Epithelial cells from diverse tissues, including the enterocytes that line the intestinal tract, remodel their apical surface during differentiation to form a brush border: an array of actin-supported membrane protrusions known as microvilli that increases the functional capacity of the tissue. Although our understanding of how epithelial cells assemble, stabilize, and organize apical microvilli is still developing, investigations of the biochemical and physical underpinnings of these processes suggest that cells coordinate cytoskeletal remodeling, membrane-cytoskeleton cross-linking, and extracellular adhesion to shape the apical brush border domain.

Biologists have long appreciated the intimate connection between morphology and function. Powerful examples are provided by the epithelial cells that line our hollow organs, including the gut, kidney, lung, and cochlea. The intestinal tract provides one of the most striking cases. Here, morphological adaptations at both the tissue and cellular level allow the intestinal epithelium to make close and prolonged contact with luminal contents, in turn promoting efficient uptake of available nutrients. The small intestinal mucosa features thousands of macroscopic (∼0.5 mm in length) fingerlike folds of tissue referred to as villi (Fig. 1, A, B, and D), which project into the lumen, increasing the tissue surface area available for nutrient absorption (Helander and Fändriks, 2014). The epithelium that lines each villus is composed of several cell types including goblet cells, enteroendocrine cells, Paneth cells, and enterocytes. Among these, enterocytes are by far the most abundant and are solely responsible for nutrient absorption. Because of the ever-present threat of chemical or physical damage, the intestinal epithelium is continuously renewed by stem cells found in the “crypts” at the base of each villus (Fig. 1, B and D; van der Flier and Clevers, 2009). As nascent epithelial cells emerge from the crypt, they undergo rapid differentiation (Fig. 1 C), and a subset takes on the attributes of mature enterocytes (Mariadason et al., 2005; Chang et al., 2008).

Fully differentiated enterocytes are characterized by a prominent brush border with two defining morphological features. First, microvilli demonstrate remarkably uniform length (Fig. 1 E), with little variability in dimensions within and between cells (∼100 nm diameter × ∼1–3 µm length depending on the region of the intestinal tract). Second, these protrusions exhibit highly ordered packing such that when the brush border is viewed en face hexagonal arrays of microvilli are observed (Fig. 1, C and F). The optimized packing of microvilli is a cellular level adaptation that further increases the surface area exposed to the lumen. Based on morphometric data obtained by light and electron microscopy, estimates suggest that microvilli amplify the surface area of the small intestine 9-16 fold (Helander and Fändriks, 2014). Thus, the numerous microvilli within the brush border support a membrane “reservoir,” which allows the enterocyte to enrich membrane-associated molecules needed for nutrient absorption (Maroux et al., 1988) and host defense (Koyama et al., 2002; Shifrin et al., 2012) on the apical surface.

Building core actin bundles

To generate microvilli, epithelial cells must overcome physical forces that oppose membrane deformation including membrane bending stiffness (κ, pN•nm), surface tension (Tm, pN/m), and membrane–cytoskeleton adhesion energy (γ, pN•nm/nm2; Sheetz, 2001; Nambiar et al., 2010). To this end, cells harness cytoskeletal dynamics to generate forces that “push” against cellular membranes and drive deformation (Theriot, 2000; Atilgan et al., 2006). In the case of intestinal microvilli, the origin of the deforming force remains unclear but is likely generated by the polymerization and bundling of actin filaments. In vitro biophysical studies indicate that actin filament elongation can generate forces in the pN range (Miyata et al., 1999; Cameron et al., 2000; Parekh et al., 2005; Footer et al., 2007). Interestingly, a classic set of experiments using native isolated brush borders revealed that actin polymerization preferentially occurs at the membrane-associated ends of core actin bundles (Pollard and Mooseker, 1981; Mooseker et al., 1982). In other early experiments, Tilney and Cardell (1970) observed the association of electron-dense foci just below the plasma membrane during the regrowth of microvilli that were destroyed by the application of hydrostatic pressure. Remarkably, actin filaments appear to originate from this material during microvillar regrowth. Similar structures have been observed at the tips of enterocyte microvilli (Mooseker and Tilney, 1975), stereocilia (Rzadzinska et al., 2004), and filopodia of motile cells (Svitkina et al., 2003). Although the composition of these structures is not well understood, they most likely represent protein complexes involved in the spatial and temporal control of actin polymerization, including actin nucleators and capping proteins, membrane bending and shaping proteins, and the signaling proteins or small GTPases that control them. However, the molecular machinery involved in controlling the polymerization of microvillar actin filaments in vivo is still unknown.

Because single actin filaments are not stiff enough to deform the membrane, microvillar protrusion requires that cells bundle numerous filaments together. This has the dual effect of increasing the number of putative force-producing elements (i.e., F-actin plus ends) per unit area of membrane and increasing the structural rigidity of the protrusion (Mogilner and Rubinstein, 2005; Claessens et al., 2006; Bathe et al., 2008). Recent electron tomography studies indicate that microvillar cores are composed of ∼30–40 bundled filaments that exhibit a slight clockwise-oriented twist when viewing distal tips en face (Ohta et al., 2012). Actin filaments within microvilli are bundled by villin (Bretscher and Weber, 1979; Mooseker et al., 1980), espin (Bartles et al., 1998), and fimbrin (Fig. 2 A and Fig. 3 A; Bretscher and Weber, 1980). Because the resulting bundles are composed of parallel actin filaments, these structures also hold the potential to serve as tracks for myosin motor proteins. In vivo studies examining the expression pattern of the microvillar core proteins revealed that villin, fimbrin, and actin localize to the apical cortex in enterocytes early during polarization, before the onset of protrusion (Heintzelman and Mooseker, 1990a,b). The role of filament bundling in microvillar protrusion was subsequently established in studies showing that exogenous villin could induce microvillar growth on the surface of cells that do not normally make microvilli (Franck et al., 1990). Later work demonstrated that overexpression of espin promoted microvillar elongation in an epithelial cell culture model system (Loomis et al., 2003). Fimbrin has also been implicated in physically linking microvillar rootlets to the dense network of cytokeratin filaments that comprise the terminal web (Grimm-Günter et al., 2009). Anchoring microvilli into the terminal web plays a crucial role in promoting the long-term stability of the brush border, as disruption of this functional connection leads to microvilli that are sensitive to biochemical manipulations and degradation (Grimm-Günter et al., 2009).

Although villin, espin, and fimbrin are acknowledged as the major F-actin bundling proteins in the intestinal brush border, mouse models lacking one or more of these proteins are still able to assemble microvilli. Indeed, deletion of villin in mice had a minimal impact on brush border organization under normal conditions (Ferrary et al., 1999). Even more remarkably, a mouse model lacking villin, espin, and fimbrin still assembled a functional brush border, although microvillar organization was perturbed (Revenu et al., 2012). These investigations strongly suggest that other atypical actin-bundling proteins may play a role in building core bundles. One possibility is the receptor tyrosine kinase substrate EPS8 (Fig. 2 A), which localizes to the tips of microvilli (Fig. 3 A) and is capable of both capping plus ends and bundling filaments (Croce et al., 2004; Hertzog et al., 2010). Interestingly, a knockout (KO) of EPS8 in mice led to microvilli that were shorter and more variable in length, but again, functional protrusions still formed (Tocchetti et al., 2010). Collectively, these studies indicate that brush border assembly is biologically robust, with highly effective compensatory mechanisms in place to ensure that microvillar core actin bundles are assembled during enterocyte differentiation.

Stabilizing protrusions

Although actin polymerization and bundling likely contribute forces that drive the protrusion of individual microvilli, additional physical obstacles arise when new protrusions are built in close proximity to each other. Unless prevented, membrane surface tension will promote the coalescence and fusion of adjacent protrusions to achieve an energetic minimum (Atilgan et al., 2006). This is a significant concern where the density of surface protrusions is exceedingly high, as in the intestinal brush border. To counteract such coalescence and stabilize protrusions, epithelial cells take advantage of molecules such as unconventional myosins and ERM (ezrin, radixin, moesin) family proteins, which cross-link the plasma membrane to the underlying actin cytoskeleton.

Myosin-1a.

Class I myosins are single-headed motor proteins that function at the actin cytoskeleton–plasma membrane interface in numerous cellular processes including endo- and exocytosis (Novak et al., 1995; Bose et al., 2004), the release of microvesicles (McConnell and Tyska, 2007), and the maintenance of membrane tension and cortical rigidity (Dai et al., 1999; Nambiar et al., 2009). The intestinal brush border is home to several class I myosins, with myosin-1a (myo1a) being by far the most abundant (Fig. 2 B and Fig. 3 B; McConnell et al., 2011). Expression of myo1a is limited to the intestinal tract, where it localizes almost exclusively to the brush border (Skowron et al., 1998; Skowron and Mooseker, 1999). Within microvilli, it forms the characteristic lateral bridges observed with transmission EM, which appear to cross-link the plasma membrane to the actin core (Fig. 1 G; Mooseker and Tilney, 1975; Howe and Mooseker, 1983). Myo1a is a slow motor (Wolenski et al., 1993a,b; Jontes et al., 1997) that interacts with membrane using a tail homology 1 (TH1) domain that exhibits moderate affinity for lipids with acidic head groups, such as phosphatidylserine or PI(4,5)P2 (Hayden et al., 1990; Mazerik and Tyska, 2012). TH1 contains two distinct membrane-binding motifs, the N-terminal and C-terminal targeting motifs (Mazerik and Tyska, 2012), which function to keep this motor in close proximity to the plasma membrane (Mazerik et al., 2014). Interestingly, mutations in the C-terminal motif have been linked to a loss of epithelial polarity and the development of colon cancer, which indicates that normal membrane binding is important for myo1a tumor suppressor function in vivo (Mazzolini et al., 2012). Kinetic studies indicate that the myo1a motor domain is a low-duty-ratio motor; i.e., it only spends a small fraction of its total ATPase cycle time bound to actin (Jontes et al., 1997). Although this kinetic limitation may appear to be inconsistent with a role in membrane–cytoskeleton cross-linking, each microvillus is home to thousands of asynchronously cycling myo1a molecules (Brown and McKnight, 2010), an ensemble large enough to ensure a continuous interaction between the plasma membrane and the actin core.

Mice lacking myo1a exhibit significant perturbations in brush border morphology including extensive membrane herniations and fused microvilli (Tyska et al., 2005). These abnormalities suggest that physical coupling between the plasma membrane and the actin core is disrupted in the absence of myo1a. Direct support for this proposal was eventually provided by optical trap-based measurements of membrane tension in isolated myo1a KO brush borders and in live cells where levels of myo1a were manipulated (Nambiar et al., 2009). Despite the absence of membrane–cytoskeleton cross-links, myo1a KO enterocytes still maintain functional microvilli (Tyska et al., 2005). Compensatory mechanisms in this case involve other class I myosins that redistribute to the brush border upon loss of myo1a, including myo1c and myo1d (Tyska et al., 2005; Benesh et al., 2010).

Disrupting the ability of myo1a to link plasma membrane to the actin core might also facilitate the infection of enteric pathogens. Enteropathogenic Escherichia coli (EPEC) colonize the intestinal mucosa by forming intimate attachments with enterocytes through the injection of virulence factors that remodel the apical surface (Rothbaum et al., 1983; Knutton et al., 1987). This remodeling destroys the brush border, resulting in severe watery diarrhea (Knutton et al., 1987). One such virulence factor, EspB, interacts with and inhibits actin binding of a variety of myosin family members, including myo1a (Iizumi et al., 2007). This raises the interesting possibility that EspB-mediated disruption of myo1a cross-links may be an important event in EPEC-induced remodeling of the apical surface. Indeed, a mutant strain of EPEC with a defective copy of EspB unable to interact with myosins exhibited reduced microvillar effacement in cultured cells and lower infection rates in mice (Iizumi et al., 2007). Interestingly, disrupting myo1a function during EPEC infection also prevents the shedding of antimicrobial vesicles from the tips of microvilli (Shifrin et al., 2012).

Myosin-6.

A second unconventional myosin, myosin-6 (myo6), also mediates membrane–cytoskeleton interactions in the brush border. Myo6 is unique among myosins in that it is the only minus-end-directed motor (Wells et al., 1999). Monomeric myo6 is thought to function as a mechanical tether in cells (Altman et al., 2004), whereas dimeric myo6 acts as an active transporter for numerous cargo (Buss and Kendrick-Jones, 2011). In enterocytes, myo6 localizes to the subapical terminal web region (Fig. 3 B), where it tethers the plasma membrane to the actin core bundle and regulates clathrin-dependent endocytosis (Ameen and Apodaca, 2007; Hegan et al., 2012). Loss of myo6 in mice results in lifting of the plasma membrane off the actin cytoskeleton and the fusion of adjacent microvilli (Hegan et al., 2012). Without motifs for direct membrane binding, how myo6 couples the actin cytoskeleton to the brush border membrane at the base of microvilli is unclear, but it might assemble into a multiprotein membrane-associated complex that localizes to this region. For example, in hair cells of the inner ear, myo6 exists as part of a complex containing chloride intracellular channel 5 (CLIC5), taperin, radixin, and protein tyrosine phosphatase receptor Q (PTPRQ). This complex localizes to the base of stereocilia (Salles et al., 2014), specialized actin-supported protrusions found on the apical surface of inner ear hair cells that play an essential role in hearing and balance. Stereocilia of mice null for myo6 become fused at their bases during development, resulting in profound deafness and vestibular dysfunction in these animals (Avraham et al., 1995; Self et al., 1999). A similar phenotype is observed in mice deficient in other components of this protein complex including CLIC5 (Salles et al., 2014), radixin (Kitajiri et al., 2004), and PTPRQ (Goodyear et al., 2003). Interestingly, myo6 is absent in brush borders of myo1a null mice, whereas myo1a levels are not perturbed in myo6 mutant mice (Tyska et al., 2005; Hegan et al., 2012). Whether this indicates that proper targeting of myo6 is uniquely sensitive to microvillar perturbations or if there exists a more complex functional interplay between these two motors is unknown, but further investigation will be needed to understand the significance and function of this relationship.

Ezrin.

Ezrin is the only ERM family member expressed in the intestinal epithelium (Bretscher, 1983; Berryman et al., 1993). Ezrin is composed of an N-terminal 4.1 ERM (FERM) domain that binds to membrane lipids including PI(4,5)P2 and a C-terminal ERM-association domain (C-ERMAD) that interacts with F-actin (Fig. 2 B; Algrain et al., 1993; Turunen et al., 1994; Niggli et al., 1995). The N-terminal FERM domain also contains binding pockets for a variety of protein interaction partners, including the membrane-associated scaffolding proteins ERM-binding protein 50 (EBP50; Reczek et al., 1997) and NHE3 kinase A regulatory protein (E3KARP; Yun et al., 1998). Interactions between the FERM and C-ERMAD domains hold ezrin in an inactive “closed” conformation (Fig. 3 B, i) that is unable to bind F-actin or membrane-associated binding partners (Gary and Bretscher, 1995). Activation of ezrin is proposed to occur sequentially. Ezrin first targets to the plasma membrane by interacting with PI(4,5)P2. This, in turn, leads to the phosphorylation of a key conserved threonine (T567) in the C-ERMAD, which disrupts the auto-inhibitory interaction between the N-terminal FERM and the C-ERMAD, thus activating the molecule and allowing for its interaction with F-actin and other membrane-associated binding partners (Fig. 3 B, i).

Phosphorylation of ezrin is a critical downstream event in the pathway involving the Lkb1/Strad-α/Mo25 polarization–signaling complex that is proposed to regulate the early events in brush border induction (ten Klooster et al., 2009). It is, however, still unclear which kinase is responsible for ezrin activation in intestinal brush borders, with several possible candidates including the STE20-like protein kinases Mst4, LOK, and SLK (ten Klooster et al., 2009; Viswanatha et al., 2012). In mature microvilli, ezrin undergoes constant phosphocycling, with the active phosphorylated form of the protein exhibiting a half-life of ∼2 min (Viswanatha et al., 2012). Activation of ezrin is proposed to occur at the distal tips of microvilli (Fig. 3 B; Hanono et al., 2006). While in this short-lived “on” state, ezrin functions to cross-link numerous transmembrane and membrane-associated proteins to the actin core bundle. Active ezrin eventually becomes dephosphorylated by microvillus-localized phosphatases (possibly protein phosphatase 1 [PP1]), which leads to its release from membrane and cytoskeleton (Viswanatha et al., 2012).

Several lines of evidence suggest that ezrin links the membrane to the actin cytoskeleton. Ezrin KO mice die soon after birth and exhibit defects in brush border formation, with short, fused microvilli that have disorganized rootlets (Saotome et al., 2004). Defects are even more severe when ezrin expression is lost in adult animals (Casaletto et al., 2011). Moreover, dephosphorylation of ezrin is observed as an early event in renal brush border breakdown during ischemia (Chen et al., 1995); this coincides with the dissociation of ezrin from the cytoskeleton and the appearance of apical membrane blebs from renal proximal tubule epithelial cells. Interestingly, brush borders from myo6 null mice exhibit higher levels of active ezrin, which might compensate for the loss of this myosin at the base of microvilli (Hegan et al., 2012). Studies in nonepithelial systems also suggest that ezrin stabilizes interactions between membrane and actin. Mouse lymphocytes engineered to express an active form of ezrin display an ∼70% increase in membrane tension, which leads to numerous perturbations in T lymphocyte function in vivo, including decreased migration and lower T cell count in efferent lymph (Liu et al., 2012).

In addition to linking the actin core to the overlying membrane in microvilli, ezrin plays other important roles in enterocyte polarization. During brush border formation, active ezrin functions as a signaling platform by promoting the apical localization of the Cdc42-specific guanine nucleotide exchange factor (GEF) Dbl3 (Zihni et al., 2014). Activation of Cdc42 at the apical cell margins drives the Par6–aPKC pathway toward apical differentiation and brush border induction. Coupling the activation of ezrin with the localization of a Cdc42-specific GEF involved in brush border induction might allow the early events of membrane protrusion (e.g., polymerization and bundling of actin filaments) to be coupled to the availability of membrane–cytoskeleton cross-linkers needed for stabilizing nascent protrusions.

Putting the “order” in brush border

The highly ordered packing of brush border microvilli was first visualized in electron micrographs more than six decades ago (Granger and Baker, 1950). Analysis of the mechanism driving this organization was confounded for many years by the fact that most epithelial cell culture models do not recapitulate the apical surface organization observed in vivo. The CACO-2BBE cell line, however, has proven to be a valuable tool in recent studies. Derived from a human colonic adenocarcinoma (Fogh et al., 1977; Peterson and Mooseker, 1992), these cells establish a clearly defined apical–basal axis and assemble a well-ordered brush border when cultured for two to three weeks past confluency (Peterson and Mooseker, 1993). The resulting apical surface ultrastructure is comparable to that found in vivo. CACO-2BBE cells and native enterocytes also exhibit a similar gene expression profile during differentiation (Fleet et al., 2003).

Using the CACO-2BBE model system, recent investigations demonstrated that extracellular adhesion molecules play a central role in organizing microvilli during brush border assembly. At early time points in differentiation, microvilli protrude from the cell surface and cluster together, exhibiting apparent adhesion between their distal tips (Fig. 4; Crawley et al., 2014). As brush border assembly progresses, intermicrovillar adhesion promotes the incorporation of additional microvilli into existing clusters as well as the coalescence of smaller clusters into larger structures. This process eventually leads to a single large-scale cluster on the apical surface, i.e., a mature brush border. These studies further revealed that clustering microvilli are physically connected by thread-like links composed of cadherin family members—protocadherin-24 (PCDH24) and mucin-like protocadherin (MLPCDH)—which form a trans-heterophilic adhesion complex (Fig. 2 C and Fig. 3 C; Crawley et al., 2014). Both protocadherins are highly expressed in tissues that build brush borders, including the intestine and kidney (Goldberg et al., 2002; Okazaki et al., 2002), where they localize to the tips of microvilli via interactions with the scaffolding protein, harmonin-a, and the molecular motor myosin-7b (Fig. 2 C and Fig. 3 C, ii; Crawley et al., 2014). Knockdown of PCDH24 or MLPCDH impairs brush border assembly, giving rise to apical domains with fewer protrusions that are more loosely packed and highly variable in length. Thus, although the emergence of microvilli is a stochastic process, adhesion between protrusions can be used to organize an otherwise disordered system.

Cadherins also play a crucial role in the organization and function of stereocilia of the inner ear. During hair cell development, stereocilia are organized into defined rows of graded height that form a characteristic “staircase” pattern, known as a hair bundle. Key to the assembly and maintenance of the hair bundle is a series of extracellular links that physically connect neighboring protrusions. Of particular importance are links comprised of two protocadherins—cadherin-23 and protocadherin-15—which form a strong trans-heterophilic adhesion complex (Kazmierczak et al., 2007). These protocadherins are highly expressed during early development and form transient lateral links that connect neighboring stereocilia, as well as kinociliary links that bridge the microtubule-based kinocilium to adjacent stereocilia (Siemens et al., 2004; Lagziel et al., 2005; Michel et al., 2005; Rzadzinska et al., 2005; Ahmed et al., 2006). Both forms of links have been shown to be important for the initial cohesion of the hair bundle during development (Alagramam et al., 2001; Di Palma et al., 2001). As development proceeds, these cadherins are lost from the base of hair bundle stereocilia and become restricted to the tip, forming the mature structure known as the tip link (Goodyear et al., 2005; Michel et al., 2005). Tip links connect neighboring stereocilia and directly mediate mechanotransduction (Müller, 2008). Analogous to intestinal intermicrovillar adhesion links that control brush border assembly, genetic disruption of the stereocilia adhesion links results in pronounced disorganization of hair bundles (Alagramam et al., 2001; Di Palma et al., 2001; Alagramam et al., 2011). Stereocilia protocadherins also associate with several cytosolic proteins, including the scaffolding proteins harmonin-b and sans, the calcium and integrin-binding protein CIB2, and the motor myosin-7a. Together, these molecules define the core set of genes associated with Type 1 Usher syndrome, the most common form of deafness/blindness in humans (Weil et al., 1995; Bitner-Glindzicz et al., 2000; Verpy et al., 2000; Bolz et al., 2001; Bork et al., 2001; Weil et al., 2003; Riazuddin et al., 2012). Currently, harmonin represents the only component that is common to the adhesion complexes found in the inner ear and the intestine (although different isoforms are expressed in the gut and cochlea). Interestingly, Usher syndrome patients who possess a large deletion mutation in their USH1C gene (the gene that encodes for harmonin) suffer from severe GI dysfunction in addition to the neurosensory deficits related to stereocilia dysfunction (Bitner-Glindzicz et al., 2000; Hussain et al., 2004). Consistent with this, Ush1c KO mice, which were developed to model Type 1 Usher syndrome, display significant perturbations in intestinal brush border morphology (Crawley et al., 2014). These findings provide strong support for the emerging paradigm that extracellular adhesion represents a conserved mechanism used to shape the surface of epithelial cells in tissues with divergent functions.

In addition to promoting tight packing of microvilli, intermicrovillar adhesion might also be involved in unifying microvillar length. Brush borders lacking adhesion links produce microvilli that exhibit much greater variability in length (Crawley et al., 2014). One possible explanation is that intermicrovillar adhesion complex components directly influence the polymerization dynamics of the actin core. A second possibility is that intermicrovillar adhesion introduces physical constraints that prevent an individual protrusion from growing longer than its neighbors. In an interesting parallel to the brush border, components of the stereocilia tip complex also play a role in controlling the length of these specialized protrusions. Postnatal deletion of either cadherin-23 or sans results in the regression of stereocilia from the middle and lower rows of hair bundles in mice (Caberlotto et al., 2011). Additional studies will be needed to determine the models alluded to here, or other models that might explain protrusion length control, such as the balance point model proposed to regulate the length of cilia (Chan and Marshall, 2012), are active in microvilli and stereocilia.

Microvilli in mature brush borders are interconnected by a vast network of intermicrovillar links (Crawley et al., 2014). This suggests that intermicrovillar adhesion is needed not only for brush border assembly, but also for the long-term maintenance of this organelle. Whether microvillar protocadherins play a role beyond shaping and stabilizing the apical domain, e.g., in promoting barrier function, is currently unknown. However, one can easily envision a scenario where intermicrovillar adhesion contributes to the formation of a physical barrier against luminal microbes that seek to gain access to the apical surface, such as EPEC (Nougayrède et al., 2003). Both the high packing density and the uniform length of microvilli could play into this by preventing the formation of spaces or gaps in the brush border that could act as protective niches for growth of these microbes. Adherence and internalization of typically noninvasive luminal bacteria into enterocytes can also play a role in intestinal disease and has been documented in patients with inflammatory bowel disease (Kleessen et al., 2002; Swidsinski et al., 2002) and celiac disease (Forsberg et al., 2004). This form of microbial invasion is thought to occur when the brush border barrier becomes disrupted as a result of microvillar “fanning,” a condition caused by interferon-γ–induced myosin II–dependent contraction of the terminal web (Wu et al., 2014). Brush border fanning allows bacteria access to the base of microvilli, where they are internalized by lipid raft–dependent endocytosis (Clark et al., 2005; Wu et al., 2014). One might expect intermicrovillar links to resist such mechanical disruption, but additional studies will be needed to confirm the function of brush border protocadherins in this context.

Future directions

Investigators have made significant progress over the course of the last several decades toward understanding the molecular basis of brush border assembly, yet several key questions remain unanswered. One fundamental question is how cells control microvillar dimensions such as length and diameter (i.e., the number of bundled actin filaments). Although recent discoveries of intermicrovillar adhesion provide a molecular basis for relative length control, bringing uniformity to microvillar length, the matter of how absolute protrusion length is determined is still an open question. Recent studies suggest that cells grapple with an actin allocation problem that arises from a limited pool of G-actin, which in turn limits the amount of actin available for assembling cytoskeletal structures (Burke et al., 2014). It will be interesting to determine if microvillar growth is sensitive to the size of the actin pool, and if so, how enterocytes determine how much actin they can allocate to brush border assembly. With regard to filament number in the core actin bundle, this parameter is mostly likely controlled by the nucleation machinery that polymerizes microvillar actin filaments. To date, the nucleator responsible for brush border assembly has yet to be identified. Proteomic analyses of isolated brush borders identified three candidates: cordon-bleu, diaphanous homologue 1, and the Arp2/3 complex (McConnell et al., 2011; Revenu et al., 2012). Cordon-bleu is an interesting possibility; this protein was originally characterized as a linear actin nucleator controlling neuronal morphology and development (Ahuja et al., 2007). More recently, cordon-bleu was reported to localize near the base of microvilli in JEG-3 cells (a choriocarcinoma cell line derived from human placenta), although it did not appear to regulate microvillar formation in this context (Wayt and Bretscher, 2014). Future studies will need to focus on identifying the nucleator responsible for polymerizing F-actin in the intestinal brush border.

Although this review focuses on molecules that play direct roles in shaping the brush border domain, other cellular pathways must be involved in the regulation of these components and their coordination in space and time. Broadening our understanding along these lines will require development of new model systems, which are amenable to time-lapse imaging so that the temporal component of enterocyte differentiation can be studied. Recent advances in tissue-derived cell culture models represent a promising avenue for such investigations (Sato and Clevers, 2013). Indeed, stem cell–containing crypts can be isolated from intestinal epithelial tissues and expanded in culture. In the presence of appropriate growth factors, crypts will differentiate into “mini-gut” structures that exhibit crypt–villus organization like that observed in vivo (Sato et al., 2009). Intestinal organoids have yet to be used for studies of brush border assembly and cytoskeletal dynamics, but because this model is amenable to both live cell imaging and genetic manipulation, we expect it to provide an unprecedented opportunity to probe the molecular underpinnings of enterocyte differentiation.

Future studies must also focus on clarifying mechanisms of human diseases characterized by perturbations in brush border morphology. Loss of microvilli leads to nutrient malabsorption and osmotic diarrhea, common features of several intestinal diseases that pose significant threats to human health. These include infections with attaching and effacing bacteria (EPEC), celiac disease, Usher syndrome, and microvillus inclusion disease (MVID; Bailey et al., 1989; Bitner-Glindzicz et al., 2000; Wilson et al., 2001; Vallance et al., 2002; Khubchandani et al., 2011; Crawley et al., 2014). MVID in particular has received a great deal of experimental attention recently; this inherited disease is typically observed in infants, who present with unremitting diarrhea that can only be treated with total parenteral nutrition (Davidson et al., 1978). Mutations in myosin-5b and syntaxin-3 are now established as drivers of MVID (Erickson et al., 2008; Müller et al., 2008; Wiegerinck et al., 2014). Both molecules function in the apical recycling system, which recent work suggests is required for maintenance of the brush border domain (Dhekne et al., 2014; Knowles et al., 2014; Wiegerinck et al., 2014). Thus, another exciting direction for the future will be investigating the coordination between the trafficking machinery that delivers membrane to the apical surface and cytoskeletal components and adhesion molecules that build, stabilize, and organize brush border microvilli.

Acknowledgments

The authors thank all members of the Tyska laboratory for feedback and advice.

This work was supported by National Institutes of Health grants DK075555 and DK095811 (to M.J. Tyska).

The authors declare no competing financial interests.

References

References
Ahmed
,
Z.M.
,
R.
Goodyear
,
S.
Riazuddin
,
A.
Lagziel
,
P.K.
Legan
,
M.
Behra
,
S.M.
Burgess
,
K.S.
Lilley
,
E.R.
Wilcox
,
S.
Riazuddin
, et al
.
2006
.
The tip-link antigen, a protein associated with the transduction complex of sensory hair cells, is protocadherin-15
.
J. Neurosci.
26
:
7022
7034
.
Ahuja
,
R.
,
R.
Pinyol
,
N.
Reichenbach
,
L.
Custer
,
J.
Klingensmith
,
M.M.
Kessels
, and
B.
Qualmann
.
2007
.
Cordon-bleu is an actin nucleation factor and controls neuronal morphology
.
Cell.
131
:
337
350
.
Alagramam
,
K.N.
,
C.L.
Murcia
,
H.Y.
Kwon
,
K.S.
Pawlowski
,
C.G.
Wright
, and
R.P.
Woychik
.
2001
.
The mouse Ames waltzer hearing-loss mutant is caused by mutation of Pcdh15, a novel protocadherin gene
.
Nat. Genet.
27
:
99
102
.
Alagramam
,
K.N.
,
R.J.
Goodyear
,
R.
Geng
,
D.N.
Furness
,
A.F.
van Aken
,
W.
Marcotti
,
C.J.
Kros
, and
G.P.
Richardson
.
2011
.
Mutations in protocadherin 15 and cadherin 23 affect tip links and mechanotransduction in mammalian sensory hair cells
.
PLoS ONE.
6
:
e19183
.
Algrain
,
M.
,
O.
Turunen
,
A.
Vaheri
,
D.
Louvard
, and
M.
Arpin
.
1993
.
Ezrin contains cytoskeleton and membrane binding domains accounting for its proposed role as a membrane-cytoskeletal linker
.
J. Cell Biol.
120
:
129
139
.
Altman
,
D.
,
H.L.
Sweeney
, and
J.A.
Spudich
.
2004
.
The mechanism of myosin VI translocation and its load-induced anchoring
.
Cell.
116
:
737
749
.
Ameen
,
N.
, and
G.
Apodaca
.
2007
.
Defective CFTR apical endocytosis and enterocyte brush border in myosin VI-deficient mice
.
Traffic.
8
:
998
1006
.
Atilgan
,
E.
,
D.
Wirtz
, and
S.X.
Sun
.
2006
.
Mechanics and dynamics of actin-driven thin membrane protrusions
.
Biophys. J.
90
:
65
76
.
Avraham
,
K.B.
,
T.
Hasson
,
K.P.
Steel
,
D.M.
Kingsley
,
L.B.
Russell
,
M.S.
Mooseker
,
N.G.
Copeland
, and
N.A.
Jenkins
.
1995
.
The mouse Snell’s waltzer deafness gene encodes an unconventional myosin required for structural integrity of inner ear hair cells
.
Nat. Genet.
11
:
369
375
.
Bailey
,
D.S.
,
A.R.
Freedman
,
S.C.
Price
,
D.
Chescoe
, and
P.J.
Ciclitira
.
1989
.
Early biochemical responses of the small intestine of coeliac patients to wheat gluten
.
Gut.
30
:
78
85
.
Bartles
,
J.R.
,
L.
Zheng
,
A.
Li
,
A.
Wierda
, and
B.
Chen
.
1998
.
Small espin: a third actin-bundling protein and potential forked protein ortholog in brush border microvilli
.
J. Cell Biol.
143
:
107
119
.
Bathe
,
M.
,
C.
Heussinger
,
M.M.
Claessens
,
A.R.
Bausch
, and
E.
Frey
.
2008
.
Cytoskeletal bundle mechanics
.
Biophys. J.
94
:
2955
2964
.
Benesh
,
A.E.
,
R.
Nambiar
,
R.E.
McConnell
,
S.
Mao
,
D.L.
Tabb
, and
M.J.
Tyska
.
2010
.
Differential localization and dynamics of class I myosins in the enterocyte microvillus
.
Mol. Biol. Cell.
21
:
970
978
.
Berryman
,
M.
,
Z.
Franck
, and
A.
Bretscher
.
1993
.
Ezrin is concentrated in the apical microvilli of a wide variety of epithelial cells whereas moesin is found primarily in endothelial cells
.
J. Cell Sci.
105
:
1025
1043
.
Bitner-Glindzicz
,
M.
,
K.J.
Lindley
,
P.
Rutland
,
D.
Blaydon
,
V.V.
Smith
,
P.J.
Milla
,
K.
Hussain
,
J.
Furth-Lavi
,
K.E.
Cosgrove
,
R.M.
Shepherd
, et al
.
2000
.
A recessive contiguous gene deletion causing infantile hyperinsulinism, enteropathy and deafness identifies the Usher type 1C gene
.
Nat. Genet.
26
:
56
60
.
Bolz
,
H.
,
B.
von Brederlow
,
A.
Ramírez
,
E.C.
Bryda
,
K.
Kutsche
,
H.G.
Nothwang
,
M.
Seeliger
,
M.
del C-Salcedó Cabrera
,
M.C.
Vila
,
O.P.
Molina
, et al
.
2001
.
Mutation of CDH23, encoding a new member of the cadherin gene family, causes Usher syndrome type 1D
.
Nat. Genet.
27
:
108
112
.
Bork
,
J.M.
,
L.M.
Peters
,
S.
Riazuddin
,
S.L.
Bernstein
,
Z.M.
Ahmed
,
S.L.
Ness
,
R.
Polomeno
,
A.
Ramesh
,
M.
Schloss
,
C.R.
Srisailpathy
, et al
.
2001
.
Usher syndrome 1D and nonsyndromic autosomal recessive deafness DFNB12 are caused by allelic mutations of the novel cadherin-like gene CDH23
.
Am. J. Hum. Genet.
68
:
26
37
.
Bose
,
A.
,
S.
Robida
,
P.S.
Furcinitti
,
A.
Chawla
,
K.
Fogarty
,
S.
Corvera
, and
M.P.
Czech
.
2004
.
Unconventional myosin Myo1c promotes membrane fusion in a regulated exocytic pathway
.
Mol. Cell. Biol.
24
:
5447
5458
.
Bretscher
,
A.
1983
.
Purification of an 80,000-dalton protein that is a component of the isolated microvillus cytoskeleton, and its localization in nonmuscle cells
.
J. Cell Biol.
97
:
425
432
.
Bretscher
,
A.
, and
K.
Weber
.
1979
.
Villin: the major microfilament-associated protein of the intestinal microvillus
.
Proc. Natl. Acad. Sci. USA.
76
:
2321
2325
.
Bretscher
,
A.
, and
K.
Weber
.
1980
.
Fimbrin, a new microfilament-associated protein present in microvilli and other cell surface structures
.
J. Cell Biol.
86
:
335
340
.
Brown
,
J.W.
, and
C.J.
McKnight
.
2010
.
Molecular model of the microvillar cytoskeleton and organization of the brush border
.
PLoS ONE.
5
:
e9406
.
Burke
,
T.A.
,
J.R.
Christensen
,
E.
Barone
,
C.
Suarez
,
V.
Sirotkin
, and
D.R.
Kovar
.
2014
.
Homeostatic actin cytoskeleton networks are regulated by assembly factor competition for monomers
.
Curr. Biol.
24
:
579
585
.
Buss
,
F.
, and
J.
Kendrick-Jones
.
2011
.
Multifunctional myosin VI has a multitude of cargoes
.
Proc. Natl. Acad. Sci. USA.
108
:
5927
5928
.
Caberlotto
,
E.
,
V.
Michel
,
I.
Foucher
,
A.
Bahloul
,
R.J.
Goodyear
,
E.
Pepermans
,
N.
Michalski
,
I.
Perfettini
,
O.
Alegria-Prévot
,
S.
Chardenoux
, et al
.
2011
.
Usher type 1G protein sans is a critical component of the tip-link complex, a structure controlling actin polymerization in stereocilia
.
Proc. Natl. Acad. Sci. USA.
108
:
5825
5830
.
Cameron
,
L.A.
,
P.A.
Giardini
,
F.S.
Soo
, and
J.A.
Theriot
.
2000
.
Secrets of actin-based motility revealed by a bacterial pathogen
.
Nat. Rev. Mol. Cell Biol.
1
:
110
119
.
Casaletto
,
J.B.
,
I.
Saotome
,
M.
Curto
, and
A.I.
McClatchey
.
2011
.
Ezrin-mediated apical integrity is required for intestinal homeostasis
.
Proc. Natl. Acad. Sci. USA.
108
:
11924
11929
.
Chan
,
Y.H.
, and
W.F.
Marshall
.
2012
.
How cells know the size of their organelles
.
Science.
337
:
1186
1189
.
Chang
,
J.
,
M.R.
Chance
,
C.
Nicholas
,
N.
Ahmed
,
S.
Guilmeau
,
M.
Flandez
,
D.
Wang
,
D.S.
Byun
,
S.
Nasser
,
J.M.
Albanese
, et al
.
2008
.
Proteomic changes during intestinal cell maturation in vivo
.
J. Proteomics.
71
:
530
546
.
Chen
,
J.
,
J.A.
Cohn
, and
L.J.
Mandel
.
1995
.
Dephosphorylation of ezrin as an early event in renal microvillar breakdown and anoxic injury
.
Proc. Natl. Acad. Sci. USA.
92
:
7495
7499
.
Claessens
,
M.M.
,
M.
Bathe
,
E.
Frey
, and
A.R.
Bausch
.
2006
.
Actin-binding proteins sensitively mediate F-actin bundle stiffness
.
Nat. Mater.
5
:
748
753
.
Clark
,
E.
,
C.
Hoare
,
J.
Tanianis-Hughes
,
G.L.
Carlson
, and
G.
Warhurst
.
2005
.
Interferon gamma induces translocation of commensal Escherichia coli across gut epithelial cells via a lipid raft-mediated process
.
Gastroenterology.
128
:
1258
1267
.
Crawley
,
S.W.
,
D.A.
Shifrin
Jr
,
N.E.
Grega-Larson
,
R.E.
McConnell
,
A.E.
Benesh
,
S.
Mao
,
Y.
Zheng
,
Q.Y.
Zheng
,
K.T.
Nam
,
B.A.
Millis
, et al
.
2014
.
Intestinal brush border assembly driven by protocadherin-based intermicrovillar adhesion
.
Cell.
157
:
433
446
.
Croce
,
A.
,
G.
Cassata
,
A.
Disanza
,
M.C.
Gagliani
,
C.
Tacchetti
,
M.G.
Malabarba
,
M.F.
Carlier
,
G.
Scita
,
R.
Baumeister
, and
P.P.
Di Fiore
.
2004
.
A novel actin barbed-end-capping activity in EPS-8 regulates apical morphogenesis in intestinal cells of Caenorhabditis elegans
.
Nat. Cell Biol.
6
:
1173
1179
.
Dai
,
J.
,
H.P.
Ting-Beall
,
R.M.
Hochmuth
,
M.P.
Sheetz
, and
M.A.
Titus
.
1999
.
Myosin I contributes to the generation of resting cortical tension
.
Biophys. J.
77
:
1168
1176
.
Davidson
,
G.P.
,
E.
Cutz
,
J.R.
Hamilton
, and
D.G.
Gall
.
1978
.
Familial enteropathy: a syndrome of protracted diarrhea from birth, failure to thrive, and hypoplastic villus atrophy
.
Gastroenterology.
75
:
783
790
.
Dhekne
,
H.S.
,
N.H.
Hsiao
,
P.
Roelofs
,
M.
Kumari
,
C.L.
Slim
,
E.H.
Rings
, and
S.C.
van Ijzendoorn
.
2014
.
Myosin Vb and Rab11a regulate phosphorylation of ezrin in enterocytes
.
J. Cell Sci.
127
:
1007
1017
.
Di Palma
,
F.
,
R.H.
Holme
,
E.C.
Bryda
,
I.A.
Belyantseva
,
R.
Pellegrino
,
B.
Kachar
,
K.P.
Steel
, and
K.
Noben-Trauth
.
2001
.
Mutations in Cdh23, encoding a new type of cadherin, cause stereocilia disorganization in waltzer, the mouse model for Usher syndrome type 1D
.
Nat. Genet.
27
:
103
107
.
Erickson
,
R.P.
,
K.
Larson-Thomé
,
R.K.
Valenzuela
,
S.E.
Whitaker
, and
M.D.
Shub
.
2008
.
Navajo microvillous inclusion disease is due to a mutation in MYO5B
.
Am. J. Med. Genet. A.
146A
:
3117
3119
.
Ferrary
,
E.
,
M.
Cohen-Tannoudji
,
G.
Pehau-Arnaudet
,
A.
Lapillonne
,
R.
Athman
,
T.
Ruiz
,
L.
Boulouha
,
F.
El Marjou
,
A.
Doye
,
J.J.
Fontaine
, et al
.
1999
.
In vivo, villin is required for Ca2+-dependent F-actin disruption in intestinal brush borders
.
J. Cell Biol.
146
:
819
830
.
Fleet
,
J.C.
,
L.
Wang
,
O.
Vitek
,
B.A.
Craig
, and
H.J.
Edenberg
.
2003
.
Gene expression profiling of Caco-2 BBe cells suggests a role for specific signaling pathways during intestinal differentiation
.
Physiol. Genomics.
13
:
57
68
.
Fogh
,
J.
,
J.M.
Fogh
, and
T.
Orfeo
.
1977
.
One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice
.
J. Natl. Cancer Inst.
59
:
221
226
.
Footer
,
M.J.
,
J.W.
Kerssemakers
,
J.A.
Theriot
, and
M.
Dogterom
.
2007
.
Direct measurement of force generation by actin filament polymerization using an optical trap
.
Proc. Natl. Acad. Sci. USA.
104
:
2181
2186
.
Forsberg
,
G.
,
A.
Fahlgren
,
P.
Hörstedt
,
S.
Hammarström
,
O.
Hernell
, and
M.L.
Hammarström
.
2004
.
Presence of bacteria and innate immunity of intestinal epithelium in childhood celiac disease
.
Am. J. Gastroenterol.
99
:
894
904
.
Franck
,
Z.
,
M.
Footer
, and
A.
Bretscher
.
1990
.
Microinjection of villin into cultured cells induces rapid and long-lasting changes in cell morphology but does not inhibit cytokinesis, cell motility, or membrane ruffling
.
J. Cell Biol.
111
:
2475
2485
.
Gary
,
R.
, and
A.
Bretscher
.
1995
.
Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site
.
Mol. Biol. Cell.
6
:
1061
1075
.
Goldberg
,
M.
,
M.
Wei
,
B.
Tycko
,
I.
Falikovich
, and
D.
Warburton
.
2002
.
Identification and expression analysis of the human mu-protocadherin gene in fetal and adult kidneys
.
Am. J. Physiol. Renal Physiol.
283
:
F454
F463
.
Goodyear
,
R.J.
,
P.K.
Legan
,
M.B.
Wright
,
W.
Marcotti
,
A.
Oganesian
,
S.A.
Coats
,
C.J.
Booth
,
C.J.
Kros
,
R.A.
Seifert
,
D.F.
Bowen-Pope
, and
G.P.
Richardson
.
2003
.
A receptor-like inositol lipid phosphatase is required for the maturation of developing cochlear hair bundles
.
J. Neurosci.
23
:
9208
9219
.
Goodyear
,
R.J.
,
W.
Marcotti
,
C.J.
Kros
, and
G.P.
Richardson
.
2005
.
Development and properties of stereociliary link types in hair cells of the mouse cochlea
.
J. Comp. Neurol.
485
:
75
85
.
Granger
,
B.
, and
R.F.
Baker
.
1950
.
Electron microscope investigation of the striated border of intestinal epithelium
.
Anat. Rec.
107
:
423
441
.
Grimm-Günter
,
E.M.
,
C.
Revenu
,
S.
Ramos
,
I.
Hurbain
,
N.
Smyth
,
E.
Ferrary
,
D.
Louvard
,
S.
Robine
, and
F.
Rivero
.
2009
.
Plastin 1 binds to keratin and is required for terminal web assembly in the intestinal epithelium
.
Mol. Biol. Cell.
20
:
2549
2562
.
Hanono
,
A.
,
D.
Garbett
,
D.
Reczek
,
D.N.
Chambers
, and
A.
Bretscher
.
2006
.
EPI64 regulates microvillar subdomains and structure
.
J. Cell Biol.
175
:
803
813
.
Hayden
,
S.M.
,
J.S.
Wolenski
, and
M.S.
Mooseker
.
1990
.
Binding of brush border myosin I to phospholipid vesicles
.
J. Cell Biol.
111
:
443
451
.
Hegan
,
P.S.
,
H.
Giral
,
M.
Levi
, and
M.S.
Mooseker
.
2012
.
Myosin VI is required for maintenance of brush border structure, composition, and membrane trafficking functions in the intestinal epithelial cell
.
Cytoskeleton (Hoboken).
69
:
235
251
.
Heintzelman
,
M.B.
, and
M.S.
Mooseker
.
1990a
.
Assembly of the brush border cytoskeleton: changes in the distribution of microvillar core proteins during enterocyte differentiation in adult chicken intestine
.
Cell Motil. Cytoskeleton.
15
:
12
22
.
Heintzelman
,
M.B.
, and
M.S.
Mooseker
.
1990b
.
Structural and compositional analysis of early stages in microvillus assembly in the enterocyte of the chick embryo
.
Differentiation.
43
:
175
182
.
Helander
,
H.F.
, and
L.
Fändriks
.
2014
.
Surface area of the digestive tract - revisited
.
Scand. J. Gastroenterol.
49
:
681
689
.
Hertzog
,
M.
,
F.
Milanesi
,
L.
Hazelwood
,
A.
Disanza
,
H.
Liu
,
E.
Perlade
,
M.G.
Malabarba
,
S.
Pasqualato
,
A.
Maiolica
,
S.
Confalonieri
, et al
.
2010
.
Molecular basis for the dual function of Eps8 on actin dynamics: bundling and capping
.
PLoS Biol.
8
:
e1000387
.
Howe
,
C.L.
, and
M.S.
Mooseker
.
1983
.
Characterization of the 110-kdalton actin-calmodulin-, and membrane-binding protein from microvilli of intestinal epithelial cells
.
J. Cell Biol.
97
:
974
985
.
Hussain
,
K.
,
M.
Bitner-Glindzicz
,
D.
Blaydon
,
K.J.
Lindley
,
D.A.
Thompson
,
T.
Kriss
,
K.
Rajput
,
D.G.
Ramadan
,
Z.
Al-Mazidi
,
K.E.
Cosgrove
, et al
.
2004
.
Infantile hyperinsulinism associated with enteropathy, deafness and renal tubulopathy: clinical manifestations of a syndrome caused by a contiguous gene deletion located on chromosome 11p
.
J. Pediatr. Endocrinol. Metab.
17
:
1613
1621
.
Iizumi
,
Y.
,
H.
Sagara
,
Y.
Kabe
,
M.
Azuma
,
K.
Kume
,
M.
Ogawa
,
T.
Nagai
,
P.G.
Gillespie
,
C.
Sasakawa
, and
H.
Handa
.
2007
.
The enteropathogenic E. coli effector EspB facilitates microvillus effacing and antiphagocytosis by inhibiting myosin function
.
Cell Host Microbe.
2
:
383
392
.
Jontes
,
J.D.
,
R.A.
Milligan
,
T.D.
Pollard
, and
E.M.
Ostap
.
1997
.
Kinetic characterization of brush border myosin-I ATPase
.
Proc. Natl. Acad. Sci. USA.
94
:
14332
14337
.
Kazmierczak
,
P.
,
H.
Sakaguchi
,
J.
Tokita
,
E.M.
Wilson-Kubalek
,
R.A.
Milligan
,
U.
Müller
, and
B.
Kachar
.
2007
.
Cadherin 23 and protocadherin 15 interact to form tip-link filaments in sensory hair cells
.
Nature.
449
:
87
91
.
Khubchandani
,
S.R.
,
P.
Vohra
,
A.R.
Chitale
, and
P.
Sidana
.
2011
.
Microvillous inclusion disease—an ultrastructural diagnosis: with a review of the literature
.
Ultrastruct. Pathol.
35
:
87
91
.
Kitajiri
,
S.
,
K.
Fukumoto
,
M.
Hata
,
H.
Sasaki
,
T.
Katsuno
,
T.
Nakagawa
,
J.
Ito
,
S.
Tsukita
, and
S.
Tsukita
.
2004
.
Radixin deficiency causes deafness associated with progressive degeneration of cochlear stereocilia
.
J. Cell Biol.
166
:
559
570
.
Kleessen
,
B.
,
A.J.
Kroesen
,
H.J.
Buhr
, and
M.
Blaut
.
2002
.
Mucosal and invading bacteria in patients with inflammatory bowel disease compared with controls
.
Scand. J. Gastroenterol.
37
:
1034
1041
.
Knowles
,
B.C.
,
J.T.
Roland
,
M.
Krishnan
,
M.J.
Tyska
,
L.A.
Lapierre
,
P.S.
Dickman
,
J.R.
Goldenring
, and
M.D.
Shub
.
2014
.
Myosin Vb uncoupling from RAB8A and RAB11A elicits microvillus inclusion disease
.
J. Clin. Invest.
124
:
2947
2962
.
Knutton
,
S.
,
D.R.
Lloyd
, and
A.S.
McNeish
.
1987
.
Adhesion of enteropathogenic Escherichia coli to human intestinal enterocytes and cultured human intestinal mucosa
.
Infect. Immun.
55
:
69
77
.
Koyama
,
I.
,
T.
Matsunaga
,
T.
Harada
,
S.
Hokari
, and
T.
Komoda
.
2002
.
Alkaline phosphatases reduce toxicity of lipopolysaccharides in vivo and in vitro through dephosphorylation
.
Clin. Biochem.
35
:
455
461
.
Lagziel
,
A.
,
Z.M.
Ahmed
,
J.M.
Schultz
,
R.J.
Morell
,
I.A.
Belyantseva
, and
T.B.
Friedman
.
2005
.
Spatiotemporal pattern and isoforms of cadherin 23 in wild type and waltzer mice during inner ear hair cell development
.
Dev. Biol.
280
:
295
306
.
Liu
,
Y.
,
N.V.
Belkina
,
C.
Park
,
R.
Nambiar
,
S.M.
Loughhead
,
G.
Patino-Lopez
,
K.
Ben-Aissa
,
J.J.
Hao
,
M.J.
Kruhlak
,
H.
Qi
, et al
.
2012
.
Constitutively active ezrin increases membrane tension, slows migration, and impedes endothelial transmigration of lymphocytes in vivo in mice
.
Blood.
119
:
445
453
.
Loomis
,
P.A.
,
L.
Zheng
,
G.
Sekerková
,
B.
Changyaleket
,
E.
Mugnaini
, and
J.R.
Bartles
.
2003
.
Espin cross-links cause the elongation of microvillus-type parallel actin bundles in vivo
.
J. Cell Biol.
163
:
1045
1055
.
Mariadason
,
J.M.
,
C.
Nicholas
,
K.E.
L’Italien
,
M.
Zhuang
,
H.J.
Smartt
,
B.G.
Heerdt
,
W.
Yang
,
G.A.
Corner
,
A.J.
Wilson
,
L.
Klampfer
, et al
.
2005
.
Gene expression profiling of intestinal epithelial cell maturation along the crypt-villus axis
.
Gastroenterology.
128
:
1081
1088
.
Maroux
,
S.
,
E.
Coudrier
,
H.
Feracci
,
J.P.
Gorvel
, and
D.
Louvard
.
1988
.
Molecular organization of the intestinal brush border
.
Biochimie.
70
:
1297
1306
.
Mazerik
,
J.N.
, and
M.J.
Tyska
.
2012
.
Myosin-1A targets to microvilli using multiple membrane binding motifs in the tail homology 1 (TH1) domain
.
J. Biol. Chem.
287
:
13104
13115
.
Mazerik
,
J.N.
,
L.J.
Kraft
,
A.K.
Kenworthy
, and
M.J.
Tyska
.
2014
.
Motor and tail homology 1 (Th1) domains antagonistically control myosin-1 dynamics
.
Biophys. J.
106
:
649
658
.
Mazzolini
,
R.
,
H.
Dopeso
,
S.
Mateo-Lozano
,
W.
Chang
,
P.
Rodrigues
,
S.
Bazzocco
,
H.
Alazzouzi
,
S.
Landolfi
,
J.
Hernández-Losa
,
E.
Andretta
, et al
.
2012
.
Brush border myosin Ia has tumor suppressor activity in the intestine
.
Proc. Natl. Acad. Sci. USA.
109
:
1530
1535
.
McConnell
,
R.E.
, and
M.J.
Tyska
.
2007
.
Myosin-1a powers the sliding of apical membrane along microvillar actin bundles
.
J. Cell Biol.
177
:
671
681
.
McConnell
,
R.E.
,
A.E.
Benesh
,
S.
Mao
,
D.L.
Tabb
, and
M.J.
Tyska
.
2011
.
Proteomic analysis of the enterocyte brush border
.
Am. J. Physiol. Gastrointest. Liver Physiol.
300
:
G914
G926
.
Michel
,
V.
,
R.J.
Goodyear
,
D.
Weil
,
W.
Marcotti
,
I.
Perfettini
,
U.
Wolfrum
,
C.J.
Kros
,
G.P.
Richardson
, and
C.
Petit
.
2005
.
Cadherin 23 is a component of the transient lateral links in the developing hair bundles of cochlear sensory cells
.
Dev. Biol.
280
:
281
294
.
Miyata
,
H.
,
S.
Nishiyama
,
K.
Akashi
, and
K.
Kinosita
Jr
.
1999
.
Protrusive growth from giant liposomes driven by actin polymerization
.
Proc. Natl. Acad. Sci. USA.
96
:
2048
2053
.
Mogilner
,
A.
, and
B.
Rubinstein
.
2005
.
The physics of filopodial protrusion
.
Biophys. J.
89
:
782
795
.
Mooseker
,
M.S.
, and
L.G.
Tilney
.
1975
.
Organization of an actin filament-membrane complex. Filament polarity and membrane attachment in the microvilli of intestinal epithelial cells
.
J. Cell Biol.
67
:
725
743
.
Mooseker
,
M.S.
,
T.A.
Graves
,
K.A.
Wharton
,
N.
Falco
, and
C.L.
Howe
.
1980
.
Regulation of microvillus structure: calcium-dependent solation and cross-linking of actin filaments in the microvilli of intestinal epithelial cells
.
J. Cell Biol.
87
:
809
822
.
Mooseker
,
M.S.
,
T.D.
Pollard
, and
K.A.
Wharton
.
1982
.
Nucleated polymerization of actin from the membrane-associated ends of microvillar filaments in the intestinal brush border
.
J. Cell Biol.
95
:
223
233
.
Müller
,
U.
2008
.
Cadherins and mechanotransduction by hair cells
.
Curr. Opin. Cell Biol.
20
:
557
566
.
Müller
,
T.
,
M.W.
Hess
,
N.
Schiefermeier
,
K.
Pfaller
,
H.L.
Ebner
,
P.
Heinz-Erian
,
H.
Ponstingl
,
J.
Partsch
,
B.
Röllinghoff
,
H.
Köhler
, et al
.
2008
.
MYO5B mutations cause microvillus inclusion disease and disrupt epithelial cell polarity
.
Nat. Genet.
40
:
1163
1165
.
Nambiar
,
R.
,
R.E.
McConnell
, and
M.J.
Tyska
.
2009
.
Control of cell membrane tension by myosin-I
.
Proc. Natl. Acad. Sci. USA.
106
:
11972
11977
.
Nambiar
,
R.
,
R.E.
McConnell
, and
M.J.
Tyska
.
2010
.
Myosin motor function: the ins and outs of actin-based membrane protrusions
.
Cell. Mol. Life Sci.
67
:
1239
1254
.
Niggli
,
V.
,
C.
Andréoli
,
C.
Roy
, and
P.
Mangeat
.
1995
.
Identification of a phosphatidylinositol-4,5-bisphosphate-binding domain in the N-terminal region of ezrin
.
FEBS Lett.
376
:
172
176
.
Nougayrède
,
J.P.
,
P.J.
Fernandes
, and
M.S.
Donnenberg
.
2003
.
Adhesion of enteropathogenic Escherichia coli to host cells
.
Cell. Microbiol.
5
:
359
372
.
Novak
,
K.D.
,
M.D.
Peterson
,
M.C.
Reedy
, and
M.A.
Titus
.
1995
.
Dictyostelium myosin I double mutants exhibit conditional defects in pinocytosis
.
J. Cell Biol.
131
:
1205
1221
.
Ohta
,
K.
,
R.
Higashi
,
A.
Sawaguchi
, and
K.
Nakamura
.
2012
.
Helical arrangement of filaments in microvillar actin bundles
.
J. Struct. Biol.
177
:
513
519
.
Okazaki
,
N.
,
N.
Takahashi
,
S.
Kojima
,
Y.
Masuho
, and
H.
Koga
.
2002
.
Protocadherin LKC, a new candidate for a tumor suppressor of colon and liver cancers, its association with contact inhibition of cell proliferation
.
Carcinogenesis.
23
:
1139
1148
.
Parekh
,
S.H.
,
O.
Chaudhuri
,
J.A.
Theriot
, and
D.A.
Fletcher
.
2005
.
Loading history determines the velocity of actin-network growth
.
Nat. Cell Biol.
7
:
1219
1223
.
Peterson
,
M.D.
, and
M.S.
Mooseker
.
1992
.
Characterization of the enterocyte-like brush border cytoskeleton of the C2BBe clones of the human intestinal cell line, Caco-2
.
J. Cell Sci.
102
:
581
600
.
Peterson
,
M.D.
, and
M.S.
Mooseker
.
1993
.
An in vitro model for the analysis of intestinal brush border assembly. I. Ultrastructural analysis of cell contact-induced brush border assembly in Caco-2BBe cells
.
J. Cell Sci.
105
:
445
460
.
Pollard
,
T.D.
, and
M.S.
Mooseker
.
1981
.
Direct measurement of actin polymerization rate constants by electron microscopy of actin filaments nucleated by isolated microvillus cores
.
J. Cell Biol.
88
:
654
659
.
Reczek
,
D.
,
M.
Berryman
, and
A.
Bretscher
.
1997
.
Identification of EBP50: A PDZ-containing phosphoprotein that associates with members of the ezrin-radixin-moesin family
.
J. Cell Biol.
139
:
169
179
.
Revenu
,
C.
,
F.
Ubelmann
,
I.
Hurbain
,
F.
El-Marjou
,
F.
Dingli
,
D.
Loew
,
D.
Delacour
,
J.
Gilet
,
E.
Brot-Laroche
,
F.
Rivero
, et al
.
2012
.
A new role for the architecture of microvillar actin bundles in apical retention of membrane proteins
.
Mol. Biol. Cell.
23
:
324
336
.
Riazuddin
,
S.
,
I.A.
Belyantseva
,
A.P.
Giese
,
K.
Lee
,
A.A.
Indzhykulian
,
S.P.
Nandamuri
,
R.
Yousaf
,
G.P.
Sinha
,
S.
Lee
,
D.
Terrell
, et al
.
2012
.
Alterations of the CIB2 calcium- and integrin-binding protein cause Usher syndrome type 1J and nonsyndromic deafness DFNB48
.
Nat. Genet.
44
:
1265
1271
.
Rothbaum
,
R.J.
,
J.C.
Partin
,
K.
Saalfield
, and
A.J.
McAdams
.
1983
.
An ultrastructural study of enteropathogenic Escherichia coli infection in human infants
.
Ultrastruct. Pathol.
4
:
291
304
.
Rzadzinska
,
A.K.
,
M.E.
Schneider
,
C.
Davies
,
G.P.
Riordan
, and
B.
Kachar
.
2004
.
An actin molecular treadmill and myosins maintain stereocilia functional architecture and self-renewal
.
J. Cell Biol.
164
:
887
897
.
Rzadzinska
,
A.K.
,
A.
Derr
,
B.
Kachar
, and
K.
Noben-Trauth
.
2005
.
Sustained cadherin 23 expression in young and adult cochlea of normal and hearing-impaired mice
.
Hear. Res.
208
:
114
121
.
Salles
,
F.T.
,
L.R.
Andrade
,
S.
Tanda
,
M.
Grati
,
K.L.
Plona
,
L.H.
Gagnon
,
K.R.
Johnson
,
B.
Kachar
, and
M.A.
Berryman
.
2014
.
CLIC5 stabilizes membrane-actin filament linkages at the base of hair cell stereocilia in a molecular complex with radixin, taperin, and myosin VI
.
Cytoskeleton (Hoboken).
71
:
61
78
.
Saotome
,
I.
,
M.
Curto
, and
A.I.
McClatchey
.
2004
.
Ezrin is essential for epithelial organization and villus morphogenesis in the developing intestine
.
Dev. Cell.
6
:
855
864
.
Sato
,
T.
, and
H.
Clevers
.
2013
.
Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications
.
Science.
340
:
1190
1194
.
Sato
,
T.
,
R.G.
Vries
,
H.J.
Snippert
,
M.
van de Wetering
,
N.
Barker
,
D.E.
Stange
,
J.H.
van Es
,
A.
Abo
,
P.
Kujala
,
P.J.
Peters
, and
H.
Clevers
.
2009
.
Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche
.
Nature.
459
:
262
265
.
Self
,
T.
,
T.
Sobe
,
N.G.
Copeland
,
N.A.
Jenkins
,
K.B.
Avraham
, and
K.P.
Steel
.
1999
.
Role of myosin VI in the differentiation of cochlear hair cells
.
Dev. Biol.
214
:
331
341
.
Sheetz
,
M.P.
2001
.
Cell control by membrane-cytoskeleton adhesion
.
Nat. Rev. Mol. Cell Biol.
2
:
392
396
.
Shifrin
,
D.A.
Jr
,
R.E.
McConnell
,
R.
Nambiar
,
J.N.
Higginbotham
,
R.J.
Coffey
, and
M.J.
Tyska
.
2012
.
Enterocyte microvillus-derived vesicles detoxify bacterial products and regulate epithelial-microbial interactions
.
Curr. Biol.
22
:
627
631
.
Siemens
,
J.
,
C.
Lillo
,
R.A.
Dumont
,
A.
Reynolds
,
D.S.
Williams
,
P.G.
Gillespie
, and
U.
Müller
.
2004
.
Cadherin 23 is a component of the tip link in hair-cell stereocilia
.
Nature.
428
:
950
955
.
Skowron
,
J.F.
, and
M.S.
Mooseker
.
1999
.
Cloning and characterization of mouse brush border myosin-I in adult and embryonic intestine
.
J. Exp. Zool.
283
:
242
257
.
Skowron
,
J.F.
,
W.M.
Bement
, and
M.S.
Mooseker
.
1998
.
Human brush border myosin-I and myosin-Ic expression in human intestine and Caco-2BBe cells
.
Cell Motil. Cytoskeleton.
41
:
308
324
.
Svitkina
,
T.M.
,
E.A.
Bulanova
,
O.Y.
Chaga
,
D.M.
Vignjevic
,
S.
Kojima
,
J.M.
Vasiliev
, and
G.G.
Borisy
.
2003
.
Mechanism of filopodia initiation by reorganization of a dendritic network
.
J. Cell Biol.
160
:
409
421
.
Swidsinski
,
A.
,
A.
Ladhoff
,
A.
Pernthaler
,
S.
Swidsinski
,
V.
Loening-Baucke
,
M.
Ortner
,
J.
Weber
,
U.
Hoffmann
,
S.
Schreiber
,
M.
Dietel
, and
H.
Lochs
.
2002
.
Mucosal flora in inflammatory bowel disease
.
Gastroenterology.
122
:
44
54
.
ten Klooster
,
J.P.
,
M.
Jansen
,
J.
Yuan
,
V.
Oorschot
,
H.
Begthel
,
V.
Di Giacomo
,
F.
Colland
,
J.
de Koning
,
M.M.
Maurice
,
P.
Hornbeck
, and
H.
Clevers
.
2009
.
Mst4 and Ezrin induce brush borders downstream of the Lkb1/Strad/Mo25 polarization complex
.
Dev. Cell.
16
:
551
562
.
Theriot
,
J.A.
2000
.
The polymerization motor
.
Traffic.
1
:
19
28
.
Tilney
,
L.G.
, and
R.R.
Cardell
.
1970
.
Factors controlling the reassembly of the microvillous border of the small intestine of the salamander
.
J. Cell Biol.
47
:
408
422
.
Tocchetti
,
A.
,
C.B.
Soppo
,
F.
Zani
,
F.
Bianchi
,
M.C.
Gagliani
,
B.
Pozzi
,
J.
Rozman
,
R.
Elvert
,
N.
Ehrhardt
,
B.
Rathkolb
, et al
.
2010
.
Loss of the actin remodeler Eps8 causes intestinal defects and improved metabolic status in mice
.
PLoS ONE.
5
:
e9468
.
Turunen
,
O.
,
T.
Wahlström
, and
A.
Vaheri
.
1994
.
Ezrin has a COOH-terminal actin-binding site that is conserved in the ezrin protein family
.
J. Cell Biol.
126
:
1445
1453
.
Tyska
,
M.J.
,
A.T.
Mackey
,
J.D.
Huang
,
N.G.
Copeland
,
N.A.
Jenkins
, and
M.S.
Mooseker
.
2005
.
Myosin-1a is critical for normal brush border structure and composition
.
Mol. Biol. Cell.
16
:
2443
2457
.
Vallance
,
B.A.
,
C.
Chan
,
M.L.
Robertson
, and
B.B.
Finlay
.
2002
.
Enteropathogenic and enterohemorrhagic Escherichia coli infections: emerging themes in pathogenesis and prevention
.
Can. J. Gastroenterol.
16
:
771
778
.
van der Flier
,
L.G.
, and
H.
Clevers
.
2009
.
Stem cells, self-renewal, and differentiation in the intestinal epithelium
.
Annu. Rev. Physiol.
71
:
241
260
.
Verpy
,
E.
,
M.
Leibovici
,
I.
Zwaenepoel
,
X.Z.
Liu
,
A.
Gal
,
N.
Salem
,
A.
Mansour
,
S.
Blanchard
,
I.
Kobayashi
,
B.J.
Keats
, et al
.
2000
.
A defect in harmonin, a PDZ domain-containing protein expressed in the inner ear sensory hair cells, underlies Usher syndrome type 1C
.
Nat. Genet.
26
:
51
55
.
Viswanatha
,
R.
,
P.Y.
Ohouo
,
M.B.
Smolka
, and
A.
Bretscher
.
2012
.
Local phosphocycling mediated by LOK/SLK restricts ezrin function to the apical aspect of epithelial cells
.
J. Cell Biol.
199
:
969
984
.
Wayt
,
J.
, and
A.
Bretscher
.
2014
.
Cordon Bleu serves as a platform at the basal region of microvilli, where it regulates microvillar length through its WH2 domains
.
Mol. Biol. Cell.
25
:
2817
2827
.
Weil
,
D.
,
S.
Blanchard
,
J.
Kaplan
,
P.
Guilford
,
F.
Gibson
,
J.
Walsh
,
P.
Mburu
,
A.
Varela
,
J.
Levilliers
,
M.D.
Weston
, et al
.
1995
.
Defective myosin VIIA gene responsible for Usher syndrome type 1B
.
Nature.
374
:
60
61
.
Weil
,
D.
,
A.
El-Amraoui
,
S.
Masmoudi
,
M.
Mustapha
,
Y.
Kikkawa
,
S.
Lainé
,
S.
Delmaghani
,
A.
Adato
,
S.
Nadifi
,
Z.B.
Zina
, et al
.
2003
.
Usher syndrome type I G (USH1G) is caused by mutations in the gene encoding SANS, a protein that associates with the USH1C protein, harmonin
.
Hum. Mol. Genet.
12
:
463
471
.
Wells
,
A.L.
,
A.W.
Lin
,
L.Q.
Chen
,
D.
Safer
,
S.M.
Cain
,
T.
Hasson
,
B.O.
Carragher
,
R.A.
Milligan
, and
H.L.
Sweeney
.
1999
.
Myosin VI is an actin-based motor that moves backwards
.
Nature.
401
:
505
508
.
Wiegerinck
,
C.L.
,
A.R.
Janecke
,
K.
Schneeberger
,
G.F.
Vogel
,
D.Y.
van Haaften-Visser
,
J.C.
Escher
,
R.
Adam
,
C.E.
Thöni
,
K.
Pfaller
,
A.J.
Jordan
, et al
.
2014
.
Loss of syntaxin 3 causes variant microvillus inclusion disease
.
Gastroenterology.
147
:
65
: e10
.
Wilson
,
W.
,
R.B.
Scott
,
A.
Pinto
, and
M.A.
Robertson
.
2001
.
Intractable diarrhea in a newborn infant: microvillous inclusion disease
.
Can. J. Gastroenterol.
15
:
61
64
.
Wolenski
,
J.S.
,
R.E.
Cheney
,
P.
Forscher
, and
M.S.
Mooseker
.
1993a
.
In vitro motilities of the unconventional myosins, brush border myosin-I, and chick brain myosin-V exhibit assay-dependent differences in velocity
.
J. Exp. Zool.
267
:
33
39
.
Wolenski
,
J.S.
,
S.M.
Hayden
,
P.
Forscher
, and
M.S.
Mooseker
.
1993b
.
Calcium-calmodulin and regulation of brush border myosin-I MgATPase and mechanochemistry
.
J. Cell Biol.
122
:
613
621
.
Wu
,
L.L.
,
W.H.
Peng
,
W.T.
Kuo
,
C.Y.
Huang
,
Y.H.
Ni
,
K.S.
Lu
,
J.R.
Turner
, and
L.C.
Yu
.
2014
.
Commensal bacterial endocytosis in epithelial cells is dependent on myosin light chain kinase-activated brush border fanning by interferon-γ
.
Am. J. Pathol.
184
:
2260
2274
.
Yun
,
C.H.
,
G.
Lamprecht
,
D.V.
Forster
, and
A.
Sidor
.
1998
.
NHE3 kinase A regulatory protein E3KARP binds the epithelial brush border Na+/H+ exchanger NHE3 and the cytoskeletal protein ezrin
.
J. Biol. Chem.
273
:
25856
25863
.
Zihni
,
C.
,
P.M.
Munro
,
A.
Elbediwy
,
N.H.
Keep
,
S.J.
Terry
,
J.
Harris
,
M.S.
Balda
, and
K.
Matter
.
2014
.
Dbl3 drives Cdc42 signaling at the apical margin to regulate junction position and apical differentiation
.
J. Cell Biol.
204
:
111
127
.

    Abbreviations used in this paper:
     
  • EPEC

    enteropathogenic Escherichia coli

  •  
  • KO

    knockout

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