Actin-based protrusions can form prominent structures on the apical surface of epithelial cells, such as microvilli. Several cytoplasmic factors have been identified that control the dynamics of actin filaments in microvilli. However, it remains unclear whether the plasma membrane participates actively in microvillus formation. The function of Drosophila melanogaster cadherin Cad99C in the microvilli of ovarian follicle cells has been analyzed. Cad99C contributes to eggshell formation and female fertility and is expressed in follicle cells, which produce the eggshells. Cad99C specifically localizes to apical microvilli. Loss of Cad99C function results in shortened and disorganized microvilli, whereas overexpression of Cad99C leads to a dramatic increase of microvillus length and results in large bundles of microvilli. Altered microvilli morphology correlates with defects in the assembly of the vitelline membrane, an extracellular layer secreted by follicle cells that is part of the eggshell. Cad99C that lacks most of the cytoplasmic domain, including potential PDZ domain-binding sites, still promotes excessive microvillus outgrowth, suggesting that the amount of the extracellular domain determines microvillus length. Cad99C is thus a critical regulator of microvillus length, the first example of a transmembrane protein that is involved in this process (D'Alterio, 2005; Schlichting, 2006).

Microvilli are fingerlike protrusions on the apical surface of epithelial cells, where they can form a dense brush border. Microvilli are also used by several sensory cells as a basic module to form specialized structures that engage in the transduction of light and mechanosensory stimuli. Stereocilia of vertebrate inner ear hair cells are a prominent example. The core of a microvillus consists of a bundle of cross-linked parallel actin filaments, which have their barbed (+) ends inserted at the microvillus tip and their pointed (–) ends anchored in a terminal web of actin filaments. The actin filament bundle undergoes constant turnover through treadmilling. Growth of microfilaments at barbed ends is thought to push the membrane envelope forward, lengthening the protrusion (D'Alterio. 2005).

Among the molecules that serve a critical function in the formation and regulation of microvillus growth are several F-actin cross-linking proteins, including villin, epsin, fimbrin, and fascin. Epsin can induce microvillus elongation in vitro probably by affecting the actin treadmilling process, and epsin mutant deaf jerker mice have shortened hair cell stereocilia. Additional actin-binding factors that control microvillus size colocalize with the tip complex, which is thought to nucleate the microfilament bundle and to regulate actin polymerization at barbed ends. They comprise myosin XVa and its binding partner whirlin, which promote concentration-dependent elongation of hair cell stereocilia. EPS-8, another actin-binding protein located at a microvillus tip, regulates microvillus length through its barbed-end capping function in the intestine of Caenorhabditis elegans. In the early Drosophila melanogaster embryo, absence of the Abelson kinase causes abnormally long microvilli. This correlates with an ectopic accumulation of F-actin and its growth promoting factor Enabled in the apical cell cortex (D'Alterio, 2005 and references therein).

Observations like these suggested that microfilaments and their binding factors may be sufficient for microvillus formation and elongation and that the plasma membrane may serve only as an anchor for the core bundle. That coupling to the plasma membrane is important is suggested by the finding that ezrin, an ERM (ezrin-radixin-Moesin) protein that likely forms a link between actin filaments and the plasma membrane, induces microvilli in cell culture. Deficiency of ezrin, which appears to organize the terminal web of microvilli, causes shortened, irregular intestinal microvilli in mice. Similarly, the terminal web-associated ERM protein Moesin in Drosophila is required for normal organization of microvilli in rhabdomeres, and an excessive formation of irregular microvilli results from expressing constitutively active Moesin. However, there has been no evidence so far that would implicate specific integral membrane proteins as regulators of microvillus growth (D'Alterio, 2005 and references therein).

Some proteins that have received considerable attention in recent years as important organizers of hair cell stereocilia are protocadherin 15 (PCDH15), cadherin 23 (CDH23), myosin VIIa, harmonin, and SANS. Mutations in these genes are responsible for Usher syndrome type 1 (USH1), a genetic disorder that combines congenital deafness, vestibular dysfunction, and retinitis pigmentosa in humans. The phenotype of mice mutant for any USH1 gene is characterized by splayed and disorganized stereocilia and consequently it was proposed that the two USH1 cadherins may contribute to the links that visibly connect neighboring stereocilia. For CDH23, this model is supported by the observations that it colocalizes with lateral and tip links, is needed for tip link integrity, and can mediate homophilic adhesion. The molecular function of PCDH15, however, has not been elucidated. Cad99C, the fly orthologue of PCDH15, is a component of microvilli; loss- and gain-of-function analyses show that Cad99C promotes microvillus elongation in a concentration-dependent manner. The data also suggest that Cad99C acts through a mechanism that does not involve adhesion between microvilli (D'Alterio, 2005).

Loss of Cad99C results in shorter microvilli and overexpression in longer microvilli than in wild type, indicating that the concentration of Cad99C is positively correlated with the length of microvilli in follicle cells. Interestingly, modifications in the Cad99C mRNA expression level during oogenesis appear to be good indicators for changes in microvilli. During mid-oogenesis, prominent expression of Cad99C is seen in follicle cells that show forming and growing apical microvilli, and Cad99C expression culminates when microvilli reach their maximum extension. The following drop of mRNA levels in most follicle cells coincides with a regression of microvillus size, whereas centripetal cells express Cad99C strongly, consistent with the delayed formation of a microvillus brush border by these follicle cells. It is therefore proposed that transcriptionally regulated changes in the concentration of Cad99C are critically involved in the dynamic remodeling of follicle cell microvilli (D'Alterio, 2005).

The loss of Cad99C, which results in microvilli defects, also leads to defects in eggshell formation, suggesting that normal microvilli have an important function in eggshell development. Interestingly, the dynamic regulation of Cad99C expression correlates well temporally and spatially with described phases of eggshell secretion and with morphogenetic movements of follicle cells; this raises the question of how these processes are interrelated. Follicle cells undergo multiple morphogenetic movements to reach positions from which they secrete eggshell material, either while the movement is being completed or immediately afterwards. Therefore, the striking correlation between the Cad99C expression profile and morphogenetic movements likely reflects the close association between those movements and eggshell secretion (D'Alterio, 2005).

Cad99C specifically localizes to the plasma membrane of microvilli and is distributed throughout their entire length. PCDH15 shows a similar subcellular distribution in stereocilia on the surface of hair cells in the cochlea (Ahmed, 2003). In PCDH15 mutant mice (Ames waltzer) and zebrafish (orbiter), stereocilia are splayed and their arrangement is severely disturbed, causing deafness (Alagramam, 2001a; Raphael, 2001; Seiler, 2005). The function of PCDH15 in stereocilia, however, has remained unclear. This study indicates that its Drosophila orthologue Cad99C is a potent regulator of microvillus size. Interestingly, PCDH15 is found at a higher concentration in the longer stereocilia of a staircase-like bundle of hair cell stereocilia (Ahmed, 2003), and an irregular shortening of stereocilia in Ames waltzer mice has been described (Alagramam, 2001a; Raphael, 2001). This raises the possibility that PCDH15 regulates the length of stereocilia similar to Cad99C. In addition to being required for size regulation, Cad99C is also important for the normal shape and arrangement of microvilli. It will be interesting to determine in future studies whether the effect on size and on shape and arrangement of microvilli/stereocilia reflects two distinct functions of Cad99C/PCDH15 or whether they are two consequences of the same molecular function. Together, it is proposed that Cad99C/PCDH15-type cadherins have an evolutionarily conserved role in microvillus biogenesis (D'Alterio, 2005).

During the course of evolution, Cad99C/PCDH15 cadherins have adapted to act in apparently very different apical actin-based protrusions, such as the follicle cell microvilli of fly ovaries and the complex stereocilia of the vertebrate cochlea. Moreover, loss of Cad99C causes subtle defects in eye rhabdomeres and mechanosensory bristles, indicating that Cad99C is also required for other actin-based protrusions. Similar to PCDH15, which is more widely expressed in epithelial tissues (Murcia, 2001; Ahmed, 2003), Cad99C is found on the apical surface of several ectodermal epithelia during D. melanogaster development, including the imaginal discs (Schlichting, 2005). In wing imaginal discs as in the follicular epithelium, overexpression of Cad99C induces the formation of very large apical protrusions. However, there are epithelial tissues that possess a microvillus brush border but do not express Cad99C at detectable levels, including the midgut. Cad99C is therefore not a general component of microvilli and may serve a biological function that is specifically required in a subset of microvilli. Alternatively, another member of the cadherin superfamily or other membrane protein might take the place of Cad99C in the microvilli of tissues that lack this cadherin (D'Alterio, 2005).

It is speculated that, as a cadherin, Cad99C may mediate homophilic adhesion between microvilli. However, follicle cell microvilli in wild type and after overexpression of Cad99C are clearly separated from each other, implying that Cad99C mediates neither homophilic nor heterophilic interactions between microvilli while promoting their outgrowth. This conclusion is corroborated by the behavior of cell clones that either lack or overexpress Cad99C. In imaginal discs where Cad99C is concentrated at the apical interface between cells, cell clones with reduced or increased levels of Cad99C expression have wiggly boundaries, indicating that they do not sort out from wild-type cells (Schlichting, 2005). Furthermore, Cad99C located ectopically in the lateral membrane of follicle cells when overexpressed is not enriched at the border between Cad99C overexpressing cells compared with borders between wild-type and overexpressing cells as would be expected from a homophilic adhesion molecule. Similarly, in imaginal discs, the distribution of Cad99C along cell boundaries is uniform and independent of the concentration of Cad99C in neighboring cells. Hence, these findings argue against a function of Cad99C in adhesion between adjacent plasma membranes (D'Alterio, 2005).

A parallel bundle of actin filaments and associated factors that control their cross-linking and turnover are instrumental for the formation and stability of microvilli. To what extent the plasma membrane of microvilli contributes to microvillus morphogenesis either by linking to the actin core or independent of it remains largely unexplored. PCDH15 appears to influence the actin cytoskeleton of stereocilia as the amount of F-actin in stereocilia of PCDH15 mutant hair cells was reduced (Raphael, 2001). This effect is possibly mediated through its proposed interactions with the PDZ domain protein harmonin (Adato, 2005; Reiners, 2005). Among the USH1-associated proteins, harmonin has a central function, as it can also bind to CDH23 (Siemens, 2002), myosin VIIa, and SANS, and is able to interact with actin filaments promoting their bundling in cell culture, thereby potentially linking cadherins on the cell surface to the actin cytoskeleton (D'Alterio, 2005).

Unexpectedly, it was found that a truncated form of Cad99C that lacks most of the cytoplasmic tail, including the putative PDZ domain-binding sites, causes the same excessive lengthening of microvilli as the full-length protein, even in a Cad99C mutant background. This shows that the PDZ domain-binding sites do not have an essential positive regulatory function in microvillus outgrowth. It cannot be rule out that the remaining 31 juxtamembrane cytoplasmic amino acids interact with a cytoplasmic factor, but this short sequence contains no known motifs and is not conserved. Therefore, it seems likely that the membrane-bound extracellular domain of Cad99C is sufficient to promote microvillus extension independent of endogenous Cad99C. How does the extracellular domain of Cad99C control microvillus size? The data are consistent with two attractive models for Cad99C activity. The cadherin domains of Cad99C could influence the microvillus actin core by binding to an extracellular ligand, which directly or indirectly connects to the actin bundle promoting polymerization. Alternatively, the Cad99C extracellular domain might stabilize the plasma membrane of a microvillus. This may be important because to lengthen a cellular protrusion, the force created by actin polymerization has to overcome the counteracting force caused by tension in the plasma membrane envelope while it is pushed outward. Stabilization of the plasma membrane might alleviate the tension, allowing actin polymerization to proceed. The cadherin domains of Cad99C could stabilize the plasma membrane either by binding to the extracellular matrix or by self-assembling into an extracellular meshwork that forms a supporting scaffold surrounding the microvillus. The striking conservation of the Cad99C/PCDH15 extracellular domains may reflect the geometric constraints imposed by such a scaffold. The latter model in particular would be consistent with the concentration dependency of Cad99C activity and with its effect on the size and shape of microvilli (D'Alterio, 2005).

GENE STRUCTURE

cDNA clone length - 5674

Bases in 5' UTR - 139

Exons - 10

Bases in 3' UTR - 414

PROTEIN STRUCTURE

Amino Acids - 1706

Structural Domains

Gene annotation and cDNA analysis predict that Cad99C (CG310034) encodes a single-pass transmembrane protein with 11 cadherin domains (CDs). The cytoplasmic tail of Cad99C is asparagine rich and contains the motif SEVETTTEL at the COOH terminus, in which the underlined residues fit the consensus S/T-X-L/V of class 1 PDZ domain-binding sites. Cad99C is conserved in Anopheles gambiae and Apis mellifora, showing 62% and 52% identity in the extracellular region and 44 and 32% in the cytoplasmic tail, respectively. The closest relative in humans is PCDH15. Cad99C and PCDH15 have very similar protein architectures. They show 30% identity across the 11 extracellular CDs (expect value is e^-121). In comparison, there is 24% identity between the 11 CDs of Cad99C and the first 11 CDs or next 11 CDs of human fat3 (e^-57 and 3e^-63, respectively), a cadherin that gives the second best score in a Blast search. PCDH15 also contains a COOH-terminal PDZ-binding site (SQSTSL; Adato, 2005); otherwise, no significant similarity was identified in the cytoplasmic tail. It is noteworthy that the cytoplasmic tail is substantially diverged even between mouse and human PCDH15, showing only 57% identity, in contrast to 94% identity in the extracellular portion (D'Alterio. 2005).

In classic cadherins, binding of Ca²⁺ leads to dimerization and rigidification of CDs and is essential for the function of cadherins as adhesion molecules. Ca²⁺ associates with the linker region between CDs, requiring specific amino acid residues from both neighboring CDs. Interestingly, Cad99C and PCDH15 lack consensus sites for Ca²⁺-binding at conserved positions, between CDs 5/6 and 9/10, which are among the best-conserved CDs (36%-39% identity). This suggests that the extracellular region of Cad99C/PCDH15 is composed of two extended rodlike parts (CDs 1-5 and 6-9) connected by a potentially flexible hinge. CD 9 is followed by another hinge region that links to a pair of Ca²⁺-associated CDs. The similarities in protein structure strongly suggest that Cad99C is the orthologue of PCDH15 (D'Alterio. 2005).

The predicted Cad99C amino acid sequence contains the characteristic features common to members of the cadherin superfamily including a signal peptide, cadherin domains flanked by Ca⁺⁺-binding sites, and a transmembrane domain, defining Cad99C as a member of this superfamily. A β-catenin binding site, which is present in the cytoplasmic domain of type-I and type-II cadherins providing a link to the actin cytoskeleton, was not identified. However, a conserved type I PDZ-binding sequence was identified at the C-terminus of Cad99C. PDZ domain-containing proteins commonly act as linkers that facilitate the assembly of large molecular complexes within cells, suggesting that Cad99C physically interacts with a cytosolic protein complex. Only few other cadherins have been reported to contain C-terminal PDZ-binding sequences. Cadherin 23, for example, a vertebrate cadherin lacking the consensus R1 and R2 β-catenin binding sites, associates with its C-terminal PDZ-binding site with the PDZ domain-containing protein harmonin b. Interestingly, harmonin b has been shown to be able to bundle actin filaments, providing a potential link between cadherin 23 and the actin cytoskeleton independent of β-catenin. It is therefore conceivable that Cad99C interacts with a PDZ domain-containing protein that provides a link to the actin cytoskeleton independent of β-catenin. The PDZ-binding sequence of Cad99C is adjacent to and overlapping with two predicted Ser/Thr kinase phosphorylation sites. The interaction between a PDZ-binding peptide and a PDZ domain can be disrupted by phosphorylation of the PDZ-binding sequence by Ser/Thr kinases, suggesting that the potential binding of Cad99C to a PDZ domain-containing protein could be regulated by phosphorylation (Schlichting, 2005).

To analyze further the relationship between Cad99C and PCDH15, the domain organization of Drosophila and Anopheles Cad99C was compared with that of the human and mouse proteins. The four proteins share a similar structural organization, with an extracellular region containing 11 cadherin repeats (however, the available Anopheles Cad99C sequence is N-terminally truncated and has only 10 cadherin repeats), a single-span transmembrane domain, and a cytoplasmic region with a conserved C-terminal class I PDZ domain-binding site. Cad99C is the only one among the 17 cadherin-like sequences in Drosophila that has 11 cadherin repeats (Schlichting, 2006).

To estimate the evolutionary relationship between Cad99C and PCDH15, a phylogenetic tree was built based on the alignment of the full-length sequences of the cadherins most closely related to Cad99C. Drosophila and Anopheles Cad99C and mouse Pcdh15 and human PCDH15 segregate into the same clade, suggesting that Cad99C and PCDH15 are closely related. Thus, based on BLAST analysis, the conserved protein domain organization and phylogenetic analysis, it is suggested that Cad99C and PCDH15 are homologs (Schlichting, 2006).

date revised: 2 July 2006

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