Gene name - Merlin
Cytological map position - 18E1--18E2
Function - membrane-cytoskeleton scaffolding protein
Symbol - Mer
Genetic map position - 1-
Classification - ERM family, protein 4.1 superfamily
Cellular location - cytoplasm
Merlin (from ‘moesin-ezrin-radixin-like’) is a novel member of the ERM family of proteins (named after the founding members of the family: ezrin, radixin and moesin). Interest in the ERM family has been stimulated by the discovery that human neurofibromatosis 2 (NF2) is caused by mutations in merlin protein. The hallmark of NF2 is the presence of bilateral acoustic schwannomas (acoustic neuromas) affecting the eighth cranial nerve, in addition to other neurally associated tumors, such as meningiomas and ependymomas. Identified Drosophila proteins that have sequence resemblance to ERM family members include Moesin-like, Coracle, Expanded, and Inscuteable (McCartney, 1996).
The cellular functions of the ERM family in vertebrates are thought to be performed in two specific cellular domains: the microvilli, where ezrin is a component, and the adherens junction, from which radixin was first isolated. McCartney and Fehon (1996) have characterized two Drosophila ERM-like proteins, Merlin and Moesin-like. Although Merlin and Moesin-like proteins are frequently coexpressed in developing tissues, they display distinct subcellular localizations. In polarized epithelia, such as the embryonic hindgut, salivary gland, or the imaginal disc, both Moesin-like and Merlin are found in the highest concentration in the most apical part of the cell. Colocalization experiments in wing imaginal discs demonstrate that at least part of the detected Merlin and Moesin-like protein is associated with the adherens junction as is the beta-Catenin homolog, Armadillo. Moesin-like is localized to the apical cap of imaginal disc cells, a region known to contain abundant microvilli. Both Moesin-like and filamentous actin are localized in microvilli present during cellularization in the preblastoderm embryo. Merlin does not localize with actin or Moesin-like in apical buds. While Moesin-like is observed in continuous association with the plasma membrane, as is typical for an ERM family protein, Merlin is found in punctuate structures at the membrane and in the cytoplasm. Investigation of Merlin distribution in cultured cells demonstrates that it is associated with endocytic compartments. Merlin may not interact with cytoskeletal actin in the same way that has been proposed for other members of the ERM family. It is thought that Merlin is associated with membrane internalized from the cell surface. As a result of these studies, it is proposed that whereas Moesin-like may fulfill all the functions of vertebrate ERM proteins, Merlin protein has unique functions in the cell which differ from those of other ERM family members, perhaps being involved in endocytosis. It is concluded that Merlin is a functionally distinct ERM family member (McCartney, 1996).
Genetically marked clones, mutant for Merlin, were generated during the larval period. Comparison of wild-type (control) and mutant clones in the eye suggested that the mutant clones are consistently larger than their wild-type sisters. The number of ommatidia within each mutant clone was compared to the number of ommatidia in its sister wild-type clone and a ratio of mutant clone size to wild-type sister clone size was generated. Merlin mutant clones range from 2.1 to 2.7 times the size of their wild-type sisters, depending on the allele of Merlin examined. This observation suggests that the Merlin mutant cells either proliferate more rapidly than their wild-type neighbors or that they continue proliferating later in development. Another possible explanation is that the mutant cells have a defect in cell death leading to apparent overproliferation. Acridine orange staining of Merlin mutant imaginal discs did not indicate any changes in the level of cell death, however. Because loss of Merlin function in clones results in overproliferation without any gross morphological defects, it is concluded that Merlin functions in a process that specifically affects the regulation of proliferation (LaJeunesse, 1998).
Besides the overproliferation observed in Mer eye clones, a second Mer phenotype has been observed. A single viable allele, Mer3 is due to a missense mutation of Met177 to Isoleucine. Flies homozygous for Mer3 survive as viable, sterile adults and display a broadened wing phenotype along with low and variably penetrant expression of weakly roughened eyes and the development of abnormal head cuticle structures. The broadening of the wing is characteristic of mutations that increase cell proliferation in the wing. Further analysis was carried out to determine the structural domains within Merlin required for proper function, both in terms of the subcellular distribution of Merlin and the overproliferation phenotype (LaJeunesse, 1998).
Mutational analysis of Merlin protein has been carried out to try to understand how Merlin functions to restrain cell proliferation. The molecular organization of Merlin is similar to that of ERM family members and consists of a tripartite domain structure: an NH2-terminal protein 4.1 domain, a putative coiled-coil domain, and a COOH-terminal region. Because of the apparent modular nature of Merlin, several Merlin truncations were generated that contain portions of the NH2- or COOH-terminal halves of the protein and a truncation containing only the central coiled-coiled region. Wild-type Merlin is initially targeted to the membrane, and within 3 h, much of the protein localizes to punctate cytoplasmic structures. A similar pattern (membrane-associated and cytoplasmic staining) is also observed in endogenously expressed Merlin in S2 cells and within the imaginal disc epithelium (McCartney, 1996). Removal of the conserved COOH-terminal 35 amino acids results in a protein, mycMer1-600, that is localized almost exclusively at the plasma membrane and does not appear to internalize. This observation suggests that the COOH-terminal 35 amino acid residues play a role in Merlin internalization. As will be described below, internalization is likely to be important in Mer function. In the imaginal epithelia of transgenic larvae, this truncated Merlin protein is localized to the plasma membrane (similar to the localization of wild-type Merlin) but did not display the punctate cytoplasmic localization characteristic of the wild-type protein. It is concluded that components of both the NH2- and COOH-terminal halves of Merlin are required for its correct targeting to the plasma membrane (LaJeunesse, 1998).
The conserved NH2-terminal region (CNTR) was examined in greater detail. The CNTR is nearly 60% identical between Merlin and the ERM proteins; however, a closer examination of this region reveals seven amino acids (170YQMTPEM177) that are identical in human and Drosophila Merlin but are divergent from the ERM proteins. The possible functional significance of this region is further supported by the presence of the Mer3 missense mutation (the viable allele described above) at amino acid 177. This region has been named "the Blue Box" (BB). To investigate the functional significance of the BB, two Merlin proteins were generated, one lacking the BB region (mycMerdeltaBB) and one in which the BB is replaced by a polyalanine stretch (mycMerBBA). Removal or replacement of the entire BB results in proteins that are properly targeted to the plasma membrane but are not internalized. It is concluded that the BB region is required for internalization of Merlin, but is not required for association with the plasma membrane. Genetic rescue of Mer-lethality by truncated proteins indicate that all essential Merlin functions reside in the NH2-terminal 350 amino acids of the protein (LaJeunesse, 1998).
Nonmutant class siblings that carried ubiquitously expressing Mer transgenes were examined for dominant phenotypes. None of the NH2- or COOH-terminal deletions display any dominant phenotype in a Mer+ background. In contrast, dominant phenotypes, suggesting hyperproliferation, results from expression of the Merlin BB mutant proteins. The dominant phenotype includes a broadening of the wing blade, variably penetrant ectopic wing vein material (primarily around the second wing vein), anterior and posterior cross vein defects, and ectopic bristles or sense organs. The most striking quality of this dominant phenotype is the enlargement of the wing blade, resulting in a curvature of the wing surface. High levels of expression of the Merlin BB mutant proteins in the wing blade under two different Gal4 drivers (engrailedciBeGal4, apterousmd5 Gal4) result in an outheld wing phenotype associated with alterations in the wing hinge morphology (LaJeunesse, 1998).
To examine the wing blade phenotype in more detail, the wing surface areas were compared for the expression of Merlin BB proteins or wild-type Merlin under the engrailed GAL4 driver. As expected of an overproliferation phenotype, a significant increase in the total surface area of wings expressing BB mutant proteins was observed. The area of overgrowth is localized to the posterior half of the wing where the engrailed Gal4 specifically drives expression, although a decrease in surface area is observed between wing veins 2 and 3, a region anterior to the expression of the transgene. To determine whether the observed increase in area is due to increased cell number or increased cell size, the densities of wing hairs were measured at several positions within the wing blade. (Each wing blade cell secretes a single hair). The overall wing hair density in wings expressing BB Merlin proteins is indistinguishable from that in wings expressing a wild-type protein, indicating that the broadening of the wing results from an increase in cell number rather than in cell size. As shown in the somatic mosaic analysis, loss of Mer function results in overproliferation of mutant cells. Similarly, ectopic expression of BB mutant proteins resulted in increased proliferation. These results suggest that the BB mutant proteins act in a dominant-negative manner and therefore interfere with the activity of wild-type protein (LaJeunesse, 1998).
MycMer1-600, a truncated Merlin protein missing the C-terminal 35 amino acids acts as an activated protein that displays greater rescuing activity and suppresses the phenotypes produced by a dominant-negative form of Merlin. Pulsed expression of Mer1-600 in S2 cultured cells under an inducible promoter indicates that the levels of expression and the stability of this protein are not significantly different from wild-type Merlin. The simplest explanation for this phenomenon is that the COOH-terminal region contains a domain important for reducing the activity of Merlin. Recent studies demonstrate that ERM protein activity is regulated by a Rho-based signaling pathway (Takaishi, 1995, Hirao, 1996 and Mackay, 1997). Rho-Kinase has been shown to phosphorylate a conserved threonine within the COOH-terminal 35 amino acids of all ERM proteins and thereby regulate the conformational change that occurs during a putative transition from an inactive to an active state (Matsui, 1998). Although the COOH-terminal 35 amino acids of Merlin are divergent from those of the ERM proteins, the threonine residue phosphorylated by Rho-Kinase is conserved, suggesting that Merlin could use a similar mechanism for switching between an inactive and an active state. Creation of an activated Merlin protein by the removal of the COOH-terminal regulatory region is consistent with this notion, but further studies are required to confirm this regulatory role (LaJeunesse, 1998).
Two Merlin mutations described here, the dominant-negative MerDeltaBB and the activated Mer1-600, are found primarily at the plasma membrane. This apparently contradictory result, that two mutant forms of Merlin with opposite functions both localize to the plasma membrane, indicates that localization to the plasma membrane is not sufficient for Merlin function, that is to say, internalization is likely to be important in Mer function. The requirement for internalization distinguishes Mer from other ERM family members, Moesin-like for example, which are exclusively located at junctions. It is proposed that Merlin normally undergoes an activation process that occurs at the cytoplasmic face of the plasma membrane. In this model, not only is MerDeltaBB refractory to activation, but it also inhibits activation of endogenous wild-type Merlin, thus causing wild-type Merlin to accumulate at the plasma membrane in a nonfunctional state and producing a dominant phenotype. In contrast, Mer1-600 exists in a constitutively activated state, thereby evading the block presented by MerDeltaBB and suppressing the dominant BB mutant phenotype. The two functional states of wild-type Merlin may represent two distinct conformations of the protein, as has been shown for ERM proteins. Alternatively, Merlin may associate with two distinct binding partners at the membrane. In either case, one state may serve to associate Merlin with the membrane, and the second may be required for Merlin activation. (LaJeunesse, 1998). Apparently, internalized Mer functions to regulate cell proliferation, but its regulatory targets are as yet unknown.
Merlin is 55% identical to human merlin, while only 45% identical to human ezrin and mouse radixin, and 39% identical to human moesin. The similarity between Drosophila and human Merlin extends over the entire lingth of the two sequences, and is greatest in the N-terminus, where all ERM proteins share high identity. In this region, however, areas can be identified where the Merlin sequences, while similar to each other, diverge from the human and Drosophila Moesin sequences. The similarity between Drosophila Merlin and human merlin is particularly notable at the C-terminus, where the merlin proteins diverge from the other ERM family members. Sequences from the second Drosophila ERM family member, Moesin-like, are found to share 58% overall identity with both human moesin and human ezrin, 57% identity with mouse radixin, and only 41% identity with human merlin. Designation of this gene as Moesin-like, rather than as ezrin or radixin, is based primarily on its lack of the polyproline tract characteristic of the latter two proteins. furthermore, Moesin-like shares a greater degree of identity in the C-terminal divergent region with human moesin (26%) than with human ezrin (25%) or mouse radixin (22%) (McCartney, 1996).
date revised: 5 August 98
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