Developmental, Cell and Molecular Biology Group, Department of Zoology, Duke University, Durham, North Carolina 27708-1000
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Abstract |
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Merlin, the product of the Neurofibromatosis type 2 (NF2) tumor-suppressor gene, is a member of the protein 4.1 superfamily that is most closely related to ezrin, radixin, and moesin (ERM). NF2 is a dominantly inherited disease characterized by the formation of bilateral acoustic schwannomas and other benign tumors associated with the central nervous system. To understand its cellular functions, we are studying a Merlin homologue in Drosophila. As is the case for NF2 tumors, Drosophila cells lacking Merlin function overproliferate relative to their neighbors. Using in vitro mutagenesis, we define functional domains within Merlin required for proper subcellular localization and for genetic rescue of lethal Merlin alleles. Remarkably, the results of these experiments demonstrate that all essential genetic functions reside in the plasma membrane- associated NH2-terminal 350 amino acids of Merlin. Removal of a seven-amino acid conserved sequence within this domain results in a dominant-negative form of Merlin that is stably associated with the plasma membrane and causes overproliferation when expressed ectopically in the wing. In addition, we provide evidence that the COOH-terminal region of Merlin has a negative regulatory role, as has been shown for ERM proteins. These results provide insights into the functions and functional organization of a novel tumor suppressor gene.
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Introduction |
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RECENT studies have identified a rapidly growing
number of tumor suppressor genes whose normal
function in cells directly or indirectly regulates cellular proliferation. Not surprisingly, many of these genes,
such as Rb and p53, have been shown to encode proteins
that regulate aspects of the cell cycle (for review see Brown, 1997). However, other tumor suppressor genes appear to function in processes less clearly related to control
of the cell cycle. One of the most intriguing genes of this
latter class is the Neurofibromatosis type 2 (NF2)1 gene,
which encodes a member of the protein 4.1 superfamily, Merlin (Rouleau et al., 1993
; Trofatter et al., 1993
). Members of the protein 4.1 superfamily, a large group of membrane-associated cytoplasmic proteins, include protein 4.1;
talin; the ezrin, radixin, moesin (ERM) proteins; Merlin;
Drosophila Expanded; several protein phosphatases; and
at least two nonmuscle myosins (for review see McCartney
and Fehon, 1997
). The defining feature of this superfamily is a conserved region of 200-300 amino acids usually located in the NH2 terminus of the protein. This region is
particularly well conserved between Merlin and the ERM
proteins. The ERM proteins appear to function as molecular linkers by binding to transmembrane proteins through
the NH2-terminal domain and linking them to the cortical
actin cytoskeleton through a COOH-terminal actin-binding domain. Consistent with this role, ERM proteins localize to actin-rich structures such as the adherens junction
and microvilli (Franck et al., 1993
; Takeuchi et al., 1994
).
Although the structural similarities between Merlin and
the ERM proteins suggest that they may have similar functions, the exact nature of Merlin's cellular functions has
not been defined. Recent studies in cultured cells indicate
that expressed Merlin protein accumulates in some actin-rich membrane domains, such as membrane ruffles at the
leading edge of migrating cells (Gonzalez-Agosti et al.,
1996; Sainio et al., 1997
), consistent with the localization of
ERM proteins. However, several lines of evidence indicate that Merlin has functions that are clearly distinct from
those of ERM proteins. First, although the NH2-terminal
domains of Merlin and the ERM proteins are similar (the
protein 4.1 superfamily domain), Merlin lacks the well-
defined COOH-terminal actin-binding domain found in
ERM proteins (Turunen et al., 1994
; Gary and Bretscher, 1995
). In addition, while the ERM proteins are functionally redundant (Takeuchi et al., 1994
), there is no evidence
for redundancy between the ERM proteins and Merlin.
The NF2/Merlin gene can be mutated to lethality in both
Drosophila and mouse (Fehon et al., 1997
; McClatchey et
al., 1997
). Finally, in vivo studies of subcellular localization
of Merlin and Moesin reveal that they can be distinct, again supporting the idea that these proteins have different functions (McCartney and Fehon, 1996
).
To define the cellular functions of Merlin and the ERM
proteins, we have isolated and characterized two Drosophila genes, Moesin, the sole ERM gene in Drosophila,
and Merlin, a well conserved NF2 homologue (McCartney
and Fehon, 1996). In the present study, we show that Merlin is essential for viability in Drosophila and is required
for the proper regulation of cell proliferation. Furthermore, our analysis indicates that essential Merlin functions
occur at the cytoplasmic face of the cell membrane and
that all of these functions can be mediated by the conserved NH2-terminal region.
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Materials and Methods |
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Drosophila Cultures and Stocks Used
All Drosophila cultures were maintained on standard corn meal, yeast,
molasses, and agar medium. w1118 stocks were used for the transformation
of UASMerlin transgenes. The Merlin alleles used in this study are described in Fehon et al. (1997).
Somatic Mosaic Analysis and Histology of Adult Eyes
Fly stocks capable of producing clones were generated by crossing w1118
sn3 Mer* P{neoFRT}19A/FM6 with y w P{w[+mC] = PiM}5A
P{w[+mC] = PiM}10D P{neoFRT}19A/Y;P{hs-FLP}, Sb/TM6B. Offspring from this cross were heat shocked at either 36 or 72 h after egg laying (AEL) using one of two different heat shock regimens: 30 min at 38°C,
60 min at 25°C, and 30 min at 38°C or 60 min at 38°C, 60 min at 25°C, and
60 min at 38°C. In the adult, mutant clones were marked with w1118 in the
eye and sn3 in the thorax; wild-type sister clones were marked in the eye with four copies of the mini-white transgene and in the thorax with the yellow mutation. To analyze clone size in the eye, flies were placed in vented
Eppendorf tubes (Madison, WI) and flash frozen in liquid nitrogen. The
treated flies were dried with dry carbon dioxide, and the heads were then
dissected with a razor blade and mounted on a glass slide on double stick
tape. Eyes were examined and ommatidia counted with the compound microscope using transmitted light and the 10× objective. Fixation and sectioning of adult eyes was performed as previously described (Tomlinson and Ready, 1987), with the exception that the tissue was postfixed in 2%
OsO4 and embedded in Araldite resin.
Wing Measurements
Wing cuticles were prepared by first incubating the entire fly in 70% ethanol and then submerging it in a drop of water on a siliconized slide, where the wings were dissected away and mounted in ~30 µl Aquamount (BDH Laboratory Supplies, Poole, England) on a glass slide. Only wings that had been well flattened during the mounting process were used for further analysis. Camera lucida drawings were made of the wing perimeter, wing veins, and any ectopic veination. These drawings were then scanned at 75 dots per inch using a flatbed scanner and analyzed using the "measure" tool in NIH Image. The area of the entire wing or the individual areas between the wing veins (the intervein regions) were determined and expressed in square millimeters.
Sequencing of Mutant Merlin Alleles
Genomic DNA was obtained from single first instar larvae hemizygous for each Merlin allele (marked with yellow). Merlin genomic DNA was amplified using intron-specific primers. The resulting PCR products were sequenced using the AmpliCycle sequencing kit (Perkin-Elmer Corp., Branchburg, NJ).
Construction of Truncated Merlin Proteins
A Bluescript shuttle vector was generated with an NH2-terminal myc
epitope tag by annealing two primers, consmyc S (sense) 5' AAT TCA
CCA TGG AGC AAA AGC TCA TTT CTG AAG AGG ACT TGA
GGC CTA A and consmyc A (antisense) 5' GAT CTT AGG CTT CAA
GTC CTC TTC AGA AAT GAG CTT TTG CTC CAT GGT G, which
produce a duplex with over-hanging EcoRI and BglII ends. This fragment
was subsequently cloned into an EcoRI/BamHI-cut Bluescript SK plasmid. (All restriction enzymes were obtained from New England Biolabs,
Beverly, MA.) Digestion of the modified vector with StuI allowed PCR-generated Merlin transgenes to be cloned in-frame immediately downstream of the myc epitope.
All Merlin constructs were generated by PCR amplification from a full-length Merlin cDNA clone (McCartney and Fehon, 1996). The sequence
of all PCR-amplified regions was confirmed by sequencing using standard
methods. To make upstream activation sequences of the yeast Gal4 transcription factor (UAS) expression constructs, a XhoI/XbaI fragment from
the BSSK-myc shuttle vector was then cloned into an XhoI/XbaI-prepared pUASt transformation vector (Brand and Perrimon, 1993
). Transformation of these constructs was performed as described (Rebay et al.,
1993
). 4-10 independent lines were established for each construct.
To construct BSSK-myc MerBB, the 5' half of Merlin was amplified using the M13 Universal Primer of the BSSK
vector, and an antisense
Merlin primer within which a PvuII site was engineered (underlined in
primer sequence): 5' GCG TCA TCT GCA GCT GAT GCG. This product was then digested with EcoRI and PvuII. The 3' Mer
BB region was
similarly generated using M13 reverse and an internal Merlin sense primer
whose 5' sequence began at codon 177: 5' TGG GAG GAA CGG ATC
AAG ACA TGG; this product was digested with PstI and ligated together
with the 5' piece into an EcoRI/PstI cut BSSK-mycMer+. Positive clones
were sequenced to verify the presence of the deletion. The BSSK-mycMerBBA vector was constructed in a similar manner using sense (5' TAC CAG ATG ACC GCG GCA GCG TGG GAG GAA CGG) and antisense (5' CTC CCA CAT TTC CGC GGC TGC TGC GGC CTG ATC
GGT CAC TCC) primers that changed amino acids 171-177 to alanines
and contained a SacII site for cloning (underlined in primer sequence). To
make BSSK-mycMer3, a genomic PCR fragment containing the Mer3 mutation was digested with NheI and NspI. A BSSK-mycMer+ vector was
prepared by a complete NheI digest and a partial NspI digest. (The site is
unique in Merlin, but another site is in the BSSK
vector.) A ligation was
performed using the prepared PCR product and the NheI/NspI-prepared
BSSK
myc tag vector. Positive clones were sequenced to detect the presence of the point mutation.
To make Merlin green fluorescent protein (GFP) fusion proteins,
UASmycMer+, UASmycMerBB, UASmycMerBBA, and UASmycMer3
were digested with BglII and SacI. The SacI site is unique in Merlin and is
located in the last codon of the Merlin open reading frame. A GFP fragment was excised from pGEM7GFPRS (a gift of Kevin Edwards and Dan
Kiehart, Duke University) using SacI and XbaI. The SacI site lies eight
codons 5' to the start ATG of the GFP open reading frame and is in-frame
to the Merlin open reading frame. A pCaSpeR-hs vector was prepared using BglII and XbaI. A three-piece ligation was performed using gel-purified fragments of Merlin, GFP, and the prepared pCaSpeR-hs vector.
Transfection of Schneider 2 Cells and In Vivo Competition Experiments
For expression of the truncated Merlin fragments in Schneider line 2 (S2)
cultured cells, two sets of expression constructs were used: pCaSpeR-hs, a
heat shock inducible vector, and pRmHa-3, an inducible metallothionein
promoter vector. For the heat shock vectors, a BglII/XbaI fragment from
the UAS Merlin truncation constructs was cloned into a BglII/XbaI-prepared pCaSpeR-hs vector. To generate pRmHa-3, a BglII/BamHI fragment from pCaSpeR-hs Merlin constructs was cloned into BamHI-prepared pRmHa-3. The maintenance, induction, and immunofluorescent
analysis of S2 cells was performed as previously described (Fehon et al.,
1990). In the time course experiments, transfected S2 cell cultures were induced with a 20 min/38°C heat shock; samples were then collected at time
points 1, 3, and 6 h after heat shock (AHS) induction. The samples were fixed and stained with a mouse anti-myc antibody 9c10.
For the in vivo competition experiment, we cotransfected S2 cells with
two different plasmids: a pRmHa-3 myc-tagged Merlin construct (either
wild-type Merlin, Merlin1-600, or MerlinBB) and a pCaSpeR-hs non-myc-tagged Merlin GFP fusion protein construct. The transfected cells were
treated with a CuSO4 solution (final concentration 0.6 mM) to induce the
metallothionein promoter, incubated for 3 h, and heat shocked for 20 min/
38°C to induce expression of the heat shock construct. Samples were collected 5 h AHS, fixed, and stained with a mouse anti-myc antibody.
Determination of In Vivo Activity
To determine the rescue activity of our UASMer transgenes, y1 w1118 Mer/ FM7; T80Gal4/T80Gal4 females were crossed to UASmycMer transgenic males. Four equal classes of flies would be expected from this cross, including males hemizygous for a Merlin mutation that lack endogenous Merlin function. To determine the percentage of rescue, the number of F1 progeny excluding the rescued hemizygous males was divided by three to yield the predicted number of mutant offspring. The number of observed mutant progeny (marked by yellow) was then divided by this number to yield the percentage of rescue. The numbers were then normalized to the rescue provided by Merlin1-600, which provided the greatest rescue.
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Results |
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Characterization of Mutations within the Drosophila Merlin Gene
The Drosophila Merlin gene is located on the X chromosome at cytological position 18D-E. Four mutations within
Merlin have been previously identified (Fehon et al.,
1997). Although none of these alleles causes embryonic
lethality, three of these mutations cause larval and pupal
lethality without strong distinguishing phenotypes. The
three lethal Merlin alleles encode putative truncated proteins as a result of nonsense mutations: Merlin1 (Mer1),
Gln324 to stop; Mer2, Gln318 to stop; Mer4, Gln170 to
stop. A single viable allele, Mer3, was recovered from
these screens and is due to a missense mutation of Met177
to Ile. 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 (data not shown). Although no genetic deficiency exists to test for residual function of these alleles,
we conclude that Mer4 is likely a null mutation based on
the severity of the truncation predicted for this allele.
To characterize the cellular phenotypes of Merlin, we
performed a somatic mosaic analysis using the yeast 2 micron Flipase enzyme/Flipase Recognition Target site
(FLP/FRT) system (Golic and Lindquist, 1989; Xu and
Rubin, 1993
). The parental chromosome of all four Mer alleles carries an FRT site at cytological position 19A. Although previous experiments mapped Merlin distal to this FRT at 18D-E, we confirmed that Mer
clones were generated after heat shock FLP induction by staining mosaic
tissue with the Merlin antibody (data not shown). In the adult eye, mutant clones were identified by the lack of pigment due to the presence of the white mutation; wild-type
sister clones were marked by a dark orange eye color, and
heterozygous ommatidia were pale orange. The overall
morphology of the eye within the mutant clone appeared
normal (Fig. 1 B), although mutant clones occasionally
displayed a very weak roughened phenotype (data not shown). Histological sections of these clones revealed essentially normal differentiation of ommatidia with only a
few disruptions in the organization of ommatidia when
compared with the neighboring wild-type cells (Fig. 1 C).
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Comparison of genetically marked, wild-type (control),
and Mer mutant clones in the eye suggested that the mutant clones were consistently larger than their wild-type
sisters. To address this possibility, we compared the number of ommatidia within each mutant clone to the number
of ommatidia in its sister clone and generated a ratio of
mutant clone size to wild-type sister clone size. When a
control clone was generated, the area of the white
-marked clone was equal to that of its white+ sister (Fig. 1
A and D). In contrast, Merlin mutant clones ranged from
2.1 to 2.7 times the size of their wild-type sisters, depending on the allele of Merlin examined (Fig. 1, B and D). 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 (data not
shown). Because loss of Merlin function in clones results in
overproliferation without any gross morphological defects, we conclude that Merlin functions in a process that specifically affects the regulation of proliferation.
In Vitro Mutagenesis
To further investigate its cellular roles, we generated NH2-
and COOH-terminal truncations of the Merlin protein
(Fig. 2 A). These mutations were used to perform two sets
of experiments: First, we examined the subcellular localization of these mutant Merlin proteins when expressed in
cultured cells and in tissue, and second, we examined their
in vivo genetic functions. The fragments of Merlin generated for the structure/function analysis were selected based on comparisons with human Merlin and with the
ERM proteins (McCartney and Fehon, 1996), with the assumption that regions that are highly conserved are likely
to have functional significance. The molecular organization of Merlin is similar to that of ERM family members
and consists of an NH2-terminal protein 4.1 domain, a putative coiled-coil domain, and a COOH-terminal region
(Fig. 2 A). Because of the apparent modular nature of
Merlin, we generated several Merlin truncations that contained portions of the NH2- or COOH-terminal halves of
the protein and a truncation containing only the central
coiled-coiled region. The size and stability of each Merlin
truncation was verified by immunoblot (Fig. 2 B). In addition, we examined the conserved NH2-terminal region
(CNTR) in greater detail. The CNTR is nearly 60% identical between Merlin and the ERM proteins; however, a
closer examination of this region revealed seven amino acids (170YQMTPEM177) that are identical in human and
Drosophila Merlin but are divergent from the ERM proteins (McCartney and Fehon, 1996
). The possible functional significance of this region is further supported by the presence of the Mer3 missense mutation at amino acid
177. We have named this region "the Blue Box" (BB). To
investigate the functional significance of the BB, we engineered two Merlin proteins, one lacking the BB region
(mycMer
BB) and one in which the BB is replaced by a
polyalanine stretch (mycMerBBA). Furthermore, to define
the nature and the activity of the original Mer3 allele, we
generated a myc-tagged version of this allele (mycMer3).
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The Subcellular Localization of Merlin In Vitro Mutants
We examined the subcellular localization of these Merlin
fragments in Drosophila S2 cultured cells and in the imaginal disc epithelium to identify regions within Merlin that
are required for proper subcellular localization. Experiments in cultured cells allowed us to examine both the
temporal patterning and the subcellular localization of
Merlin. As previously reported (McCartney and Fehon,
1996), wild-type Merlin is initially targeted to the membrane, and within 3 h, much of the protein localizes to
punctate cytoplasmic structures (Fig. 3, A-C). A similar
pattern (membrane-associated and cytoplasmic staining)
was also observed in endogenously expressed Merlin in S2
cells (data not shown) and within the imaginal disc epithelium (McCartney and Fehon, 1996
; Fig. 3 D). Removal of
the conserved COOH-terminal 35 amino acids resulted in
a protein, mycMer1-600, that was localized almost exclusively at the plasma membrane and did not appear to internalize (Fig. 3, E-G). This observation suggests that the
COOH-terminal 35 amino acid residues play a role in
Merlin internalization. In the imaginal epithelia of transgenic larvae, this truncated Merlin protein was 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 (Fig. 3
H). More severe COOH-terminal truncations of Merlin,
mycMer1-350 (Fig. 3, I-L) and mycMer1-375 (data not
shown) appeared to associate less strongly with the plasma
membrane, suggesting that within the COOH terminus
there are sites required for proper localization. Consistent
with this notion, mycMer351-635 (Fig. 3, M-P) and
mycMer351-600 (data not shown), which retain most of the
COOH-terminal half of Merlin but none of the CNTR,
were highly membrane associated in both cultured cells
and imaginal tissue. Of the other Merlin constructs,
mycMer1-330 localized diffusely throughout the cytoplasm
with no distinct subcellular location, while mycMer1-169
and mycMer601-635 had little or no expression (data not
shown), owing possibly to their inherent instability. We
conclude from these results that components of both the
NH2- and COOH-terminal halves of Merlin are required
for its correct targeting to the plasma membrane.
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To assess the role of the Blue Box (BB) region in Merlin's subcellular localization, we examined the subcellular
distribution of wild-type and mutant proteins fused to
green fluorescent protein (GFP). Wild-type Merlin-GFP
(GFPMer+) retains full wild-type rescue function; thus,
the GFP moiety does not interfere with Merlin function
(data not shown). In S2 cells, GFPMer3 remained associated with the membrane 3-5 h after heat shock (Fig. 4,
E-G), unlike GFPMer+, which had all internalized by this
time (Fig. 4, A-C). Removal or replacement of the entire
BB resulted in proteins that were properly targeted to the
plasma membrane but were not internalized (Fig. 4, I-K).
Similarly, in imaginal epithelia, MerBB was exclusively
plasma membrane associated (Fig. 4 L).
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Characterization of In Vivo Function
To assay the in vivo function of the mutant Merlin proteins, we used the Gal4/UAS system (Brand and Perrimon, 1993) to express Merlin truncations in mutant and
wild-type genetic backgrounds. In these studies, we addressed two questions: First, what is the minimal protein
region required for Merlin function, and second, do any
mutant Merlin proteins produce antimorphic (dominant-negative) phenotypes when expressed in vivo? In the
genetic rescue experiments, UASMerlin constructs were
driven under a ubiquitously expressing Gal4 enhancer trap
(T80Gal4) in a Mer4 mutant background and tested for
their ability to rescue Mer4 lethality. Complementation of
the other lethal Merlin alleles (Mer1 and Mer2) was also
performed with similar results (data not shown). To test
for possible dominant activity of a Merlin truncation, we examined the nonmutant class flies from these same experiments for phenotypes similar to those expressed by
Merlin mutants.
In the control experiments, we observed almost complete genetic rescue with the mycMer+ transgene (Fig. 2
A). Surprisingly, even stronger genetic rescue was observed with a truncated Merlin protein missing the
COOH-terminal 35 amino acid residues (mycMer1-600).
This improved genetic rescue over mycMer+ was consistently observed with independent transgenic lines, suggesting that the truncated form has increased in vivo function. We also observed partial rescue of Mer4 lethality with two
even shorter NH2-terminal Merlin transgenes, mycMer1-350
and mycMer1-375 (Fig. 2 A). All of the rescued flies were
phenotypically wild-type and did not possess any of the
characteristic Mer3 phenotypes. Expression of mycMerBB
and mycMerBBA failed to rescue Mer4 lethality (Fig. 2 A),
indicating that this region has essential functions. In contrast, expression of mycMer3 partially rescued Mer4 lethality. However, unlike rescued flies from other experiments, all of the mycMer3 rescued flies expressed wing phenotypes similar to those expressed by flies carrying the original Mer3 allele. No rescue was observed with expression of
any other Merlin truncation. Taken together, the results
indicate that all essential Merlin functions reside in the
NH2-terminal 350 amino acids of the protein.
In the same genetic experiments, nonmutant class siblings that carried ubiquitously expressing Mer transgenes were examined for dominant phenotypes. None of the NH2- or COOH-terminal deletions displayed any dominant phenotype in a Mer+ background. In contrast, we observed dominant phenotypes resulting from expression of the Merlin BB mutant proteins. This dominant phenotype included 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 (Fig. 5, C and D and data not shown). The most striking quality of this dominant phenotype was the enlargement of the wing blade, resulting in a curvature of the wing surface. Furthermore, high levels of expression of the Merlin BB mutant proteins in the wing blade under two different Gal4 drivers (engrailedciBeGal4, apterousmd5 Gal4) resulted in an outheld wing phenotype associated with alterations in the wing hinge morphology (data not shown).
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The broadening of the wing blade we observed with expression of Merlin BB mutations is characteristic of mutations that cause increased cell proliferation in the wing
(Mahoney et al., 1991; Boedigheimer and Laughon, 1993
;
Bryant et al., 1993
). To examine this phenotype in more
detail, we compared the surface area of wings expressing
Merlin BB proteins or wild-type Merlin under the engrailed GAL4 driver. As expected of an overproliferation phenotype, we observed a significant increase in the total
surface area of wings expressing BB mutant proteins (Fig.
5 E). The area of overgrowth was localized to the posterior
half of the wing where the engrailed Gal4 specifically
drives expression (Fig. 5 B, shaded area), although a decrease in surface area was observed between wing veins 2 and 3, a region anterior to the expression of the transgene.
To determine whether the increase in area we observed
was due to increased cell number or increased cell size, we
measured the densities of wing hairs 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 was indistinguishable from that in wings
expressing a wild-type protein (data not shown), 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 resulted 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.
To confirm that the BB mutations have dominant-negative activity, we examined the modification of the dominant MerBB wing phenotype in response to alteration of
endogenous gene dose. As would be expected for an antimorphic allele, dominant Mer
BB wing phenotypes were
enhanced by a reduction of endogenous gene dose (Mer4/+)
and were suppressed in response to an increase in the level of endogenous Merlin (+/+;P{cos Mer+}; Table I). These
results confirm that Merlin BB proteins act as dominant-negative proteins.
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Based on the in vivo activity assay, mycMer1-600 appears
to behave as an activated protein. An additional way to
test this proposal is to determine whether mycMer1-600 is
able to rescue the dominant-negative activity of the BB
mutant proteins. We examined the BB phenotype of wings
coexpressing MerBB and either wild-type Merlin (mycMer+)
or the putative activated form (mycMer1-600). Flies expressing mycMer
BB under the apterous Gal4 driver display wings held at an average angle of 45° from the body
axis (Fig. 6 A). This phenotype appears to be caused by increased proliferation in the wing hinge region (data not
shown). Coexpression of mycMer+ with mycMer
BB resulted in a slight suppression in the degree of outheld
wings (Fig. 6 B). However, when mycMer1-600 was coexpressed with the mycMer
BB, we observed a dramatic suppression of the dominant BB wing phenotype (Fig. 6 C). In
a control experiment, the COOH-terminal half of Merlin
was coexpressed with mycMer
BB, and no suppression of
the phenotype was observed (Fig. 6 D). The ability of
mycMer1-600 to suppress the dominant BB phenotype supports the idea that this is an activated form of the Merlin
protein.
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Competition Experiments
To directly observe the dominant-negative behavior of
mycMerBB, we examined the localization of wild-type
Merlin protein in the presence of mycMer
BB. We cotransfected Drosophila S2 cells with GFPMer+ and either
mycMer+ or mycMer
BB and examined the localization of
both proteins. In S2 cells cotransfected with either the
mycMer+ or mycMer1-600 competitor proteins, the majority of GFPMer+ in these cells was associated with characteristic punctate cytoplasmic structures 5 h after induction
(Fig. 7, A and D) as was shown earlier (Figs. 3 C and 4 C).
However, in almost all S2 cells cotransfected with mycMer
BB competitor, wild-type GFPMerlin remained at the
plasma membrane (Fig. 7, B-D). This result suggests that
localization to the plasma membrane is not sufficient for
Merlin activity and that mycMer
BB interferes with wild-type Merlin function by causing it to accumulate at the
plasma membrane in a nonfunctional state.
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Discussion |
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Although the protein 4.1 family has typically been characterized as a group of membrane-skeletal proteins, recent
studies have revealed a diversity of function, some of
which may not be primarily related to the canonical membrane-cytoskeletal linker function (for review see McCartney and Fehon, 1997). In addition, recent genetic
studies in particular have implicated protein 4.1 family
proteins in mediating the intercellular interactions that regulate cell proliferation. For example, mutations in
Drosophila expanded, a divergent member of the protein
4.1 superfamily that localizes to the adherens junction, result in overproliferation of the cells that form the adult
wings (Boedigheimer and Laughon, 1993
). Previous experiments have shown that proliferation in this tissue is
controlled by cell-cell interactions, mediated by at least
three known signal transduction pathways, wingless, decapentapelegic, and Epidermal Growth Factor (Edgar and
Lehner, 1996
). Studies of Drosophila Coracle, a protein
4.1 homologue, indicate that it is associated with the septate junction, a structure that has previously been implicated in the regulation of cell growth (Woods and Bryant,
1993
), and that coracle mutations interact genetically with
a hypermorphic mutation of the Drosophila Epidermal
Growth Factor-receptor homologue (Fehon et al., 1994
). In
addition, the human NF2 tumor-suppressor gene Merlin
clearly has a role in maintaining proper regulation of cell
proliferation.
In NF2, as well as in other diseases involving tumor-suppressor genes, a genetically heterozygous individual experiences somatic loss of the second copy of the gene in
certain tissues (called loss of heterozygosity or LOH), resulting in the formation of tumors in those tissues. These
individuals are then genotypically mosaic with respect to
the tumor-suppressor gene. Although NF2 was originally
characterized as a disease of the central nervous system
(CNS), somatic loss of NF2 is believed to occur in the Schwann cells that form the myelin sheath surrounding
CNS axons. The glial cells form a polarized epithelium
that isolates the axons from the surrounding environment.
Thus, although Merlin is expressed in the Drosophila CNS
(McCartney and Fehon, 1996), we have concentrated our
analysis of Drosophila Merlin function in epithelial tissues,
which also express Merlin and are readily amenable to experimental analysis.
We simulated loss of heterozygosity in Drosophila using
somatic mosaic analysis in which the effects of homozygous loss of gene function in a patch of cells (a clone) were
examined in an otherwise heterozygous background. Using this system, we demonstrated that loss of Merlin function during larval development results in a two- to threefold increase in proliferation relative to the wild-type
sister clone. This hypertrophied tissue is morphologically normal, indicating that loss of Merlin function specifically
affects proliferation rather than differentiation or morphogenesis. This conclusion is supported by the proliferation defect we observed in tissues overexpressing Merlin
Blue Box mutant proteins. Interestingly, as with human
NF2, the overproliferation we observed seems moderate and does not result in the extreme hyperplasia observed in
other Drosophila "tumor suppressors" (Mahoney et al.,
1991; Bryant et al., 1993
; Xu et al., 1995
) or in the malignant transformation associated with many human cancers.
As we demonstrated in this study, the degree of overproliferation observed was dependent on the mutant allele of
Merlin examined. Similarly, individuals with NF2 display a
range of tumor growth rates that may reflect differences in
their Merlin genotypes. In general, nonsense mutations in
human NF2 result in more severe phenotypes than missense mutations. Alternatively, it is possible that some of
the observed differences in the severity of NF2 is due to
second site modifying loci. Currently, we are using the genetic techniques available in the Drosophila system to
identify second site modifiers of Merlin mutant phenotypes.
To understand how Merlin functions in the cell and how
these functions relate to the regulation of proliferation, we
identified important functional domains within Merlin required for its activity and proper subcellular localization.
Genetic rescue experiments using a series of NH2- and
COOH-terminal truncations of Merlin indicate that all essential Merlin functions reside in the CNTR. This result,
though somewhat surprising, is consistent with recent results from other protein 4.1 superfamily members. Studies
of Drosophila Coracle, a protein 4.1 homologue, indicate
that the CNTR of this protein performs several essential
functions within the septate junction (Ward et al., 1998).
In addition, recent studies of the ERM proteins indicate
that sequences COOH-terminal to the CNTR may play a
primarily regulatory role, a model that also seems to apply
to Merlin (see below). While there are clearly family members that have a more complex functional organization, taken together these results indicate that the CNTR of
many protein 4.1 superfamily members acts as an independent functional domain. The diversity of identified protein
4.1 superfamily members suggests that during the course
of evolution, the essential function of this domain, probably to serve as a targeting sequence to a particular region
of the cell membrane, has been adapted repeatedly to different proteins (Fehon et al., 1997
).
In humans, many of the mutations associated with NF2
are predicted to generate truncated forms of Merlin as a
result of nonsense mutations (Jacoby et al., 1996; Welling
et al., 1996
). Given the results presented here, some of
these truncated products should retain partial function,
just as the mycMer1-350 allele appears to be partially functional. Although it is possible that this apparent discrepancy results from differences in human and Drosophila
Merlin function, expression of human NF2 is sufficient for
genetic rescue of lethal Drosophila Merlin alleles, indicating that their functions are conserved (McCartney, B., V. Ramesh, and R. Fehon, unpublished observations). Unlike other protein 4.1 family members (Algrain et al., 1993
;
Ward et al., 1998
), the CNTR of Merlin is poorly targeted
to the plasma membrane, thereby decreasing functional
protein levels. Thus, even when overexpressed, the CNTR
provides only partial genetic rescue. It is therefore not surprising that human NF2 alleles that truncate COOH-terminally to the CNTR produce severe phenotypes even
though the proteins they encode may retain all essential
Merlin functions. Further tests need to be performed to
confirm this hypothesis. If correct, stabilization of mutant
NF2 products containing an intact CNTR (but lacking
COOH-terminal targeting sequences) could be an effective therapeutic strategy for some patients afflicted with
NF2 because it would increase the levels of a partially
functional Merlin protein.
We have shown that mycMer1-600 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 (data not
shown). Thus, 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 et al., 1995;
Hirao et al., 1996
; Mackay et al., 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 et al., 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 (McCartney and
Fehon, 1996
), 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. It is interesting to note in this
regard that an isoform of human and mouse NF2 that alters the COOH terminus of Merlin has been identified
(isoform II; Haase et al., 1994
; Pykett et al., 1994
). Recent
studies indicate that isoform II, which retains the conserved threonine residue, is less functional than isoform I
in suppressing growth (Sherman et al., 1997
), consistent
with the idea that the COOH terminus of Merlin has regulatory functions.
Two Merlin mutations described here, the dominant-negative MerBB 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. We propose that Merlin
normally undergoes an activation process that occurs at
the cytoplasmic face of the plasma membrane (Fig. 8). In
our model, not only is Mer
BB 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 Mer
BB 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 (Berryman et al., 1995
;
Bretscher et al., 1995
). 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.
|
In this report, we have shown that Merlin is required for
the regulation of proliferation in Drosophila epithelial
cells and that it functions at the plasma membrane. The
mechanism by which Merlin functions to regulate cellular
proliferation is still unclear. Recent evidence suggests that
other proteins of the protein 4.1 superfamily operate by
organizing functional regions within the plasma membrane (Helander et al., 1996; Ward et al., 1998
). Merlin may function in a similar fashion by coordinating interactions of transmembrane signaling molecules and cytoplasmic factors that regulate cellular proliferation. We now
plan to elucidate the signaling pathways that Merlin regulates as well as identify genes involved in its regulation.
![]() |
Footnotes |
---|
Received for publication 3 March 1998 and in revised form 14 May 1998.
Address all correspondence to Richard G. Fehon, B361 LSRC, Research Drive, Duke University, Durham, NC 27708-1000. Tel.: (919) 613-8192. Fax: (919) 613-8177. E-mail: rfehon{at}acpub.duke.eduWe would like to thank Cathy Laurie for use of the camera lucida, Dan Kiehart and Kevin Edwards for providing the GFP plasmid, and the Bloomington stock center for fly stocks. We thank Team Injection for help with the generation of transgenic Drosophila lines used in this study, Stephen White for technical assistance, and our colleagues in the Fehon lab for suggestions and discussions.
This work was supported by National Institutes of Health grant (R01-NS34783) to R.G. Fehon, National Neurofibromatosis Foundation Young Investigator Awards to D.R. LaJeunesse and B.M. McCartney, and a National Research Service Award postdoctoral fellowship (F32-NS10224) to D.R. LaJeunesse.
![]() |
Abbreviations used in this paper |
---|
AEL, after egg laying;
AHS, after heat
shock;
BB, Blue Box region;
BBA, Merlin with the seven Blue Box residues changed to alanine;
CNS, central nervous system;
CNTR, conserved
NH2-terminal region;
BB, Merlin with Blue Box region removed;
ERM, ezrin-radixin-moesin;
FLP, yeast 2 micron Flipase enzyme;
FRT, Flipase
Recognition Target site;
GFP, green fluorescent protein;
Mer, Merlin;
NF2, Neurofibromatosis type 2;
S2 cells, Schneider line 2 cells;
UAS, upstream activation sequences of yeast Gal4 transcription factor.
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