par-6–aPKC), which function antagonistically through revealed the molecular mechanisms that instruct the organization of aPKC kinase to exclude the basolateral complex Lethal giant the characteristic V-shaped staircase-like architecture of stereocilia in larvae, Discs large 1 and Scribble (Lgl–Dlg1–Scrib). Basolaterally, in cochlear hair cells (see Box 1). turn, Par-1 kinase antagonizes the apical complex. aPKC and Par-1 It is now clear that assembly of actin-based protrusions requires establish phosphorylation-dependent feedback loops in order to the assembly of protein complexes, the timing and spatial exclude trespassing of proteins and to maintain membrane distribution of which are beginning to be elucidated. In the next domain identity (Benton and St Johnston, 2003). Crumbs (Crb), section, we focus on how membrane traffic provides support for a large transmembrane protein that forms the so-called Crumbs assembly of distinct membrane domains in epithelial cells. complex, participates in an essential epithelial polarity program Journal of Cell Science

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STX3, encoding the motor protein Myo5b and the SNARE protein Box 1. Emerging mechanism for the staircase-like syntaxin 3, which are involved in regulated apical transport and architecture of stereocilia membrane fusion, respectively (Müller et al., 2008; Wiegerinck Hair cells display two integrated levels of polarity. The cells are et al., 2014). A prominent feature in patients with defects in Myo5b systematically and uniformly oriented in the cochlea, thus they display is the accumulation of apical tubules and vesicles that appear to organ-wide planar cell polarity (PCP) (Goodrich and Strutt, 2011). represent Rab8 and Rab11 double-positive recycling compartments Additionally, hair cells have an intrinsic planar axis, outlined by the (Vogel et al., 2017). Myo5b interacts with Rab8 and Rab11, which direction of the staircase-like gradient of stereocilia height towards the tallest hair cell, the kinocilium, at the edge of the V-shaped bundle of are both required for transport of cargos from the trans-Golgi stereocilia (Pickles et al., 1984). Initially, epithelial cells of sensory network to the plasma membrane (Chen et al., 1998; Roland et al., epithelium are covered by microvillar precursors. During apical 2007). Rab8a-deficient mice exhibit a marked decrease in apical morphogenesis, a lateral ‘bare zone’ emerges, uniquely devoid of proteins in intestinal cells, a phenotype that leads to nutrient microvilli. The bare zone forms through apical recruitment of a complex of malabsorption and death (Sato et al., 2007). These mice also display the mammalian adaptor protein inscuteable (mInsc), Leu-Gly-Asn subapical microvillus inclusion bodies produced by endocytosis repeat-enriched protein (LGN, also known as GPSM2) and the – inhibitory heteromeric G protein Gαi (Tarchini et al., 2013). The from the apical membrane. Myo5b Rab11 interaction is required complementary medial side is enriched in atypical PKC, which is for delivery of cargo to plasma membranes in order to establish an excluded from the bare zone. The mInsc–LGN–Gαi complex expands apical membrane domain (Wakabayashi et al., 2005). Mice that are the bare zone, thus relocalizing the kinocilium from the lateral junction deficient in both Rab8a and Rab11a show a severe enteropathy and towards the cell center and shaping the stereocilia bundle (Tarchini et al., extensive formation of basolateral microvilli (Feng et al., 2017). 2013). Mice lacking mInsc display impaired lateromedial extension of the Syntaxin 3 is also known to localize apically in epithelial cells bare zone and exhibit abnormal stereociliary sub-bundles. Deletion of LGN results in disorganized or even missing bundles, displacement of where it facilitates fusion of vesicles to the plasma membrane kinocilium and a generally disrupted apical morphology, indicating that (Delgrossi et al., 1997; Sharma et al., 2006). In summary, LGN is responsible for the inward relocalization of the kinocilium along appropriate apical morphology and physiology requires an the lateromedial axis and asymmetric positioning of stereocilia (Tarchini intimate relationship between cargo trafficking, recycling and et al., 2013). Deletion of Gαi causes delocalization of LGN and results in local assembly of delivered cargo into a functional membrane misorientation of the kinocilia, suggesting that Gαi regulates the lateral domain. Defects in any of these systems result in disease. hair cell orientation through the movement of the kinocilium. LGN and Gαi, but not mInsc, are also enriched in the tip of stereocilia in the tallest row at the medial boundary of the bare zone, and LGN mutants display Tip-links in the structure and function of stereocilia shorter stereocilia and an increased number of rows in the architecture of and microvilli the staircase (Tarchini et al., 2016). Consequently, it has been suggested Analyses of the molecular basis of deafness in patients with that the mInsc–LGN–Gαi first orchestrates the formation of the bare zone hereditary Usher Syndrome 1 have been instrumental in defining a and the V-shaped stereocilia, before LGN and Gαi are localized to protein complex that links the tip of one stereocilium to the side of stereociliary tips in order to define the height of the tallest first row the adjacent one (Fig. 1A) (Pickles et al., 1984). Collectively, this (Tarchini et al., 2016). A fourth component, dishevelled-associating protein with a high frequency of leucine residues (Daple, encoded by work has shown that the MyTH4-FERM tail domain of the motor CCDC88C), couples the PCP pathway to the formation of the cell- protein Myo7a is essential to form a stable ternary complex with the intrinsic planar axis by connecting the PCP protein dishevelled to the PDZ-domain-containing scaffolding proteins USH1C (harmonin) cell-instrinsic signaling component Gαi. Mice lacking Daple exhibit and scaffold protein containing ankyrin repeats and SAM domain misoriented and misshapen staircases of stereocilia, caused by (SANS, also called USH1G) to bind the cytoplasmic tail of the α uncoupling of G i from the kinocilium (Siletti et al., 2017). cadherin CDH23 (Caberlotto et al., 2011; Grati and Kachar, 2011; Li et al., 2017). In addition, the extracellular region of the cadherin dimer interacts with the extracellular region of the protocadherin (Bachmann et al., 2001). The Crumbs complex localizes to the PCDH15 dimer on a neighboring stereocilium (Siemens et al., subapical compartment of the cell (Tepass, 1996), and its deletion 2004). There, the cytoplasmic region of PCDH15 interacts with the results in complete loss of the apical membrane domain, whereas mechano-electrical transduction (MET) channels located on the tip Crb overexpression expands the size of apical domain (Wodarz of the adjacent stereocilium. When sound induces the stereocilia to et al., 1995). The regulatory machinery of membrane polarity bend, the tip-link transmits tension that allows for the influx of K+ overlaps with trafficking of cargos to specific membrane domains. and Ca2+ (Beurg et al., 2006, 2009). The identity of MET channels For example, the GTPases Rab11 and, downstream, Rab8 cooperate that are linked to PCDH15 has not been firmly established. with the apical Par3–aPKC complex to deliver vesicles with early However, sensory transduction signals require the transmembrane apical identity markers, such as podocalyxin, to membranes for channel-like proteins (TMC) 1 and 2, which form heteromeric initiation of lumenogenesis (Bryant et al., 2010). Misregulation of complexes at stereociliary tips and allow Ca2+ permeability (Kurima the intracellular trafficking machinery that underlies cell polarity et al., 2015; Pan et al., 2013). In addition, studies in frogs have leads to disease such as microvillous inclusion disease (MVID), a shown that Pcdh15a interacts with Tmc1 and Tmc2a, orthologs of severe neonatal enteropathy that affects villus enterocytes (Cutz mammalian TMC1 and TMC2 (Maeda et al., 2014). et al., 1989; Davidson et al., 1978). Lack of absorptive channels, Remarkably, a similar type of linking system has been discovered exchangers and nutrient transporters in apical membranes of between microvilli on epithelial cells (Fig. 1B). Here, heterotypic enterocytes leads to diarrhea, life-threatening dehydration and extracellular linkages occur near the tips of microvilli between metabolic disorders (Ruemmele et al., 2006). Neonates with MVID protocadherin PCDH24 (encoded by CDHR2) and mucin-like exhibit increased numbers of subapical vesicles, a sporadic presence protocadherin MLPCDH (encoded by CDHR5) to form links of of microvilli on the basolateral surface, loss of microvilli and 50 nm width (Crawley et al., 2014a, 2016; Li et al., 2016; Yu et al., microvillous inclusions, characterized by intracellular vesicle-like 2017). The cytoplasmic tail of PCDH24 binds USH1C (harmonin) structures luminally lined by microvilli (Sherman et al., 2004). and forms a ternary linkage with the SANS homolog ANKS4B to

MVID has been attributed to mutations in the genes MYO5B and link to the MyTH4-FERM domains of Myo7b. Since USH1C is a Journal of Cell Science

3 REVIEW Journal of Cell Science (2018) 131, jcs221853. doi:10.1242/jcs.221853 common component of tip-linkages between stereocilia and microvilli, and is abundant in the intestine (Crawley et al., 2014a), Box 2. Evolutionary origins of microvilli and stereocilia this explains why a fraction of patients with mutations in the USH1C The closely related structural and tip-link components of microvilli and gene, besides suffering from hearing defects, are also affected by stereocilia suggest they could share a common evolutionary origin. As intestinal disease (Bitner-Glindzicz et al., 2000), including the gut is an evolutionary older organ than the auditory system, it is protracted diarrhea (Kobayashi et al., 1998, 1999; Powell et al., intriguing to think of microvilli as the primordial actin-based structure and a precursor to stereocilia that appeared later in evolution. In support of 1982). The membrane proteome of intestinal epithelial cells this concept, stereocilia typically first emerge as small, short microvilli of includes proteins that are involved in ion transport, nutrient uniform length, before growing into the classic pattern of rows with uptake and bacterial sensing (van der Post and Hansson, 2014) increasing heights (Tilney et al., 1992). The specialized tip links most and Ush1c-knockout mice display loss of microvilli in a fraction of likely arose through gene duplication followed by genetic divergence to intestinal epithelial cells (Crawley et al., 2014a), which explains the encode tissue-specific complexes (Peña et al., 2016). The conservation observed enteropathy. Loss of other components of the inter- of tip linkages in microvilli and stereocilia might be associated with membrane remodeling and mechanosensing in each respective organ. microvillar linkage also results in irregular microvillar lengths and Fluid shear stress has been shown to trigger microvilli formation through loss of the hexagonal packing that is typical of fully differentiated Ca2+ influx through mechanosensitive activation of the Ca2+-selective cells in culture or in the mouse intestine (Crawley et al., 2014a). transient receptor potential cation channel vanilloid 6 (TRPV6), which is Interestingly, the Drosophila ortholog of PCDH15, Cad99C, is expressed in intestinal epithelial cells (Bianco et al., 2006; Miura et al., found on the microvilli of follicle cells, and its loss results in 2015). Likewise, mechanical stimuli to deflect stereocilia result in shortened and disordered microvilli (D’Alterio et al., 2005). opening of MET channels, giving rise to an electrical output (Ohmori, 1985). Finally, both microvilli and stereocilia have to maintain tension Additionally, the adhesion molecule Fasciclin 2 has been found to generated by the formation of actin-based protrusions on the apical cell localize to the brush border of the renal tubules in Drosophila, and membrane. The closely related ERM protein family that crosslink the interference studies indicate that it is necessary for the regulation of actin core to the plasma membrane appear to contribute to this function microvilli length and organization (Halberg et al., 2016). Thus, in hair cell stereocilia and intestinal microvilli (Rouven Brückner these data suggest an emerging principle showing that correct length et al., 2015). regulation and organizational packing of microvilli requires appropriate inter-microvillar links. It is attractive to think that tip links might be a simple mechanism shortened actin protrusions are an indirect effect of diminished to automatically give rise to the beautiful hexagonal packing crosslinking and smaller actin bundle diameter remains to be characteristic of microvilli on intestinal epithelial cells. As shown determined. Fimbrin acts as an F-actin bundler in stereocilia as well in both the vertebrate and fly models, tethering microvilli to each as in microvilli (Hanein et al., 1998; Tilney et al., 1989), whereas other could also be an effective physical mechanism for regulation villin is a multifunctional protein that acts as an F-actin-severing and of microvilli length on individual cells. Whereas the length of -capping protein, as well as an actin bundler and nucleator that microvilli between adjacent cells is usually quite uniform, regulates the distribution of actin filaments (Bretscher and Weber, occasionally this can be variable, but remains strikingly uniform 1980; Khurana and George, 2008). on individual cells, which is consistent with this model (Crawley Remarkably, mice that lack espin and villin display no et al., 2014a). However, how hair cells generate rows of stereocilia particular microvillar phenotype, whereas fimbrin-knockout mice with the same length adjacent to rows with longer lengths is not yet exhibit shorter microvilli that lack rootlets (Ferrary et al., 1999; fully understood. For a discussion of a potential shared evolutionary Grimm-Günter et al., 2009; Zheng et al., 2000). Nevertheless, triple origin of microvilli and stereocilia, see Box 2. knockout mice lacking espin, fimbrin and villin are not only viable, but still develop microvilli. However, microvilli are sparse, Actin filament crosslinkers disorganized and shorter in these animals (Revenu et al., 2012). The length of microvilli varies depending on cell type. In enterocytes, Consequently, it has been proposed that one or more additional each microvillus measures ∼1–2 µm and contains a core of ∼30–40 bundling proteins participate in regulating actin dynamics and unipolar actin filaments (Mooseker, 1985). Stereocilia can be microvillus length; however, none have so far been identified considered an exaggerated form of microvilli with variable lengths (Revenu et al., 2012). A protein that has recently been implicated in based on hair cell type and localization in the cochlea or vestibular regulation of microvillar length is cordon bleu, a WH2 domain- system, as outlined above (Tilney and Saunders, 1983; Tilney et al., containing protein localized at the base of microvilli (Wayt and 1988). The length of microvilli and stereocilia is, at least in part, Bretscher, 2014). Overexpression of cordon bleu results in shorter regulated by the growth rate of the core actin bundle, where longer microvilli in human syncytiotrophoblasts in a WH2 domain- actin protrusions have faster rates of actin polymerization (Narayanan dependent manner (Wayt and Bretscher, 2014), thus suggesting a et al., 2015; Rzadzinska et al., 2004). role for the reported F-actin-severing features of these WH2 domains The actin-crosslinking proteins espin, fimbrin and villin (Jiao et al., 2014). Other studies have shown that cordon bleu usually each play a part in regulation of microvillar length (Fig. 1B) localizes to the base of microvilli, and that overexpression of cordon (Grimm-Günter et al., 2009; Loomis et al., 2003; Ubelmann et al., bleu in intestinal epithelial cells generates more and longer microvillar 2013). Mice carrying mutations in the Espn gene, so-called jerker actin bundles in a WH2-dependent manner (Grega-Larson et al., mice (Grüneberg et al., 1941; Zheng et al., 2000), which exhibit 2016). It is likely that the inconsistent data on the function of cordon characteristic head-jerking movements and rapid circling, show bleu reflects cell-specific effects. In fact, cordon bleu interacts with gradual shortening and thinning of stereocilia followed by hair cell PACSIN-2 (also known as Syndapin-2) in both syncytiotrophoblasts degeneration and vestibular dysfunction (Anniko et al., 1989; and intestinal epithelial cells (Grega-Larson et al., 2016; Wayt and Sekerková et al., 2011). The role of espin in length regulation has also Bretscher, 2014), but whereas this interaction is required for been highlighted in epithelial cells in which espin overexpression targeting PACSIN-2 to microvilli in syncytiotrophoblasts, in results in lengthening of microvilli (Loomis et al., 2003). However, intestinal epithelial cells, PACSIN-2 acts upstream of cordon bleu whether espin is directly involved in length regulation or whether for its localization at the base of microvilli. Journal of Cell Science

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Less is known about the actin-crosslinking proteins of stereocilia. to the apical domain requires local phosphorylation at the apical However, stereocilia lack villin, but utilize at least fimbrin, espin membrane, followed by dephosphorylation in the cytoplasm and fascin as F-actin crosslinkers (Fig. 1A). Fimbrin was the first (Fig. 2A). Remarkably, the basal rate of ezrin phosphocycling crosslinking protein described (Flock et al., 1982; Tilney et al., correlates with the turnover rate of fluorescently tagged ezrin in the 1989) and recent work indicates it is the most abundant one (Krey apical membrane (∼1–2 min) and is significantly faster than the et al., 2016). Espin was initially identified by analysis of the deaf turnover of the microvillus itself (∼12 min) (Garbett and Bretscher, jerker mouse (Zheng et al., 2000), and fascin was the most recently 2012; Gorelik et al., 2003). The fast cycling of ezrin activation reflects described (Chou et al., 2011). A recent study points at the important its role as a crosslinker between the continuously treadmilling actin role of barbed-end-capping proteins in stabilizing actin filaments by filaments and the numerous membrane proteins in the overlying preventing depolymerization. Here, the heterodimeric capping plasma membrane. protein CAPZ is recruited to growing actin filaments in order to prevent depolymerization, especially when the concentration of free A coincidence detection mechanism restricts active actin monomers is low (Avenarius et al., 2017). Deletion of CAPZ ezrin apically in mouse utricles results in both shorter and narrower stereocilia, Two closely related kinases, lymphocyte-oriented kinase (LOK, and diminished auditory and vestibular function. also known as STK10) and Ste20-like kinase (SLK), have been In conclusion, microvilli and stereocilia are dynamic structures that identified as the major ezrin kinases in epithelial cells (Viswanatha require a diverse set of actin-regulating proteins to provide accurate et al., 2012). Phosphorylation of ERM proteins by LOK and SLK is control over actin filament assembly, disassembly and stability. In the functionally conserved: in Drosophila, Slik is the only kinase that next section, we discuss another aspect of stereociliary and phosphorylates the single ERM protein present in flies, Moesin, microvillar dynamics, namely the physical coupling of the actin on the regulatory threonine residue that corresponds to residue bundle of microvilli and stereocilia by phosphoregulated crosslinkers. T567 in human ezrin (Carreno et al., 2008; Hipfner et al., 2004; Kunda et al., 2008). LOK-mediated phosphorylation of ezrin is an Linking the actin core to the plasma membrane intricate multistep process that culminates in T567 phosphorylation The ERM proteins belong to the 4.1 superfamily of proteins that (Pelaseyed et al., 2017). The FERM domain of ezrin contains share a 4.1 protein, ezrin, radixin and moesin (FERM) domain as a a binding pocket for phosphatidylinositol 4,5-bisphosphate common feature. The ERM proteins, each encoded by a single gene [PI(4,5)P2] (Niggli et al., 1995) located adjacent to the coiled-coil in mammals, act as tissue-specific crosslinkers between the plasma α-helix connecting the FERM domain to the CTD. It is likely that membrane and the underlying cortical F-actin cytoskeleton in many binding of PI(4,5)P2 to this particular binding site triggers a different cellular contexts (Fehon et al., 2010). Most epithelial cells conformational change that lowers the affinity between the FERM only express ezrin; stereocilia on hair cells of the cochlea contain domain and CTD. The lower-affinity FERM–CTD complex allows some ezrin, but predominantly express radixin (Kikuchi et al., 2002; for the CTD of LOK to wedge in between the FERM and CTD, thus Kitajiri et al., 2004). Consequently, ezrin-knockout mice die prying ezrin open and allowing LOK kinase domain to gain access soon after birth with short microvilli and disorganized rootlets to the phosphorylatable T567 residue (Fig. 2B). Several important (Saotome et al., 2004) and the intestinal defects are even more features of this mechanism are highlighted below: the transition of severe upon tissue-specific depletion of ezrin in adult animals the FERM–CTD complex from the high-affinity closed (Casaletto et al., 2011). The mouse radixin knockout is viable, but conformation to a lower-affinity conformation requires that the stereocilia degenerate after formation, resulting in deafness FERM and CTD domains are connected through the coiled-coil (Kitajiri et al., 2004). Thus, ERM proteins have central roles in α-helix. In fact, a FERM–CTD complex that lacks this coiled-coil the structure and function of microvilli and stereocilia. region cannot be phosphorylated by LOK, even in the presence of ERM proteins consist of an N-terminal FERM domain, a central PI(4,5)P2 (Pelaseyed et al., 2017). Thus, binding of PI(4,5)P2 to full- α-helical domain and a C-terminal domain (CTD) in which the length ezrin – a process we call ‘priming’–does not simply change F-actin-binding site lies (Turunen et al., 1994). In their inactive the conformation of the FERM domain, but requires active (closed) form, the FERM domain binds with high affinity to the involvement of the α-helical region to reduce the affinity between CTD with the α-helical region forming an anti-parallel coiled coil the FERM and CTD domains (Pelaseyed et al., 2017). In this sense, (Gary and Bretscher, 1995; Li et al., 2007). In this closed state, both the central helical region can function as a spring to reduce the the sites on the FERM domain for binding membrane proteins and affinity between the domains. Ezrin is explicitly primed by the F-actin-binding site in the CTD are masked (Li et al., 2007). PI(4,5)P2 and not by inositol trisphosphate (IP3), Phosphorylation of a conserved threonine residue (T567 in ezrin) in phosphatidylinositol 3-phosphate [PI(3)P], phosphatidylinositol the CTD lowers the affinity between the FERM domain and CTD, 4-phosphate [PI(4)P] or phosphatidylinositol (3,4,5)-trisphosphate resulting in ezrin adopting a more open configuration (Nakamura [PI(3,4,5)P3] (Pelaseyed et al., 2017). The specificity of PI(4,5)P2 for et al., 1995). Open ezrin can now act as a crosslinker by engaging ezrin is in agreement with reports showing that PI(4,5)P2 constitutes plasma membrane proteins through its N-terminal FERM domain, ∼1% of the phospholipids in the apical membrane, where it recruits whereas the CTD binds F-actin in the bundled filaments (Gary and proteins at the interface between the plasma membrane and the Bretscher, 1995) (Fig. 2B). Oscillation of ezrin between the open cytoplasm. By contrast, PI(3,4,5)P3 is found in the inner membrane and closed states is regulated by rapid cycles of phosphorylation leaflet on the basolateral surface (Blazer-Yost et al., 2004; van den and dephosphorylation (phosphocycling), exhibiting a turnover Bogaart et al., 2011). As recruited proteins engage, through time of ∼1 min in cultured cells (Viswanatha et al., 2012). If the cycle electrostatic interactions, with negatively charged phosphates in of ezrin phosphorylation and dephosphorylation is decoupled – the inositol ring of phosphoinositides (Balla, 2005), our findings either by inhibiting ezrin dephosphorylation or by expressing a suggest that the specific location of negative charges in PI(4,5)P2 constitutively active phosphomimetic ezrin mutant (T567E) – the dictates the specificity for the ezrin FERM domain. LOK is the only polarized distribution of ezrin in the apical membrane is lost known kinase to be specifically localized to the apical membrane of

(Viswanatha et al., 2012). Consequently, restricting ezrin function epithelial cells, and this is mediated through its CTD (Viswanatha Journal of Cell Science

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B Fig. 2. Model for spatial control of ezrin (+) phosphocycling required for microvilli formation. (A) Ezrin undergoes continuous cycles of local phosphorylation in the tip region of microvilli where LOK resides; this is followed by global dephosphorylation, most likely at the base of microvilli where the LOK P concentration is lower, before ezrin cycles back to its F-actin active, phosphorylated form. (B) Model of the 4 coincidence detection mechanism behind LOK- mediated phosphorylation of ezrin. (1) Inactive cytoplasmic ezrin is recruited to the first apical identity

code PI(4,5)P2. Priming by PI(4,5)P2 results in lower affinity between the FERM domain and the CTD. A (–) (2) The CTD of the second apical identity code, LOK,

LOK Ezrin Phosphorylated ezrin binds in a a wedge-like manner between the FERM Tip of microvilli domain and the CTD of ezrin. (3) LOK kinase

(localized ezrin P domain can now phosphorylate ezrin on T567. phosphorylation) (4) Phosphorylated active ezrin adopts an open 3 conformation that allows crosslinking of the plasma Base of microvilli membrane to the underlying actin cytoskeleton. (global ezrin Adapted from Pelaseyed et al. (2017), where it was dephosphorylation) published under a CC BY 4.0 license (https:// creativecommons.org/licenses/by/4.0/).

T 5 67

C LOK N

Ezrin 1 FERM CTD PI(4,5)P2

et al., 2012). Since LOK and PI(4,5)P2 are both enriched on the ezrin, but ezrin is mislocalized to basolateral membranes. apical membrane, and both are required for ezrin phosphorylation, Furthermore, intentional delocalization of LOK to basolateral this provides a powerful coincidence detection mechanism to ensure subdomains caused mislocalized ezrin enrichment at these that ezrin activation only occurs on the apical domain (Pelaseyed basolateral sites (Viswanatha et al., 2012). et al., 2017). In summary, a coincidence detection mechanism controls the Importantly, two additional features ensure LOK kinase specificity specificity of ezrin phosphorylation by a combination of two for ezrin. First, the LOK kinase domain requires a distal docking site, different identity codes – PI(4,5)P2 and LOK – both of which are situated N-terminal to T567, in order to efficiently phosphorylate spatially restricted to apical membranes (Pelaseyed et al., 2017). T567 (Pelaseyed et al., 2017). Second, LOK and SLK are highly This high specificity, coupled with dynamic ezrin phosphocycling specific kinases for ERM proteins, because they exhibit a strong involving local phosphorylation and delocalized dephosphorylation preference for substrates with a tyrosine at position −2fromT567,a (Viswanatha et al., 2012) (Fig. 2A,B), reveals that morphological residue that is conserved in all ERM proteins (Belkina et al., 2009). polarity is not a static, but rather a dynamic and spatially Replacing the tyrosine residue with a positively charged lysine controlled process. residue greatly diminishes the ability of LOK to phosphorylate ezrin (T.P., unpublished data). LOK and its relative SLK belong to the Regulation of membrane protein composition germinal center-like kinase (GCK)-V subfamily of kinases (Dan The microvilli of epithelial cells increase the interface between the et al., 2001; Delpire, 2009). They consist of a conserved N-terminal cell and the extracellular environment ∼10-fold, whereas the protein kinase domain, a less-conserved intermediate region and a content of the microvillar membrane controls the physiological moderately conserved CTD (Sabourin et al., 2000). The LOK CTD processes within the cell. Microvillar proteins include the inhibits the kinase activity of LOK, most likely through a cis intermicrovillar adhesion molecules discussed previously, as well interaction (Pelaseyed et al., 2017). In fact, even small amounts of the as specialized receptors that elicit intracellular signaling upon ligand LOK CTD prevent phosphorylation of ezrin in vitro as well as in cells, recognition, channels, exchangers and transporter of ions and where it inhibits microvilli assembly (Viswanatha et al., 2012). nutrients, and enzymes such as hydrolases that process digested Remarkably, the CTD of LOK itself localizes to the distal region of nutrients (McConnell et al., 2011; van der Post and Hansson, microvilli (Viswanatha et al., 2012), suggesting that the CTD targets 2014). Large, extended glycoproteins, such as transmembrane LOK to microvilli through an unidentified mechanism. Interestingly, mucins, constitute a glycocalyx that protects the cell against cells expressing delocalized LOK lacking the CTD still phosphorylate extracellular insults (Pelaseyed et al., 2013). Journal of Cell Science

6 REVIEW Journal of Cell Science (2018) 131, jcs221853. doi:10.1242/jcs.221853

The protein composition of the microvillar membrane is ezrin-binding site of EBP50 are crucial elements for restoration of primarily determined by sorting and delivery pathways during microvilli in EBP50-deficient cells. Surprisingly, mutations that membrane trafficking (Apodaca et al., 2012; Garcia-Castillo et al., inactivate the PDZ1 and PDZ2 of EBP50, thus precluding ligand 2017). It is believed that the unmasked FERM domain of ezrin binding, greatly diminish its in vivo exchange rate, suggesting that engages the cytoplasmic tails of a number of membrane proteins and proteins interacting with the PDZ domains in EBP50 influence its this might occur directly or through additional scaffolding proteins in vivo dynamics (Garbett and Bretscher, 2012). EBP50 undergoes (Viswanatha et al., 2013). Ezrin oscillates between open/active and phosphorylation by Cdc2 (He et al., 2001) and PKC (Fouassier closed/inactive conformations (Jayaraman and Nicholson, 2007). et al., 2005; Li et al., 2009; Raghuram et al., 2003). In Consequently, the interaction between ezrin and specific partners phosphorylated EBP50, ligand binding to PDZ2 renders PDZ1 that bind the FERM domain and CTD, respectively, depends on the inaccessible to its ligand(s), resulting in inactive EBP50. Once ‘openness’ of ezrin, a feature that is controlled in part by the PDZ2 is unoccupied, phosphorylated EBP50 can function in phosphorylation status of T567 in the CTD (Nakamura et al., 1999; microvilli assembly (Garbett et al., 2010). EBP50 is a scaffolding Pearson et al., 2000). Studies on ezrin indicate that phosphorylation protein for numerous membrane proteins, such as cystic fibrosis of T567 in CTD creates a partially open ezrin (Chambers and transmembrane conductance regulator (CFTR) and β2-adrenergic Bretscher, 2005). A fully open ezrin with an unmasked FERM receptor (β2AR)(Caoetal.,1999;Halletal.,1998a,b;Viswanatha domain requires a synthetic truncation in the CTD that completely et al., 2014), which in turn are under tight regulation (Guggino and abolishes the interaction between ezrin FERM and CTD (Reczek and Stanton, 2006; Yang et al., 2015). Therefore, it is plausible that a Bretscher, 1998). A global analysis of ezrin-binding partners has highly dynamic EBP50 reflects the need for a tunable protein identified several membrane and cytosolic proteins (Viswanatha scaffold that can be controlled through phosphorylation as well as et al., 2013) that can be classified into three groups: (1) proteins PDZ interactions in order to meet the demands from rapidly that preferentially associate with phosphorylated and/or active changing physiological cues. As noted above, EBP50 contributes ezrin [e.g. LOK, SLK and dystroglycan (DAG1)], (2) proteins that in an unknown manner to the regulation of microvilli assembly, as associate most strongly with a synthetic hyperactive ezrin variant with cultured cells with reduced EBP50 expression have greatly a fully unmasked FERM domain [e.g. ezrin-radixin-moesin-binding reduced apical microvilli number (Garbett et al., 2010). phosphoprotein 50 (EBP50)] and, (3) proteins that selectively In summary, phosphorylation-dependent regulation of the interact with non-phosphorylated and/or inactive ezrin [e.g. the interactions between ERM proteins and their binding partners F-actin-nucleating formin FHOD-1, the Ras GTPase-activating plays a central role in instructing the dynamic morphological and protein RASA1, and protein-associating with the carboxyl-terminal functional changes that take place in the apical aspects of polarized domain of ezrin (SCYL3, also known as PACE-1) (Viswanatha epithelial cells. et al., 2013)]. The comprehensive interactome of ezrin supports a ‘conformation activation model’ in which some, mostly cytoplasmic, Conclusions and perspectives proteins associate with inactive ezrin, and some microvillar proteins Ever since microvilli were first seen on intestinal epithelial cells by associate with partially active ezrin and others with fully open ezrin. electron microscopy more than 60 years ago (Granger and Baker, Fully active ezrin binds the PDZ domain-containing scaffolding 1950), their structure and function have fascinated cell biologists. In proteins of the NHE3-regulatory factor (NHERF) family, which can parallel, over the past 40 years, our understanding of the program interact with a host of other proteins. The family consists of four that specifies epithelial cell polarity has advanced immensely; members: EBP50 (also known as NHERF1 and SLC9A3R1), however, how it restricts microvilli, or stereocilia, to the apical NHE3 kinase A regulatory protein (E3KARP, also known as membrane has not been fully elucidated. Furthermore, we do not NHERF2 and SLC9A3R2), PDZ domain-containing protein 1 know how epithelial cells regulate the precise number of microvilli (PDZK1, also known as NHERF3) and intestinal and kidney- or sterocilia on their surface – in one case with structures of uniform enriched PDZ protein (IKEPP, also known as NHERF4 and length, and in the other with precisely defined different lengths. PDZD3). EBP50 and the paralog E3KARP contain two PDZ Whereas the role of protocadherins in distal tip complexes of domains, whereas PDZK1 and IKEPP have four PDZ domains in stereocilia and microvilli has been established, it remains to be tandem. EBP50 and E3KARP both have an ERM-binding domain proven whether other apical membrane proteins, such as the in their C-terminal tails (Reczek and Bretscher, 1998; Yun et al., extended transmembrane mucins of the glycocalyx, play important 1998), whereas PDZK1 and IKEPP do not. The four PDZ proteins roles in membrane bending and curvature when actin protrusions also exhibit differences in terms of tissue distribution and push through the apical plasma membrane to form tight bundles of subcellular localization. EBP50 and PDZK1 are expressed stereocilia and microvilli (Stachowiak et al., 2012). apically in polarized cells of the kidney, small intestine and the The proposed mechanism of ezrin phosphorylation, which liver, whereas E3KARP resides apically in lungs and kidney involves two apical identity codes – PI(4,5)P2 and the ERM- (Donowitz et al., 2005; Ingraffea et al., 2002; Yun et al., 1998). By specific kinases LOK and SLK – represents one aspect of the contrast IKEPP is a primarily cytoplasmic protein found in the requirements for localized microvilli assembly at apical membranes. intestine and kidneys (Scott et al., 2002). However, we still have little knowledge about how microvillar EBP50 associates with the FERM domain of ezrin at a site that is assembly is initiated at this specific membrane domain. fully masked by the CTD in the closed inactive conformation Furthermore, how do LOK and SLK localize to microvilli and (Reczek and Bretscher, 1998; Reczek et al., 1997). EBP50 how does the CTD of LOK contribute to its localization? How does colocalizes with ezrin in microvilli, but is much more dynamic the presence of microvilli impact on the steady-state apical than ezrin, exhibiting a remarkable in vivo exchange rate of ∼10 s, membrane proteome, and what role does the dynamics of ezrin compared with ∼1 min for ezrin (Garbett and Bretscher, 2012). and scaffolding proteins play in retention or regulation of membrane Knockdown of EBP50 in human syncytiotrophoblasts reduces, but proteins? Addressing both general questions regarding cell polarity does not fully eliminate, the number of cells with microvilli as well as highly specific questions regarding microvilli and

(Garbett et al., 2010; Hanono et al., 2006). The PDZ1 domain and stereocilia will be greatly facilitated by combining recent advances Journal of Cell Science

7 REVIEW Journal of Cell Science (2018) 131, jcs221853. doi:10.1242/jcs.221853 in organoid engineering with modern techniques, such as Carreno, S., Kouranti, I., Glusman, E. S., Fuller, M. T., Echard, A. and Payre, F. quantitative proteomics, genome editing with CRISPR/Cas9 and (2008). Moesin and its activating kinase Slik are required for cortical stability and microtubule organization in mitotic cells. J. Cell Biol. 180, 739-746. high-resolution in vivo imaging. Casaletto, J. B., Saotome, I., Curto, M. and McClatchey, A. I. (2011). Ezrin-mediated apical integrity is required for intestinal homeostasis. Proc. Natl. Acknowledgements Acad. Sci. USA 108, 11924-11929. We are grateful to Dr Andrew Lombardo for valuable comments on the manuscript. Chambers, D. N. and Bretscher, A. (2005). Ezrin mutants affecting dimerization and activation. Biochemistry 44, 3926-3932. Competing interests Chen, W., Feng, Y., Chen, D. and Wandinger-Ness, A. (1998). 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