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Developmental Cell, Vol. 8, 43–52, January, 2005, Copyright ©2005 by Elsevier Inc.
DOI 10.1016/j.devcel.2004.10.020
Fragile X Protein Functions with Lgl
and the PAR Complex in Flies and Mice
Daniela C. Zarnescu,1 Peng Jin,2
Joerg Betschinger,4 Mika Nakamoto,2 Yan Wang,5
Thomas C. Dockendorff,6,7 Yue Feng,3
Thomas A. Jongens,6 John C. Sisson,5
Juergen A. Knoblich,4 Stephen T. Warren,2
and Kevin Moses1,*
1
Department of Cell Biology
2
Department of Human Genetics
3
Department of Pharmacology
Emory University School of Medicine
Atlanta, Georgia 30322
4
Institute of Molecular Pathology
Dr. Bohrgasse 7
1030 Vienna
Austria
5
The Section of Molecular Cell
and Developmental Biology
The Institute for Cellular and Molecular Biology
The University of Texas at Austin
Austin, Texas 78712
6
Department of Genetics
University of Pennsylvania School of Medicine
Philadelphia, Pennsylvania 19104
Summary
Fragile X syndrome, the most common form of inherited mental retardation, is caused by loss of function
for the Fragile X Mental Retardation 1 gene (FMR1).
FMR1 protein (FMRP) has specific mRNA targets and is
thought to be involved in their transport to subsynaptic
sites as well as translation regulation. We report a
saturating genetic screen of the Drosophila autosomal
genome to identify functional partners of dFmr1. We
recovered 19 mutations in the tumor suppressor lethal
(2) giant larvae (dlgl) gene and 90 mutations at other
loci. dlgl encodes a cytoskeletal protein involved in
cellular polarity and cytoplasmic transport and is regulated by the PAR complex through phosphorylation.
We provide direct evidence for a Fmrp/Lgl/mRNA
complex, which functions in neural development in
flies and is developmentally regulated in mice. Our
data suggest that Lgl may regulate Fmrp/mRNA sorting, transport, and anchoring via the PAR complex.
Introduction
Fragile X syndrome (FraX) is the most common form of
inherited mental retardation and affects about 1/4000
males, with an average IQ below 70, attention deficit
and hyperactivity disorders, sleep disturbances, autism,
facial dysmorphia, and macroorchidism (reviewed in
O’Donnell and Warren, 2002). FraX patients have elongated neocortical dendritic spines, which represent an
*Correspondence: [email protected]
7
Present address: Department of Zoology, Miami University, Oxford,
Ohio 45056.
immature state, associated with mental retardation (Hinton et al., 1991). A similar phenotype has been reported
in FraX null mutant mice (Nimchinsky et al., 2001). The
disease is a consequence of loss of function for the
Fmr1 gene (O’Donnell and Warren, 2002). FMRP shuttles
between the nucleus and the cytoplasm and associates
with RNA via its two KH domains and an RGG box
(O’Donnell and Warren, 2002).
While mice and humans contain a three-member Fmr1
gene family, Drosophila has a single gene ortholog:
dFmr1 (also known as dfxr; for nomenclature used here,
see Experimental Procedures; Wan et al., 2000). dFmr1
mutants are viable and exhibit defects in synapse morphology and function (Lee et al., 2003; Schenck et al.,
2003; Zhang et al., 2001) and in neuronal arborization
and circadian rhythm (Gao, 2002). Fmrp acts at several
points in RNA metabolism and has been recently shown
to associate with components of the RNA interference
pathway, suggesting that it may control translation via
micro-RNAs (Brown et al., 2001; Carthew, 2002; Darnell
et al., 2001; Jin et al., 2004). Drosophila MAP1B (futsch)
mRNA is a dFmr1 target and futsch loss of function
genetically suppresses a dFmr1 loss-of-function phenotype (Zhang et al., 2001). Rac1 mRNA is also a dFmr1
target, while Rac1 protein cooperates with dFmr1 in
regulating dendrite arborization (Lee et al., 2003) as well
as synaptic morphology via its interaction with CYFIP/
Sra (Schenck et al., 2003). In summary, the modulation
of protein expression at the synapse is important for
neural plasticity, and Fmrp is thought to function in this
process as an agent for localized translational regulation.
To identify novel genes that participate in dFmr1 function, we performed a genetic screen for dominant modifiers of retinal overexpression of dFmr1 in Drosophila.
Here we report that the tumor suppressor lethal (2) giant
larvae (dlgl) interacts genetically with dFmr1 in the eye
neuroepithelium as well as at the glutamatergic neuromuscular synapse in a cooperative manner. dLgl is a
ubiquitously expressed (Strand et al., 1994b), cytoskeleton-associated protein, which contains WD-40 domains
(Betschinger et al., 2003; Plant et al., 2003). Mutations
in dlgl are lethal, and phenotypic studies have demonstrated a requirement for the gene product in various
aspects of development such as dorsal closure (Manfruelli et al., 1996), neuroblast delamination (Ohshiro et
al., 2000; Peng et al., 2000), wing development (Arquier
et al., 2001), and oogenesis (Bilder et al., 2000). dLgl
contributes to the formation of molecular scaffolds by
associating with itself (Jakobs et al., 1996) and other
molecules including nonmuscle myosin (Strand et al.,
1994a) and the PAR complex (Bilder, 2004; Strand et
al., 1994b). Not only is Lgl conserved in vertebrates, but
recent studies have demonstrated that its association
with the PAR protein complex and its phosphorylation
by atypical-PKC-zeta (aPKC-zeta) are conserved between flies and mice (Bilder, 2004). dLgl is a tumor suppressor and functions with Discs-large and Scribble in
controlling cell polarity in epithelia and neuroblasts
Developmental Cell
44
(Bilder, 2004; Klezovitch et al., 2004; Knust and Bossinger, 2002; Tepass et al., 2001). In yeast and vertebrate
cells (MDCK), Lgl homologs regulate docking and fusion
of post-Golgi vesicles with the plasma membrane
through the SNARE complex (Larsson et al., 1998; Lehman et al., 1999). In summary, Lgl is believed to contribute to cellular asymmetry via its association with (1) cell
junctional complexes and (2) the cytoplasmic transport machinery.
Here we show biochemically that Fmrp and Lgl proteins form a functional complex in flies and mice. We
also show that this complex includes specific mRNAs
and that it is developmentally regulated in the mouse.
Furthermore, we have data to suggest that the Fmrp/Lgl
complex can be regulated by the PAR protein complex.
Results
dlgl Is a Genetic Suppressor of dFmr1 Gain
of Function in the Developing
Eye Neuroepithelium
Ectopic overexpression of dFmr1 in the compound eye
under the control of a sevenless gene enhancer
(sev:dFmr1), results in a viable, dominant rough eye phenotype accompanied by retinal disorganization (Figures
1B and 1F; see Wan et al., 2000). To identify novel contributors to Fmrp function, we performed a chemical
mutagenesis screen for autosomal dominant modifiers
of the sev:dFmr1 phenotype. We screened 51,150 F1
flies and isolated 109 dominant suppressor and enhancer mutations. Of these, 63 mutations fall into 63
single-hit complementation groups and 17 are allelic to
Star (a haploinsufficient locus, often identified in dominant modifier screens based on eye phenotypes). The
rest (29 out of 109) fall into five complementation groups
with more than one allele each. Four of these complementation groups are not yet characterized. The fifth
group is notable in that we recovered 19 independent
alleles, all of which are dominant suppressors of
sev:dFmr1 and also recessive lethal. We mapped this
group to the dlgl locus (Figures 1C and 1G; Mechler et
al., 1985). As we conducted the screen, we also recovered 17 mutations in the white gene as a monitor of
genetic saturation (see Experimental Procedures). These
frequencies suggest that the screen was effectively saturating for loci at which a simple loss-of-function mutation interacts reliably with sev:dFmr1. dlgl is located
close to a telomere, and it could be that the large number
of dlgl alleles recovered in the screen might be due to
subtelomeric deletion, but DNA gel blots reveal no large
rearrangements at the locus in our lines (data not
shown). Furthermore, we confirmed that extant dlgl mutations also suppress sev:dFmr1 (Figure 1D), as quantified by scoring the number of visible photoreceptor cells
per ommatidium (see Experimental Procedures; Figures 1E–1H).
Given the known roles for Fmrp in mRNA regulation
and for Lgl in cell junctions and transport, the genetic
interaction between dFmr1 and dlgl could suggest two
possible, not mutually exclusive, models: Fmrp could
act as an intermediary between specific target mRNAs
and Lgl to mediate their targeted transport and/or anchoring at specific sites (such as synapses).
Figure 1. dlgl Mutations Are Dominant Suppressors of sev:dFmr1
Genotypes as indicated, anterior right, dorsal up.
(A–D) Adult compound eyes, scanning electron micrographs.
(E–G) Retinal sections.
(H) Histogram of rhabdomere numbers per ommatidium, percentage
of ommatidia against number of rhabdomeres as indicated. Error
bars: standard error of the mean.
(I–K) Confocal sections at the same depth within larval eye discs
stained for dFmr1 antigen, anterior right, arrowheads indicate morphogenetic furrow. Bracket below (J) and (K) indicates domain of
transgene expression as denoted by elevated dFmr1 in sev:dFmr1
(J). dFmr1 overexpression is reduced by removing dlgl dosage (K).
(L) Histogram of dFmr1 levels. Scale bars equal 50 ␮m in (A), 10 ␮m
in (E), and 20 ␮m in (I).
We first asked how we recovered dlgl alleles in the
screen: i.e., does dlgl heterozygous, loss-of-function
mitigate dFmr1 overexpression? As expected, dFmr1
protein is elevated posterior to the morphogenetic furrow in sev:dFmr1 compared to wild-type (the domain of
sevenless enhancer activity, compare Figures 1I to 1J).
When dlgl dosage is reduced, this ectopic dFmr1 protein
is reduced (Figure 1K; compare domains of transgene
expression, posterior to the morphogenetic furrow). Indeed, quantification of dFmr1 staining intensity confirmed that reduction of dlgl dosage in a sev:dFmr1
background results in a 22% decrease in signal posterior
Fmr Protein Acts with Lgl and the PAR Complex
45
to the furrow, where the transgene is expressed, normalized as an internal control to the anterior domain where
only endogenous dFmr1 is present (compare left boxes
in Figure 1J [more] to Figure 1K [less] and Figure 1L; see
Experimental Procedures). These data do not suggest a
mechanism, but show that the phenotypic suppression
observed in the screen is simply due to a quantitative
reduction in the ectopic dFmr1 protein, consistent with
dlgl acting as a dominant suppressor of sev:dFmr1.
dlgl Is a Dominant Enhancer of dFmr1
in Synapse Biogenesis
FraX patients exhibit synaptic morphology defects,
which may be interpreted as an increase in synaptic
area, consistent with a reduced ability to modify synapses with learning (O’Donnell and Warren, 2002). Similarly, in flies, dFmr1 mutations affect the neuromuscular
junction (NMJ) by increasing the synaptic area (Schenck
et al., 2003; Zhang et al., 2001). We examined the muscle
6/7 NMJ synaptic surface (Rohrbaugh et al., 2000) and
found that dlgl loss of function dominantly interacts with
dFmr1. Thus, while dlgl/⫹ and dFmr1/⫹ synapses are
essentially indistinguishable from wild-type, dlgl/⫹;
dFmr1/⫹ larvae exhibit synaptic hyperplasia (see Figures 2A–2E). This enhancement was confirmed by
counting the number of synaptic boutons, which rises
more than 2-fold in trans-heterozygote individuals
(mean 133 boutons, n ⫽ 16) compared to wild-type
(mean 61 boutons, n ⫽ 10). This interaction is statistically
significant (t test: p ⬍⬍ .001). Furthermore, these genetics are consistent with our screen. This effect is not due
to an increase in muscle size, which is unchanged (see
Experimental Procedures; data not shown). Thus, while
the genetic screen is based on a deliberate genetic artifact (the overexpression of dFmr1 in the eye), this interaction at the NMJ supports the notion that dlgl and
dFmr1 do function together in vivo.
Although dFmr1 is ubiquitously expressed, dFmr1
function is required on the presynaptic side of the NMJ
(Zhang et al., 2001). We asked if we can bypass the
requirement for dFmr1 at the NMJ by providing additional dlgl function (see Experimental Procedures). Overexpression of a full-length dlgl cDNA, either pre- or postsynaptically, results in no significant change in the
number of synaptic boutons (presynaptic: 92.7 ⫾ 2.9
[n ⫽ 10]; postsynaptic: 107.5 ⫾ 4.1 [n ⫽ 6]). This is
consistent with dlgl functioning genetically either upstream or parallel to dFmr1.
Is this epistatic relationship between dFmr1 and dlgl
also true at the molecular level? In most places, dFmr1
is uniformly expressed at low levels (D.C.Z. and K.M.,
unpublished data; Wan et al., 2000). However, in the
developing egg chamber, dFmr1 is upregulated in oocytes up to stage 9 (Figures 2F–2H). To avoid early developmental effects, we used a hypomorphic, temperaturesensitive allele of dlgl (dlglts3) to transiently reduce dlgl
function (24 hr at restrictive temperature; De Lorenzo et
al., 1999) and find that this accumulation of elevated
levels of dFmr1 protein is affected (compare arrows to
arrowheads in Figures 2F–2K). This is consistent with
the previous finding that reduction in dlgl dosage can
affect the accumulation of dFmr1 in sev:dFmr1 eye discs
(Figures 1I–1L) and that dlgl acts genetically upstream
or in parallel to dFmr1 function.
Figure 2. dlgl Acts Genetically Upstream or in Parallel to dFmr1
(A–D) Neuromuscular junctions (NMJs), segment 3, muscle 6/7.
Large (arrow) and small (arrowhead) boutons, genotypes indicated.
dlgl null allele is a significant dominant enhancer of dFmr1 (compare
[B], [C], and [D]).
(E) Histogram: number of boutons per junction, error bars: standard
error of the mean. dlgl4/⫹; dFmr13/⫹ is very highly significantly different to dlgl4/⫹ or dFmr13/⫹ alone (p ⬍⬍ .001, asterisk), while dlgl4/⫹
and dFmr13/⫹ are not statistically significant from wild-type.
(F–K) Developing egg chambers stained for dFmr1 (F, H, I, K) and
dLgl (G, H, J, K). dFmr1 accumulates in wild-type (F–H) but not in
all dlglts3 (I–K) oocytes (compare arrows to arrowheads in [F]–[K]).
Note in (J) and (K), some dLgl perdurance after 24 hr at restrictive
temperature. Scale bars equal 20 ␮m in (A) and 60 ␮m in (F).
Fmrp and Lgl Associate In Vivo to Form
a Developmentally Regulated Complex
In order to avoid potential artifacts associated with overexpression in tissue culture, we used immunoprecipitation experiments of endogenous proteins from living
animals to show that, indeed, dLgl and dFmr1 form a
protein complex in vivo (Figure 3A, asterisks). We can
immunoprecipitate each protein with an antibody to the
other, showing that the experiment is reciprocally replicable. However, as these experiments are not quantitative, we can draw no firm conclusions as to the stoichiometry of the complex. The presence of a dFmr1/dLgl
Developmental Cell
46
but considerably stronger in 2-week-old individuals, despite the overall levels of mLgl and mFmrp levels remaining apparently constant. This is the time at which
mFmrp is thought to maximally regulate synaptogenesis
in the brain (Nimchinsky et al., 2001), and thus our data
are consistent with a dynamic Fmrp/Lgl complex function in neural development and demonstrate a developmental component of Fmrp.
In order to estimate an approximate size of the dFmr1/
dLgl complex, we subjected Drosophila extracts to sucrose fractionation (see Figure 3E and Experimental Procedures). dFmr1 and dLgl comigrate in several fractions,
and based on the migration of molecular standards (data
not shown), we estimate that dFmr1 associates with
dLgl to form a large macromolecular complex (several
hundred kilo-Daltons).
Taken together, our biochemical data show that Lgl
and Fmrp are present in a common complex, in vivo, in
flies and mice.
Figure 3. Fmrp and Lgl Proteins Coimmunoprecipitate and Cofractionate with aPKC-zeta and PAR6
Visualizing antibodies indicated right.
(A) Immunoprecipitation from Drosophila heads. Input (10% of total
extract), supernatant (loaded 10%), pellet, precipitating antibody
indicated above. Note: anti-dFmr1 precipitates dLgl and anti-dLgl
precipitates dFmr1 (asterisks). Note: for clarity, five times more
dFmr1 pellet than the dLgl pellet was loaded for the dLgl blot and
five times more dLgl pellet than dFmr1 pellet was loaded for the
dFmr1 blot.
(B) FLAG-dFmr1 precipitates dLgl from fly extracts and this is reduced by competing FLAG peptide.
(C and D) Precipitating antibody indicated above; anti-mFmrp coprecipitates with mLgl in the developing mouse brain, weakly at birth
(asterisk) and more strongly at 2 weeks (two asterisks).
(E) Western blot of cellular fractions from a sucrose gradient show
dFmr1, aPKC-zeta, and PAR6 comigrating in several fractions with
and without dLgl (fractions 6–10 and 11–17, respectively). Molecular
weights: dFmr1 ⵑ82 kDa, dLgl ⵑ127 kDa, aPKC-zeta ⵑ75 kDa,
PAR6 ⵑ45 kDa.
complex was further confirmed in vitro by anti-FLAG
epitope immunoprecipitation using in vitro translated
FLAG-dFmr1 protein to specifically bring down dLgl
from a Drosophila extract (Figure 3B). A pull-down experiment using in vitro translated, tagged proteins failed
to show that dFmr1 and dLgl bind directly (data not
shown), suggesting that other (unknown) molecules may
lie between Fmrp and Lgl in vivo.
Next, we asked if this interaction identified in Drosophila is conserved in a mammal. Just as in the fly, we
can reciprocally immunoprecipitate both endogenous
mLgl and mFmrp from mouse brain extracts (Figures
3C and 3D, asterisks). Significantly, this association is
developmentally regulated: it is weak in newborn mice
Fmrp and Lgl Have Overlapping
Expression Patterns
We used immunohistochemistry to show that endogenous dFmr1 is uniformly cytoplasmic throughout the
developing fly retinal neuroepithelium (Figures 4A and
4C) while dLgl is overlapping but accumulates to higher
levels at the periphery of cells (Figures 4B and 4C).
We have also examined other tissues (ovaries, larval
neuromuscular junctions) and observed a similar result
(Figure 2 and data not shown). Thus, while the endogenous dFmr1 and dLgl proteins do not have identical
distributions, they are both present throughout the cytoplasm, at this level of resolution. It may be that some
fraction of the cellular pool of each protein (dLgl and
dFmr1) act together, while the remainder of the pool of
each protein may be in some transitional state or may
be contributing to some other cellular function(s).
Endogenous Lgl Is Reorganized
in Response to Fmrp
To further test the physical interaction between Fmrp
and Lgl in vivo, we used Mouse Embryonic Fibroblasts
(MEFs). Endogenous mouse Fmrp (mFmrp) and mouse
Lgl (mLgl) are expressed at low levels in the cytoplasm of
MEFs (Plant et al., 2003). We expressed Red Fluorescent
Protein fused to mFmrp (RFP-mFmrp) in MEFs (Experimental Procedures) and, as previously reported (De
Diego Otero et al., 2002; Mazroui et al., 2002), we find
that RFP-mFmrp assembles into perinuclear and cytoplasmic granules (arrow in Figures 4D, 4F, 4G, and 4I).
Consequently, endogenous mLgl becomes concentrated in the same particles (arrow in Figures 4E, 4F,
4H, and 4I) with most RFP-mFmrp and mLgl granules
clustered into mosaic-like complexes (asterisk in Figures 4G–4I). These data suggest that Lgl may act as a
molecular scaffold for Fmrp granules. This shows that
the endogenous dLgl protein distribution is reorganized
in response to an increased level of dFmr1 protein. In
converse experiments, Lgl overexpression has no detectable effect on Fmrp distribution (data not shown).
These data are consistent with Fmrp being normally
limiting in both systems and with Lgl protein being in
Fmr Protein Acts with Lgl and the PAR Complex
47
granules in vertebrate cells but not in the fly, possibly
because the vertebrate cultured cells are four to five
times larger, allowing the better resolution of intracellular structures, or because we may be expressing the
transgenes to different levels in the two systems.
dLgl Cofractionates with dFmr1 in a Golgi
Membrane-Associated Fraction
Mammalian Lgl plays a role in the establishment of apical-basal polarity in epithelia by sorting basolateral-specific proteins at the level of the Golgi apparatus (Bilder,
2004). In addition, Fmrp has been found to associate
with the endoplasmic reticulum (ER) in vertebrate cells
(Ohashi et al., 2002). Given our observation of mFmrp/
mLgl-containing granules in the perinuclear area of
MEFs and CAD cells, we investigated the distribution of
dFmr1 and dLgl in a membrane fractionation experiment
(see Experimental Procedures). While dLgl is widely distributed across the gradient, it cofractionates with
dFmr1 in the Golgi-associated but not ER- plasma membrane-associated fractions as determined by the presence or the absence of specific markers, respectively
(Figure 5A). These data are consistent with a dFmr1/
dLgl complex in association with the Golgi apparatus,
although a direct link remains to be proven.
Figure 4. dFmr1 and dLgl Proteins Have Overlapping Expression
Proteins below, genotypes/cell type right.
(A–C) Single confocal sections of developing eye, endogenous expression.
(D–I) RFP-mFmrp in MEFs.
(D–F) Arrow, transfected; arrowhead, untransfected cell.
(G–I) Projections of seven consecutive sections (each 1 ␮m thick)
showing that the most intensely staining mFmrp granules contain
mLgl (arrows), while more diffuse mFmrp granules do not (arrowheads); asterisk indicates a mosaic of both types of granules.
(J–O) Mouse CNS catecholaminergic (CAD) cells. Arrows indicate
colocalization of RFP-mFmrp and mLgl in granules trafficking within
developing neurites.
Scale bars equal 10 ␮m in (A), 20 ␮m in (D) and (J), and 1 ␮m in (G)
and (M).
excess (see Discussion below). We also used a cell
model for vertebrate neurogenesis (CAD cells, a CNS
catecholaminergic cell line; Qi et al., 1997). As in MEFs,
RFP-mFmrp expression results in reorganization of the
endogenous mLgl into Fmrp-containing granules (Figures 4J–4O). mFmrp/mLgl colocalizing granules are visualized both in the perinuclear region and within developing neurites, suggesting a role for Lgl in Fmrp
trafficking in these mouse cultured cells (arrows in Figures 4J–4O). Taken together, these data show that Fmrp
can recruit Lgl to a granular complex in vivo (at least
in this overexpression condition). We can detect these
dLgl Associates with a Small Subset
of mRNAs via dFmr1
It has been previously reported that mFmrp granules
colocalize with various proteins including microtubuleassociated motors as well as RNA (De Diego Otero et
al., 2002). Taken together with our data, this suggests
the possibility that Lgl might associate with at least
a subset of Fmrp containing RNPs and thus regulate
specific mRNA targets. We used Affymetrix microarrays
to identify Drosophila mRNAs that are candidate cargo
in the dFmr1/dLgl protein complex. First, we selected
those mRNAs that are consistently enriched by dFmr1mediated immunoprecipitated complex (compared to
input mRNA). To control for specificity, we eliminated
those that are equally well precipitated by an anti-dFmr1
antibody from wild-type and dFmr1 null extracts. This
list of specific dFmr1-associated target mRNA consists of 83 transcripts and includes some (but not all)
known targets of dFmr1 (see Figure 5A; Experimental
Procedures and Supplemental Data at http://www.
developmentalcell.com/cgi/content/full/8/1/43/DC1/).
Next, we selected mRNAs that are enriched in Lgl-mediated immunoprecipitation (compared to input) and we
identified a total of 78 Lgl-associated mRNAs. Of these,
9 were enriched in both dFmr1 and dLgl immunoprecipitations. Interestingly, none of the 9 dFmr1/dLgl shared
mRNAs can be precipitated by anti-dLgl antibodies from
dFmr1 null extracts, demonstrating that their association with dLgl depends genetically upon dFmr1 (see
Table 1). This experimental approach is limited by absolute mRNA abundance, and thus we are less able to
detect cargo mRNAs that are expressed at relatively low
levels (there is a detection sensitivity threshold), so we
expect that the true list of in vivo cargo mRNAs is longer.
In summary, we have detected 9 mRNAs that associate specifically in a dFmr1/dLgl complex via the dFmr1
Developmental Cell
48
Taken together, our data are consistent with dLgl as a
regulator of a specific set of dFmr1 functions.
Figure 5. dFmr1 and dLgl Cosediment with Golgi Membranes in
Density Gradients and Associate with mRNA
(A) Western blots of fractions from a linear Optiprep density gradient
show that the majority of membrane-associated Lgl and Fmr cosediment with the Golgi-associated protein Lava lamp (Lva). Portions of
membrane-associated Lgl also cosediments with the endoplasmic
reticulum (ER) marker BiP and the plasma membrane marker Toll,
fractions 2–5 (data not shown). Equal volumes of each fraction and
50 ␮g of the gradient load (P100) were analyzed.
(B) Venn diagram shows the total number of genes represented on
the chips (large square: 14,010), the number of genes present in
input (small square: ⵑ6,250), the number of mRNAs specifically
associated with dFmr1 in wild-type (83), and the number of mRNAs
enriched in dLgl IP from wild-type versus input (78). Nine mRNAs
are shared between dFmr1 and dLgl IP but unchanged in dLgl IP
versus input from dFmr1 nulls (see Table 1).
partner. Although most of these 9 genes remain to be
characterized, two of them (CG9681 and CG3348) have
been previously shown to cycle in synchrony with the
circadian clock (McDonald and Rosbash, 2001). This is
particularly interesting because dFmr1 has been demonstrated to play a role in circadian rhythms (Gao, 2002).
Lgl Functions with Fmrp in the PAR Complex
The PAR complex is essential for cellular polarity in
many taxa. Furthermore,the PAR complex acts with dLgl
in neuroblast polarity and asymmetric cell division in
Drosophila (Ohno, 2001). Within the PAR complex,
aPKC-zeta phosphorylates Lgl, thus rendering it inactive
and changing its binding properties (Betschinger et al.,
2003; Plant et al., 2003). mLgl and mPAR-6 colocalize
at the Golgi in MEFs, and in response to wounding, mLgl
and aPKC-zeta colocalize at the leading edge of the
cells where they regulate polarized migration (Plant et
al., 2003).
Does the PAR complex interact genetically and/or
physically with Fmrp? In sucrose gradients of larval extracts, aPKC-zeta and PAR-6 migrate in fractions that
contain dFmr1 and not dLgl (Figure 3E, fractions 6–10)
as well as in fractions that contain both dFmr1 and dLgl
together (Figure 3E, fractions 11–17). This is consistent
with a PAR complex association with dFmr1 and dLgl
in the higher fractions.
During oogenesis, dFmr1 accumulates in the developing oocytes, particularly at the posterior pole (Figures
2F and 6A) and colocalizes with Bazooka (Baz/PAR3;
Figures 6A and 6B). aPKC-zeta is also enriched at the
oocyte posterior, albeit in a manner restricted to the
membrane rather than throughout the oocyte cytoplasm
(data not shown; Cox et al., 2001). Furthermore, heterozygous loss-of-function mutations in both Baz (Baz4)
and PAR6 (PAR6Delta226) act as dominant enhancers of
sev:dFmr1 (Figures 6D–6F), while having no dominant
eye defects on their own (data not shown). This suggests
that PAR3/PAR6 antagonize dFmr1 function.
aPKC-zeta is a core regulator of the PAR complex
and controls synapses in Drosophila by regulating microtubule dynamics (Ruiz-Canada et al., 2004). A lossof-function allele for aPKC-zeta (aPKCk06403) genetically
suppresses the sev:dFmr1 rough eye phenotype just as
does dlgl (Figure 6D, compare with Figure 1). At the
NMJs, aPKC genetic loss of function can dominantly
suppress the synaptic hyperplasia exhibited by dFmr13
homozygote nulls (Figures 6G–6J). This result is the reverse of the gain-of-function suppression interaction
from overexpression observed in the eye and demonstrates that aPKC and dFmr1 function together in vivo.
Table 1. mRNAs Associated with dLgl via dFmr1
wt
wt/dfmr13
wt
dfmr13
Gene
dFmr1
IP/Input
dFmr1
IP
dLgl
IP/Input
dLgl
IP/Input
Biological Process
CG16969
CG4101
CG6136
CG9293
CG9681
CG14210
CG3348
CG6444
Cbl
7.6
15.0
18.3
12.0
8.2
3.3
9.1
5.3
4.14
3.5
2.5
2.5
3
2.1
2.3
2.6
2.3
2.3
4.5
6.7
8.1
6.1
5.73
2.5
5.1
3.5
7.7
NC
NC
NC
NC
NC
NC
NC
NC
NC
unknown
unknown
ion transport
apoptosis/cell cycle
immunity/circadian rhythm
unknown
circadian rhythm
unknown
cell cycle
Enrichment is shown in actual numbers (not log scale) as an average from two different experiments. NC, not changed. Biological process
as predicted or annotated in Flybase (http://www.flybase.org/).
Fmr Protein Acts with Lgl and the PAR Complex
49
We used immunoprecipitation experiments to directly
detect an aPKC-zeta/mFmrp/mLgl triple complex in the
mouse brain (Figure 6K). aPKC-zeta also associates with
dFmr1 and dLgl in flies, albeit more weakly (data not
shown).
Fmrp function can be regulated by phosphorylation
of three serine residues, which are conserved between
flies and mammals (Ceman et al., 2003; Siomi et al.,
2002). In vertebrates, phosphorylated Fmrp is associated with stalled polyribosomes, and thus phosphorylation is believed to modulate translation regulation of
target mRNAs (Ceman et al., 2003). We used a kinase
assay to show that aPKC-zeta has the ability to phosphorylate full-length dFmr1 in vitro (Figure 6L). Although
dFmr1 possesses four aPKC-zeta phosphorylation consensus sites, when aPKC-zeta is reduced more than
90%, we cannot detect clear mobility shifts in dFmr1
protein (data not shown). It remains formally possible
that aPKC-zeta acts a kinase for dFmr1 in vivo, within
special developmental or cellular contexts. This is consistent with previous work showing that aPKC-zeta can
phosphorylate Lgl as well as Crumbs protein in independent complexes (Betschinger et al., 2003; Sotillos et al.,
2004). Taken together, these genetic and biochemical
data suggest that the Fmrp/Lgl function may be in part
regulated by the interaction with the PAR complex, although the dynamics of the Fmrp/Lgl/PAR complex remains to be established.
Discussion
Here we report the identification of Lgl as a functional
partner of the Fragile X protein, Fmrp. Lgl forms a large
macromolecular complex with Fmrp, which is developmentally regulated and modulates the architecture of
the neuromuscular junction in the fly. At the cellular level,
Lgl and Fmrp are temporally and spatially coexpressed
during development and colocalize in granules in the
soma and the developing neurites of mouse cultured
cells. Fractionation experiments show that Fmrp and Lgl
comigrate with Golgi membrane-associated complexes.
Furthermore, the Fmrp/Lgl complex contains a subset
of mRNAs and interacts physically and genetically with
the PAR complex, an essential component of the cellular
polarization pathway. Our results suggest that Lgl functions with Fmrp to regulate a subset of target mRNAs
during synaptic development and/or function. We propose that Lgl may regulate Fmrp/mRNA containing
RNPs by (1) sorting at the Golgi, (2) transport in neurites,
and (3) anchoring at specific membrane domains, such
Figure 6. Fmrp Interacts with the PAR Complex
(A and B) Immunolocalization of dFmr1 and Baz show upregulation
in the developing oocyte (see arrows).
(C–F) Adult compound eyes, scanning electron micrographs (genotypes as indicated).
(G–I) Neuromuscular junctions (NMJs), segment 3, muscle 6/7. aPKC
null allele is a significant dominant suppressor of dFmr1 (compare
[G], [H], and [I]).
(J) Histogram: number of boutons per junction, error bars: standard
error of the mean. dFmr13 (102 boutons, n ⫽ 13) and aPKC06403/⫹
(81 boutons, n ⫽ 12) both show elevated bouton numbers relative
to wild-type (see Figure 2A), but this elevation is largely rescued in
aPKC/⫹; dFmr13 (66 boutons, n ⫽ 8); the difference between dFmr13
and aPKC/⫹; dFmr13 is statistically significant (p ⬍ 0.001, asterisk).
(K) aPKC-zeta associates with mFmrp and mLgl in developing
mouse brains. Precipitating antibody indicated to the left, blotting
antibody to the right.
(L) aPKC-zeta phosphorylates dFmr1 in vitro. dFmr1 expressed as
fusion with Maltose Binding Protein (MBP). Coomassie-stained gel
on top, autoradiograph on the bottom, lanes loaded as indicated.
Scale bars equal 20 ␮m in (A), 50 ␮m in (C), and 15 ␮m in (G).
Developmental Cell
50
as subsynaptic sites. The neurite transport function may
involve molecular motors such as myosin II and kinesin,
previously shown to associate with Lgl and Fmrp, respectively (Bilder, 2004; Ohashi et al., 2002). The anchoring mechanism may involve the PAR complex, which
has a demonstrated role in defining membrane domains
and has recently been shown to generate asymmetry in
the C. elegans embryo by stabilizing RNPs at the posterior pole (Cheeks et al., 2004).
Lgl Interacts Genetically and Forms a Functional
Complex with Fmrp in Neural Development
Our data demonstrate that Fmrp and Lgl form a functional complex in living neurons, and this is conserved
in flies and mice. In the mouse brain, mFmrp associates
with mLgl preferentially at a time of increased synaptogenesis, demonstrating a developmentally regulated interaction between Lgl and Fmrp. Our data suggest that
dLgl acts to regulate a subset of dFmr1-associated
mRNAs with some encoding circadian regulated molecules (CG3348 and CG9681) and some encoding secreted or transmembrane proteins (CG6136, CG4101,
CG9681) among others. dLgl also associates with mRNA
independent of dFmr1 (see Supplemental Data); thus, it
is formally possible that dLgl interacts with other RNA
binding proteins, which remain to be determined.
Fmrp has been implicated in the translational regulation of specific target mRNAs, perhaps via the RNAi
pathway (Carthew, 2002; Jin et al., 2004). Also, it has
been proposed that Fmrp is involved in the transport
and localization of mRNAs: cellular fractionation and
immunolocalization data revealed the association of
Fmrp-containing complexes with molecular motors
such as kinesin and myosin V (De Diego Otero et al.,
2002; Ohashi et al., 2002). Lgl functions in cellular polarity via regulating myosin motor activity and/or vesicle
transport (Bilder, 2001) and has been shown to regulate
polarized delivery by sorting at the Golgi (Bilder, 2004).
Taken together, these concepts and our data suggest
that Lgl may act as a scaffold for Fmrp granules, possibly
at the Golgi, and perhaps aids in carrying specific mRNA
targets to sites of locally controlled translation (such as
subsynaptic sites).
A Fmrp/Lgl/PAR Complex May Function
as a Synaptic Tag in Neural Development
It is also possible that Lgl anchors Fmrp at specific
membrane domains such as synapses, perhaps via the
PAR complex. Lgl function is regulated by the PAR complex, specifically via phosphorylation by aPKC-zeta and
by direct binding to PAR6 (Betschinger et al., 2003; Plant
et al., 2003). Our genetic interaction data suggest that
PAR6 and Baz antagonize dFmr1 function, which is in
accordance with previously published work showing
that the PAR complex inhibits dlgl (Betschinger et al.,
2003) and with our data that demonstrate that dlgl functions cooperatively with dFmr1. aPKC-zeta was shown
to antagonize most dLgl functions with the exception
of its role in regulating neuroblast apical size (Bilder,
2004), and we find our data to be consistent with such
reports. In our hands, loss of function for aPKC-zeta
suppresses gain of function sev:dFmr1 as well as the
loss-of-function phenotype of dFmr1 at the NMJs.
Taken together, these data suggest a dynamic relationship between the various members of the complex. One
possible interpretation of our results is that aPKC/PAR
can act on Fmrp directly or via Lgl depending on the
developmental and/or cellular context. Furthermore, this
is consistent with our sucrose fractionation experiments, which suggest the existence of at least two complexes comprising dFmr1/PAR proteins and dFmr1/
dLgl/PAR proteins.
The PAR complex not only functions in cell polarity,
but also at the synapse, where it is believed to function
in synaptic tagging (Martin and Kosik, 2002). Synaptic
tags have been proposed to transiently mark a synapse
after activation in a way that will translate the local
events into persistent functional changes (such as longterm depression), processes in which Fmrp is also
thought to act (Huber et al., 2002). Thus, the Fmrp/Lgl/
PAR complex may act in synaptic plasticity linking synaptic input to the remodeling of the cytoskeleton and
mediating required translational changes.
Experimental Procedures
Nomenclature
Naming conventions used here are as follows: (1) human: FMR1
(gene), FMRP (protein); (2) fly: dFmr1 (for Fmr1 gene, also known
as dfxr), dFmr1 (protein), dlgl (for l(2)gl gene), dLgl (protein); (3)
mouse: mFmrp (protein), mLgl (protein); (4) generic: Fmrp (protein),
Lgl (protein).
Drosophila Stocks, Genetic Screen, and Mapping
Genotypes: w1118; cn1; P(w, ry)E)7(94D), w1118; l(2)DTS911 nocSco/
sev:dFmr1 CyO, w1118; dlgl4/kr:GFP CyO; dFmr13/TM6B, w1118; UASdFmr1 UAS-dlgl/TM6B, baz4/FM7i, par6⌬226/FM7i, daPKC06403/kr:GFP
CyO. dlgl overexpression: UAS-dlgl/⫹; dFmr1 driven by: BG 487
(postsynaptic), elav-Gal4 (presynaptic). Genetic screen: isogenic
w1118; cn1; (P(w, ry)E)7, 25 mM EMS (Sigma), treated males crossed
to autosomally isogenic w1118; l(2)DTS911 nocSco/sev:dFmr1 CyO. New
dlgl alleles named: dlgla1 - dlgla19, a single lethal complementation
group, maps left of P(w⫹mC⫽lacW)l(2)k11324 (by meiotic recombination), left of EP(2)456 (by P-induced male recombination), fails to
complement Df(2L)TE21A, dlgl334 and dlgl275. Lethality rescued by
dlgl transgene (Ins(2R) p(l(2)gl⫹ neo30)53DE); Mechler et al., 1985).
Temperature shift for oocyte morphology: wild-type and lglts3 shifted
to 29⬚C for 24.
Microscopy and Immunohistochemistry
Statistics as previously described (Kumar et al., 2003). Wild-type:
mean rhabdomeres/facet ⫽ 6.99, standard deviation (SD) ⫽ 0.07
(188 ommatidia counted in 3 eyes), sev: dFmr1/⫹: mean ⫽ 6.06,
SD ⫽ 0.13 (331, in 3 eyes), sev: dFmr1/dlgl334 mean ⫽ 6.60, SD ⫽
0.14 (281, in 3 eyes). Polyclonal anti-Lgl antibodies as described
(Ohshiro et al., 2000). Quantitative analysis in Figure 1 by Photoshop
(Adobe): mean and SD of signal for three 50 square pixel areas
anterior to the furrow (endogenous), compared to three 100 pixel
square areas, posterior (ectopic). Reduction of dlgl dosage: 22%
decrease in signal (149.3 ⫾ 2.9 in Figure 1J, reduced to 116.3 ⫾ 2.4
in Figure 1K). Anti-dFmr1 6A15, anti-mLgl, anti-CSP, anti-mFmrp,
anti-Baz antibodies as described (Plant et al., 2003; Wan et al., 2000;
Wodarz et al., 2000; Zinsmaier et al., 1994). Rhodamine-, FITC-, and
Cy5-conjugated secondary antibodies (Jackson ImmunoResearch).
Eye discs, ovaries, and NMJs prepared as described (Kumar et al.,
2003; Rohrbaugh et al., 2000; Wodarz et al., 2000; Zarnescu and
Thomas, 1999). Analysis of boutons adapted from Rohrbaugh et al.
(2000). Surface areas of muscles 6/7 were measured by Photoshop.
Cloning, IP, and Pull-Down Experiments
IP: w1118 flies frozen in liquid nitrogen, heads collected, homogenization and processing adapted from Brown et al. (2001). Final washes
in 50 mM NaCl, 50 mM Tris (pH 7.5). Pull-down: FLAG-dFmr1 (TnT,
Fmr Protein Acts with Lgl and the PAR Complex
51
Promega), incubated with M2 Sepharose (Sigma) and fly extract,
nonspecific interactions competed with FLAG peptide (Sigma).
Mouse brain IPs as described (Brown et al., 2001). SmaI-XbaI dFmr1
cDNA fragment inserted into pCMF (Invitrogen). CFP-mlgl: gift from
T. Pawson (University of Toronto).
References
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We thank the Bloomington Drosophila stock center; V. Budnik, C.
Doe, J. Fawcet, F. Matsuzaki, T. Pawson, K. Zinsmaier, A. Wodarz,
and especially B. Mechler for reagents; R. Ordway, S. Ceman, and
especially V. Faundez for technical advice; and S. Cook, R. Airinei, D.
Saiman, M. Dawson, W. Pierre, and the Emory IM&MF for technical
assistance. We thank K. Moberg and members of the Moses lab for
comments on the manuscript. This paper is dedicated to the memory
of M. Diaconu. D.C.Z. and T.C.D. were supported by fellowships
from FRAXA. T.C.D. and T.A.J. were supported by NIH HD33834.
P.J. is supported by Rett Syndrome Research Foundation, Y.F. by
NIH 5 PO1 HD355765, and S.T.W. by NIH R37HD20521,
P01HD35576, and P30HD024064. J.C.S. supported by a March of
Dimes Basil O’Connor Award and RO1 GM067013 from the NIH.
J.A.K.’s lab supported by Boehringer Ingelheim, the Austrian Academy of Sciences, and the Wiener Wirtschaftsfoerderungsfonds.
D.C.Z. is supported by NIH F32 NS046880. This work was chiefly
supported by NIH R21 NS43536.
Received: June 23, 2004
Revised: September 19, 2004
Accepted: October 22, 2004
Published: January 3, 2005
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