Select autosomal dominant DFNA11 deafness mutations activate Myo7A in epithelial cells.

Myosin-7A (Myo7A) is a motor protein crucial for the organization and function of stereocilia, specialized actin-rich protrusions on the surface of inner ear hair cells that mediate hearing. Mutations in Myo7A cause several forms of genetic hearing loss, including autosomal dominant DFNA11 deafness. Despite its importance, the structural elements of Myo7A that control its motor activity within cells are not well understood. In this study, we used cultured kidney epithelial cells to screen for mutations that activate the motor-dependent targeting of Myo7A to the tips of apical microvilli on these cells. Our findings reveal that Myo7A is regulated by specific IQ motifs within its lever arm, and that this regulation can function at least partially independent of its tail sequence. Importantly, we demonstrate that many of the DFNA11 deafness mutations reported in patients activate Myo7A targeting, providing a potential explanation for the autosomal dominant genetics of this form of deafness.

The microvillar protocadherin CDHR5 associates with EBP50 to promote brush border assembly.

Transporting epithelial cells of the gut and kidney interact with their luminal environment through a densely packed collection of apical microvilli known as a brush border (BB). Proper brush border assembly depends on the intermicrovillar adhesion complex (IMAC), a protocadherin-based adhesion complex found at the distal tips of microvilli that mediates adhesion between neighboring protrusions to promote their organized packing. Loss of the IMAC adhesion molecule Cadherin-related family member 5 (CDHR5) results in significant brush border defects, though the functional properties of this protocadherin have not been thoroughly explored. Here, we show that the cytoplasmic tail of CDHR5 contributes to its correct apical targeting and functional properties in an isoform-specific manner. Library screening identified the Ezrin-associated scaffolds EBP50 and E3KARP as cytoplasmic binding partners for CDHR5. Consistent with this, loss of EBP50 disrupted proper brush border assembly with cells exhibiting markedly reduced apical IMAC levels. Together, our results shed light on the apical targeting determinants of CDHR5 and further define the interactome of the IMAC involved in brush border assembly.

Comparative analysis of the MyTH4-FERM myosins reveals insights into the determinants of actin track selection in polarized epithelia.

MyTH4-FERM (MF) myosins evolved to play a role in the creation and function of a variety of actin-based membrane protrusions that extend from cells. Here we performed an analysis of the MF myosins, Myo7A, Myo7B, and Myo10, to gain insight into how they select for their preferred actin networks. Using enterocytes that create spatially separated actin tracks in the form of apical microvilli and basal filopodia, we show that actin track selection is principally guided by the mode of oligomerization of the myosin along with the identity of the motor domain, with little influence from the specific composition of the lever arm. Chimeric variants of Myo7A and Myo7B fused to a leucine zipper parallel dimerization sequence in place of their native tails both selected apical microvilli as their tracks, while a truncated Myo10 used its native antiparallel coiled-coil to traffic to the tips of filopodia. Swapping lever arms between the Class 7 and 10 myosins did not change actin track preference. Surprisingly, fusing the motor-neck region of Myo10 to a leucine zipper or oligomerization sequences derived from the Myo7A and Myo7B cargo proteins USH1G and ANKS4B, respectively, re-encoded the actin track usage of Myo10 to apical microvilli with significant efficiency.

A heterologous in-cell assay for investigating intermicrovillar adhesion complex interactions reveals a novel protrusion length-matching mechanism.

Solute transporting epithelial cells build arrays of microvilli on their apical surface to increase membrane scaffolding capacity and enhance function potential. In epithelial tissues such as the kidney and gut, microvilli are length-matched and assembled into tightly packed "brush borders," which are organized by ∼50-nm thread-like links that form between the distal tips of adjacent protrusions. Composed of protocadherins CDHR2 and CDHR5, adhesion links are stabilized at the tips by a cytoplasmic tripartite module containing the scaffolds USH1C and ANKS4B and the actin-based motor MYO7B. Because several questions about the formation and function of this "intermicrovillar adhesion complex" remain open, we devised a system that allows one to study individual binary interactions between specific complex components and MYO7B. Our approach employs a chimeric myosin consisting of the MYO10 motor domain fused to the MYO7B cargo-binding tail domain. When expressed in HeLa cells, which do not normally produce adhesion complex proteins, this chimera trafficked to the tips of filopodia and was also able to transport individual complex components to these sites. Unexpectedly, the MYO10-MYO7B chimera was able to deliver CDHR2 and CDHR5 to distal tips in the absence of USH1C or ANKS4B. Cells engineered to localize high levels of CDHR2 at filopodial tips acquired interfilopodial adhesion and exhibited a striking dynamic length-matching activity that aligned distal tips over time. These findings deepen our understanding of mechanisms that promote the distal tip accumulation of intermicrovillar adhesion complex components and also offer insight on how epithelial cells minimize microvillar length variability.

A cryptic sequence targets the adhesion complex scaffold ANKS4B to apical microvilli to promote enterocyte brush border assembly.

Nutrient-transporting enterocytes interact with their luminal environment using a densely packed collection of apical microvilli known as the brush border. Assembly of the brush border is controlled by the intermicrovillar adhesion complex (IMAC), a protocadherin-based complex found at the tips of brush border microvilli that mediates adhesion between neighboring protrusions. ANKS4B is known to be an essential scaffold within the IMAC, although its functional properties have not been thoroughly characterized. We report here that ANKS4B is directed to the brush border using a noncanonical apical targeting sequence that maps to a previously unannotated region of the scaffold. When expressed on its own, this sequence targeted to microvilli in the absence of any direct interaction with the other IMAC components. Sequence analysis revealed a coiled-coil motif and a putative membrane-binding basic-hydrophobic repeat sequence within this targeting region, both of which were required for the scaffold to target and mediate brush border assembly. Size-exclusion chromatography of the isolated targeting sequence coupled with in vitro brush border binding assays suggests that it functions as an oligomer. We further show that the corresponding sequence found in the closest homolog of ANKS4B, the scaffold USH1G that operates in sensory epithelia as part of the Usher complex, lacks the inherent ability to target to microvilli. This study further defines the underlying mechanism of how ANKS4B targets to the apical domain of enterocytes to drive brush border assembly and identifies a point of functional divergence between the ankyrin repeat-based scaffolds found in the IMAC and Usher complex.

The small EF-hand protein CALML4 functions as a critical myosin light chain within the intermicrovillar adhesion complex.

Specialized transporting and sensory epithelial cells employ homologous protocadherin-based adhesion complexes to remodel their apical membrane protrusions into organized functional arrays. Within the intestine, the nutrient-transporting enterocytes utilize the intermicrovillar adhesion complex (IMAC) to assemble their apical microvilli into an ordered brush border. The IMAC bears remarkable homology to the Usher complex, whose disruption results in the sensory disorder type 1 Usher syndrome (USH1). However, the entire complement of proteins that comprise both the IMAC and Usher complex are not yet fully elucidated. Using a protein isolation strategy to recover the IMAC, we have identified the small EF-hand protein calmodulin-like protein 4 (CALML4) as an IMAC component. Consistent with this finding, we show that CALML4 exhibits marked enrichment at the distal tips of enterocyte microvilli, the site of IMAC function, and is a direct binding partner of the IMAC component myosin-7b. Moreover, distal tip enrichment of CALML4 is strictly dependent upon its association with myosin-7b, with CALML4 acting as a light chain for this myosin. We further show that genetic disruption of CALML4 within enterocytes results in brush border assembly defects that mirror the loss of other IMAC components and that CALML4 can also associate with the Usher complex component myosin-7a. Our study further defines the molecular composition and protein-protein interaction network of the IMAC and Usher complex and may also shed light on the etiology of the sensory disorder USH1H.

Muscle-specific stress fibers give rise to sarcomeres in cardiomyocytes.

The sarcomere is the contractile unit within cardiomyocytes driving heart muscle contraction. We sought to test the mechanisms regulating actin and myosin filament assembly during sarcomere formation. Therefore, we developed an assay using human cardiomyocytes to monitor sarcomere assembly. We report a population of muscle stress fibers, similar to actin arcs in non-muscle cells, which are essential sarcomere precursors. We show sarcomeric actin filaments arise directly from muscle stress fibers. This requires formins (e.g., FHOD3), non-muscle myosin IIA and non-muscle myosin IIB. Furthermore, we show short cardiac myosin II filaments grow to form ~1.5 µm long filaments that then 'stitch' together to form the stack of filaments at the core of the sarcomere (i.e., the A-band). A-band assembly is dependent on the proper organization of actin filaments and, as such, is also dependent on FHOD3 and myosin IIB. We use this experimental paradigm to present evidence for a unifying model of sarcomere assembly.

Shear stress induces noncanonical autophagy in intestinal epithelial monolayers.

Flow of fluids through the gut, such as milk from a neonatal diet, generates a shear stress on the unilaminar epithelium lining the lumen. We report that exposure to physiological levels of fluid shear stress leads to the formation of large vacuoles, containing extracellular contents within polarizing intestinal epithelial cell monolayers. These observations lead to two questions: how can cells lacking primary cilia transduce shear stress, and what molecular pathways support the formation of vacuoles that can exceed 80% of the cell volume? We find that shear forces are sensed by actin-rich microvilli that eventually generate the apical brush border, providing evidence that these structures possess mechanosensing ability. Importantly, we identified the molecular pathway that regulates large vacuole formation downstream from mechanostimulation to involve central components of the autophagy pathway, including ATG5 and LC3, but not Beclin. Together our results establish a novel link between the actin-rich microvilli, the macroscopic transport of fluids across cells, and the noncanonical autophagy pathway in organized epithelial monolayers.

Myosin-7b Promotes Distal Tip Localization of the Intermicrovillar Adhesion Complex.

Transporting epithelial cells interact with the luminal environment using a tightly packed array of microvilli known as the brush border. During intestinal epithelial differentiation, microvillar packing and organization are driven by cadherin-dependent adhesion complexes that localize to the distal tips of microvilli, where they drive physical interactions between neighboring protrusions. Although enrichment of the "intermicrovillar adhesion complex" (IMAC) at distal tips is required for proper function, the mechanism driving tip accumulation of these factors remains unclear. Here, we report that the actin-based motor myosin-7b (Myo7b) promotes the accumulation of IMAC components at microvillar tips. Myo7b is highly enriched at the tips of microvilli in both kidney and intestinal brush borders, and loss of Myo7b in differentiating intestinal epithelial cells disrupts intermicrovillar adhesion and, thus, brush border assembly. Analysis of cells lacking Myo7b revealed that IMAC components and the resulting intermicrovillar adhesion links are mislocalized along the microvillar axis rather than enriched at the distal tips. We also found that Myo7b motor domains are capable of supporting tip-directed transport. However, motor activity is supplemented by other passive targeting mechanisms that together drive highly efficient IMAC accumulation at the tips. These findings illuminate the molecular basis of IMAC enrichment at microvillar tips and hold important implications for understanding apical morphogenesis in transporting and sensory epithelial tissues.

Impact of cordon-bleu expression on actin cytoskeleton architecture and dynamics.

Cordon-bleu (COBL) is a multifunctional WASP-Homology 2 (WH2) domain-containing protein implicated in a wide variety of cellular functions ranging from dendritic arborization in neurons to the assembly of microvilli on the surface of transporting epithelial cells. In vitro biochemical studies suggest that COBL is capable of nucleating and severing actin filaments, among other activities. How the multiple activities of COBL observed in vitro contribute to its function in cells remains unclear. Here, we used live imaging to evaluate the impact of COBL expression on the actin cytoskeleton in cultured cells. We found that COBL induces the formation of dynamic linear actin structures throughout the cytosol. We also found that stabilizing these dynamic structures with the parallel actin-bundling protein espin slows down their turnover and enables the robust formation of self-supported protrusions on the dorsal cell surface. Super-resolution imaging revealed a global remodeling of the actin cytoskeleton in cells expressing these two factors. Taken together, these results provide insight as to how COBL contributes to the assembly of actin-based structures such as epithelial microvilli. © 2016 Wiley Periodicals, Inc.

Structure of the Single-lobe Myosin Light Chain C in Complex with the Light Chain-binding Domains of Myosin-1C Provides Insights into Divergent IQ Motif Recognition.

Myosin light chains are key regulators of class 1 myosins and typically comprise two domains, with calmodulin being the archetypal example. They bind IQ motifs within the myosin neck region and amplify conformational changes in the motor domain. A single lobe light chain, myosin light chain C (MlcC), was recently identified and shown to specifically bind to two sequentially divergent IQ motifs of the Dictyostelium myosin-1C. To provide a molecular basis of this interaction, the structures of apo-MlcC and a 2:1 MlcC·myosin-1C neck complex were determined. The two non-functional EF-hand motifs of MlcC pack together to form a globular four-helix bundle that opens up to expose a central hydrophobic groove, which interacts with the N-terminal portion of the divergent IQ1 and IQ2 motifs. The N- and C-terminal regions of MlcC make critical contacts that contribute to its specific interactions with the myosin-1C divergent IQ motifs, which are contacts that deviate from the traditional mode of calmodulin-IQ recognition.

ANKS4B Is Essential for Intermicrovillar Adhesion Complex Formation.

Transporting and sensory epithelial cells shape apical specializations using protocadherin-based adhesion. In the enterocyte brush border, protocadherin function requires a complex of cytoplasmic binding partners, although the composition of this complex and logic governing its assembly remain poorly understood. We found that ankyrin repeat and sterile α motif domain containing 4B (ANKS4B) localizes to the tips of adherent brush border microvilli and is essential for intermicrovillar adhesion. ANKS4B interacts with USH1C and MYO7B, which link protocadherins to the actin cytoskeleton. ANKS4B and USH1C also bind to the MYO7B cargo-binding tail at distinct sites. However, a tripartite complex only forms if ANKS4B and MYO7B are first activated by USH1C. This study uncovers an essential role for ANKS4B in brush border assembly, reveals a hierarchy in the molecular interactions that drive intermicrovillar adhesion, and informs our understanding of diseases caused by mutations in USH1C and ankyrin repeat proteins, such as Usher syndrome.

Cordon bleu promotes the assembly of brush border microvilli.

Microvilli are actin-based protrusions found on the surface of diverse cell types, where they amplify membrane area and mediate interactions with the external environment. In the intestinal tract, these protrusions play central roles in nutrient absorption and host defense and are therefore essential for maintaining homeostasis. However, the mechanisms controlling microvillar assembly remain poorly understood. Here we report that the multifunctional actin regulator cordon bleu (COBL) promotes the growth of brush border (BB) microvilli. COBL localizes to the base of BB microvilli via a mechanism that requires its proline-rich N-terminus. Knockdown and overexpression studies show that COBL is needed for BB assembly and sufficient to induce microvillar growth using a mechanism that requires functional WH2 domains. We also find that COBL acts downstream of the F-BAR protein syndapin-2, which drives COBL targeting to the apical domain. These results provide insight into a mechanism that regulates microvillar growth during epithelial differentiation and have significant implications for understanding the maintenance of intestinal homeostasis.

Shaping the intestinal brush border.

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.

Dynamics of brush border remodeling induced by enteropathogenic E. coli.

Enteropathogenic Escherichia coli (EPEC) induces dramatic remodeling of enterocyte brush borders, a process that includes microvillar effacement and actin pedestal formation. Although the Arp2/3 complex is involved in formation of a branched actin network within pedestals, the fate of parallel actin bundles in microvilli during infection remains unclear. Here, we find that in polarized intestinal epithelial cells, EPEC stimulates long-range microvillar dynamics, pulling protrusions toward sites of bacterial attachment in a process mediated by the adhesion molecule protocadherin-24. Additionally, retraction of the EPEC bundle forming pilus stimulates directed elongation of nearby microvilli. These processes lead to coalescence of microvilli and incorporation of the underlying parallel actin bundles into pedestals. Furthermore, stabilization of microvillar actin bundles delays pedestal formation. Together, these results suggest a model where EPEC takes advantage of pre-existing actin filaments in microvillar core bundles to facilitate pedestal formation.

Structure of the small Dictyostelium discoideum myosin light chain MlcB provides insights into MyoB IQ motif recognition.

Dictyostelium discoideum MyoB is a class I myosin involved in the formation and retraction of membrane projections, cortical tension generation, membrane recycling, and phagosome maturation. The MyoB-specific, single-lobe EF-hand light chain MlcB binds the sole IQ motif of MyoB with submicromolar affinity in the absence and presence of Ca(2+). However, the structural features of this novel myosin light chain and its interaction with its cognate IQ motif remain uncharacterized. Here, we describe the NMR-derived solution structure of apoMlcB, which displays a globular four-helix bundle. Helix 1 adopts a unique orientation when compared with the apo states of the EF-hand calcium-binding proteins calmodulin, S100B, and calbindin D9k. NMR-based chemical shift perturbation mapping identified a hydrophobic MyoB IQ binding surface that involves amino acid residues in helices I and IV and the functional N-terminal Ca(2+) binding loop, a site that appears to be maintained when MlcB adopts the holo state. Complementary mutagenesis and binding studies indicated that residues Ile-701, Phe-705, and Trp-708 of the MyoB IQ motif are critical for recognition of MlcB, which together allowed the generation of a structural model of the apoMlcB-MyoB IQ complex. We conclude that the mode of IQ motif recognition by the novel single-lobe MlcB differs considerably from that of stereotypical bilobal light chains such as calmodulin.

Intestinal brush border assembly driven by protocadherin-based intermicrovillar adhesion.

Transporting epithelial cells build apical microvilli to increase membrane surface area and enhance absorptive capacity. The intestinal brush border provides an elaborate example with tightly packed microvilli that function in nutrient absorption and host defense. Although the brush border is essential for physiological homeostasis, its assembly is poorly understood. We found that brush border assembly is driven by the formation of Ca(2+)-dependent adhesion links between adjacent microvilli. Intermicrovillar links are composed of protocadherin-24 and mucin-like protocadherin, which target to microvillar tips and interact to form a trans-heterophilic complex. The cytoplasmic domains of microvillar protocadherins interact with the scaffolding protein, harmonin, and myosin-7b, which promote localization to microvillar tips. Finally, a mouse model of Usher syndrome lacking harmonin exhibits microvillar protocadherin mislocalization and severe defects in brush border morphology. These data reveal an adhesion-based mechanism for brush border assembly and illuminate the basis of intestinal pathology in patients with Usher syndrome. PAPERFLICK:

PakB binds to the SH3 domain of Dictyostelium Abp1 and regulates its effects on cell polarity and early development.

Dictyostelium p21-activated kinase B (PakB) phosphorylates and activates class I myosins. PakB colocalizes with myosin I to actin-rich regions of the cell, including macropinocytic and phagocytic cups and the leading edge of migrating cells. Here we show that residues 1-180 mediate the cellular localization of PakB. Yeast two-hybrid and pull-down experiments identify two proline-rich motifs in PakB-1-180 that directly interact with the SH3 domain of Dictyostelium actin-binding protein 1 (dAbp1). dAbp1 colocalizes with PakB to actin-rich regions in the cell. The loss of dAbp1 does not affect the cellular distribution of PakB, whereas the loss of PakB causes dAbp1 to adopt a diffuse cytosolic distribution. Cosedimentation studies show that the N-terminal region of PakB (residues 1-70) binds directly to actin filaments, whereas dAbp1 exhibits only a low affinity for filamentous actin. PakB-1-180 significantly enhances the binding of dAbp1 to actin filaments. When overexpressed in PakB-null cells, dAbp1 completely blocks early development at the aggregation stage, prevents cell polarization, and significantly reduces chemotaxis rates. The inhibitory effects are abrogated by the introduction of a function-blocking mutation into the dAbp1 SH3 domain. We conclude that PakB plays a critical role in regulating the cellular functions of dAbp1, which are mediated largely by its SH3 domain.

Identification of calmodulin and MlcC as light chains for Dictyostelium myosin-I isozymes.

Dictyostelium discoideum express seven single-headed myosin-I isozymes (MyoA-MyoE and MyoK) that drive motile processes at the cell membrane. The light chains for MyoA and MyoE were identified by expressing Flag-tagged constructs consisting of the motor domain and the two IQ motifs in the neck region in Dictyostelium. The MyoA and MyoE constructs both copurified with calmodulin. Isothermal titration calorimetry (ITC) showed that apo-calmodulin bound to peptides corresponding to the MyoA and MyoE IQ motifs with micromolar affinity. In the presence of calcium, calmodulin cross-linked two IQ motif peptides, with one domain binding with nanomolar affinity and the other with micromolar affinity. The IQ motifs were required for the actin-activated MgATPase activity of MyoA but not MyoE; however, neither myosin exhibited calcium-dependent activity. A Flag-tagged construct consisting of the MyoC motor domain and the three IQ motifs in the adjacent neck region bound a novel 8.6 kDa two EF-hand protein named MlcC, for myosin light chain for MyoC. MlcC is most similar to the C-terminal domain of calmodulin but does not bind calcium. ITC studies showed that MlcC binds IQ1 and IQ2 but not IQ3 of MyoC. IQ3 contains a proline residue that may render it nonfunctional. Each long-tailed Dictyostelium myosin-I has now been shown to have a unique light chain (MyoB-MlcB, MyoC-MlcC, and MyoD-MlcD), whereas the short-tailed myosins-I, MyoA and MyoE, have the multifunctional calmodulin as a light chain. The diversity in light chain composition is likely to contribute to the distinct cellular functions of each myosin-I isozyme.

Autophosphorylation activates Dictyostelium myosin II heavy chain kinase A by providing a ligand for an allosteric binding site in the alpha-kinase domain.

Dictyostelium discoideum myosin II heavy chain kinase A (MHCK A), a member of the atypical α-kinase family, phosphorylates sites in the myosin II tail that block filament assembly. Here we show that the catalytic activity of A-CAT, the α-kinase domain of MHCK A (residues 552-841), is severely inhibited by the removal of a disordered C-terminal tail sequence (C-tail; residues 806-841). The key residue in the C-tail was identified as Thr(825), which was found to be constitutively autophosphorylated. Dephosphorylation of Thr(825) using shrimp alkaline phosphatase decreased A-CAT activity. The activity of a truncated A-CAT lacking Thr(825) could be rescued by P(i), phosphothreonine, and a phosphorylated peptide, but not by threonine, glutamic acid, aspartic acid, or an unphosphorylated peptide. These results focused attention on a P(i)-binding pocket located in the C-terminal lobe of A-CAT. Mutational analysis demonstrated that the P(i)-pocket was essential for A-CAT activity. Based on these results, it is proposed that autophosphorylation of Thr(825) activates ACAT by providing a covalently tethered ligand for the P(i)-pocket. Ab initio modeling studies using the Rosetta FloppyTail and FlexPepDock protocols showed that it is feasible for the phosphorylated Thr(825) to dock intramolecularly into the P(i)-pocket. Allosteric activation is predicted to involve a conformational change in Arg(734), which bridges the bound P(i) to Asp(762) in a key active site loop. Sequence alignments indicate that a comparable regulatory mechanism is likely to be conserved in Dictyostelium MHCK B-D and metazoan eukaryotic elongation factor-2 kinases.