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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Neurosci. Author manuscript; available in PMC 2010 October 14.
Published in final edited form as:
PMCID: PMC2881940
NIHMSID: NIHMS195663

Importin-β11 Regulates Synaptic pMAD and Thereby Influences Synaptic Development and Function at the Drosophila Neuromuscular Junction

Abstract

Importin proteins act both at the nuclear pore to promote substrate entry and in the cytosol during signal trafficking. Here, we describe mutations in the Drosophila gene importin-β11 which has not previously been analyzed genetically. Mutants of importin-β11 died as late pupae from neuronal defects and neuronal importin-β11 was present not only at nuclear pores but also in the cytosol and at synapses. Neurons lacking importin-β11 were viable and properly differentiated but exhibited discrete defects. Synaptic transmission was defective in adult photoreceptors and at larval neuromuscular junctions. Mutant photoreceptor axons formed grossly normal projections and synaptic terminals in the brain, but synaptic arbors on larval muscles were smaller while still containing appropriate synaptic components. BMP signaling was the apparent cause of the observed NMJ defects. Importin-β11 interacted genetically with the BMP pathway and at mutant synaptic boutons, a key component of this pathway, phosphorylated Mothers Against Decapentaplegic (pMAD), was reduced. Neuronal expression of an importin-β11 transgene rescued this phenotype as well as the other observed neuromuscular phenotypes. Despite the loss of synaptic pMAD, pMAD persisted in motor neuron nuclei, suggesting a specific impairment in the local function of pMAD. Restoring levels of pMAD to mutant terminals via expression of constitutively active type I BMP receptors or by reducing retrograde transport in motor neurons, also restored synaptic strength and morphology. Thus, importin-β11 function interacts with the BMP pathway to regulate a pool of pMAD that must be present at the presynapse for its proper development and function.

Keywords: Drosophila, neuromuscular junction, synapse development, importin, BMP, pMAD

Introduction

The complex and dynamic nature of neuronal circuits necessitates well-orchestrated signals to direct the development, maintenance and plasticity of synapses. These signals include both local responses to a synapse's milieu and nuclear alterations in gene transcription and neurons therefore require that the nucleus receive and interpret signals from the cells periphery (Goodman and Shatz, 1993; Greer and Greenberg, 2008).

One potential nexus for the interaction of local and nuclear signaling is the importin protein family. These proteins have a canonical function as nuclear import receptors that regulate transport from the cytoplasm to the nucleus, thereby fulfilling the need of proteins larger than 30-60 kDa to transit the nuclear pore in an actively catalyzed manner (Paine et al., 1975; Peters, 1983; Görlich and Kutay, 1999). Because nuclear entry can be a carefully regulated process for many signaling molecules that govern transcription, importins are critical to regulating neuronal properties (Otis et al., 2006; Ting et al., 2007; Perry and Fainzilber, 2009).

There are over 20 importin genes in mammals. This diversity permits the specialization of importins for particular cargo classes and the independent and tissue-specific regulation of their nuclear import (Fried and Kutay, 2003). In the classical nuclear import pathway, a cargo containing a nuclear localization signal (NLS) binds to an importin-α which in turn binds to an importin-β. The latter, via its interactions with nucleoporins, shuttles the heteromer-cargo complex into the nucleus (Görlich et al., 1995; Moroianu et al., 1995). Importin-βs however, can also promote nuclear entry by direct interaction with cargo independent of an importin-α (Görlich and Kutay, 1999). Directionality is tightly regulated by the small GTPase Ran which is GTP-bound in the nucleus where it dissociates importins from their cargo (Rexach and Blobel, 1995; Görlich and Kutay, 1999).

Importins, however, are not spatially restricted to the nucleus and can occupy roles beyond that of nuclear gatekeeper (Jakel et al., 2002; Vrailas et al., 2006; James et al., 2007). In the nervous system, particularly, they have gained significant attention for their role in synapse targeting (Ting et al., 2007) and signaling from the periphery to the nucleus (Otis et al., 2006; Perry and Fainzilber, 2009). Axonal injury causes the local translation of importin-β, which then translocates with its cargo to the soma to promote neurite re-outgrowth (Hanz et al., 2003; Perlson et al., 2005). Similarly, synaptic stimuli cause importins to move from dendrites to nuclei to effect forms of synaptic plasticity (Thompson et al., 2004; Lai et al., 2008). Importins are thus emerging as fundamental regulators of neuronal function (Otis et al., 2006; Perry and Fainzilber, 2009).

In a forward genetic screen for defective synaptic transmission, mutants were isolated in Drosophila importin-β11 (impβ11), also called ran binding-protein 11, one of 12 importins identified in the Drosophila genome. In mammalian cells, importin-β11 is required for nuclear import of the ubiquitin conjugating enzyme UbcM2 (Plafker and Macara, 2000; Plafker et al., 2004) and the ribosomal protein rpL12 (Plafker and Macara, 2002), but no neuronal functions are known. Because only one importin-β has been characterized at the Drosophila larval neuromuscular junction (NMJ), importin-β13 (Giagtzoglou et al., 2009), we undertook to characterize the impβ11 mutants. We found that loss of impβ11 caused a local misregulation of BMP signaling at the larval NMJ, which resulted in a reduction in both the number of boutons and synaptic transmission.

Materials and Methods

Fly stocks

impβ11 alleles were isolated as previously described (Dickman et al., 2005). The y,w;FRT42D GMR-hid y+ cl 2R/CyO;EGUF/EGUF parental stock used in the mutagenesis was previously described (Stowers and Schwarz, 1999).

Five independent alleles of impβ11 were isolated and mapped using the deficiencies Df(2R)ΔM22-103 (PoxnΔM22-103), a generous gift from M. Noll and W. Boll (Boll and Noll, 2002), Df(2R)Jp1 and Df(2R)Jp5 (Bloomington Stock Center, Bloomington, IN) followed by genomic sequencing to identify the precise molecular lesions. For all experiments, the control genotype was y,w; FRT42D (Xu and Rubin, 1993) while for impβ11 mutants, the impβ1170 allele was used in trans to the genomic deficiency Df(2R)ΔM22-103.

UAS-impβ11-eGFP was generated by cloning the full-length cDNA (EST, LD41918; Invitrogen, Carlsbad, CA) into the pUAST vector (Brand and Perrimon, 1993) using PCR-added BglII and KpnI sites. eGFP was ligated in frame into the pUAST-impβ11 construct using engineered KpnI and XbaI sites. Transgenic flies were generated by standard transformation methods (Rubin and Spradling, 1982).

All fly stocks were raised in humidified incubators at 25°C. Third-instar larvae were grown in low-density cages at 25°C on agar grape plates with yeast paste.

The following stocks were also used: elav-Gal4 (Luo et al., 1994); da-Gal4 (Wodarz et al., 1995); G14-Gal4, OK6-Gal4, witA12, witB11, witHA1 (Aberle et al., 2002); nsybF-33R (Deitcher et al., 1998); MAD10 (Sekelsky et al., 1995); UAS-gbb9.1 (S. Thor; Wharton et al., 1999); UAS-tkvQ253D (mobilized onto X by D. Allan; Nellen et al., 1996); UAS-sax Q263D (mobilized onto X by D. Allan; Haerry et al., 1998); UAS-GFP-MAD (Dudu et al., 2006); UAS-dynamitin (D. Allan; Duncan and Warrior, 2002); tubulin-GAL4 (Lee and Luo, 1999); UAS-wit (Marques et al., 2002). Stocks were obtained from the Bloomington Stock Center (Bloomington, IN) where available.

Antibody Production

A PCR fragment with flanking 5′ EcoRI and 3′ AvaI sites encoding importin-β11 amino acids 923 to 1075 was amplified and cloned into the pGEX-4T-1 vector (Amersham Biosciences, Fairfield, CT) using the following primers: 5′ GST-RanB2- GAA TTC GGC GAA GTG ATG GAC AA 3′ GST-RanB2- GAG CTC CGG CCT GAG GTG GAC AA. The fidelity of the clone was verified by sequencing throughout the entire open reading frame. Subsequently, the 51 kDa GST fusion protein was expressed in and purified from E. coli BL21 cells using the Bulk GST Fusion Purification Module according to the manufacturer's protocols (Amersham Biosciences, Fairfield, CT). The resultant protein was injected into naïve New Zealand white rabbits (Covance, Denver, PA). Crude antisera were then affinity purified over an Affi-Prep 10 column (Bio-Rad, Hercules, CA) containing the original fusion protein to which the antibody was raised. Affinity-purified antibody was eluted from the column by standard methods (Harlow and Lane, 1988) in 100 mM glycine, pH 2.3 and neutralized in 1 M Tris-Base, pH 7.5. The antibodies were then dialyzed with PBS containing 5% BSA. Antibodies were tested by recognition of a 110 kDa band by Western blot corresponding to the predicted molecular weight of importin-β11 that was absent in null mutants. One antibody, 5157, was found to recognize endogenous and exogenous importin-β11 and was used for subsequent studies.

Germline clones

The following stocks and genotypes were used.

  • y,w, hs-FLP; FRT42D Imp-β1170 / CyO
  • y,w, hs-FLP; FRT42D
  • y,w; FRT42D Imp-β11 70 / Cyo, GFP [CyO, P(ActGFP)]
  • y,w; FRT42D
  • w[*]; P{w[+mW.hs]=FRT(w[hs])}G13 P{w[+mC]=ovoD1-18}2R/Dp(?;2)bw[D], S[1] wg[Sp-1] Ms(2)M[1] bw[D]/CyO; +; + (BL 2125)
  • y,w, hs-FLP / y,w; FRT42B ovoD / FRT42D Imp-β1170
  • y,w, hs-FLP / y,w; FRT42B ovoD / FRT42D

Germline clones were generated using the dominant female sterile (DFS) technique using the ovoD mutation and a source of FLPase under the control of a heat-shock promoter (Chou and Perrimon, 1992, 1996). In experimental conditions, FRT42B ovoD bearing males were crossed to y,w, hs-FLP; FRT42D impβ1170/CyO or virgins. The progeny were heat-shocked at 37°C starting in the second instar for 1 hour a day for 6 consecutive days to induce recombination. Though the recombination of 42B and 42D is expected to cause a chromosomal deletion between these two loci, this deletion has been previously shown to have no impact on the production of viable embryos (Murthy et al., 2005). Indeed, control crosses using the parental 42D chromosome resulted in embryos that developed into normal adults. Resultant virgin female adults of the genotype y,w, hs-FLP/y,w; FRT42B, ovoD/FRT42D, impβ1170 were then mated to y,w; FRT42D, impβ11 70/Cyo, GFP [CyO, P{ActGFP}] with the intent to select against GFP-bearing balancer chromosomes to ensure both a germline null and a zygotic null. Results were confirmed independently using the impβ1120 allele.

Immunostaining

Third-instar larval fillets were fixed in 4% paraformaldehyde for 20 minutes. Antibody staining was performed in PBS containing 0.3% Triton-X-100 and 5% normal donkey serum. Fillets were mounted in Vectashield (Vector Labs; Burlingame, CA). Adult recombinant heads were prepared as described (Stowers et al., 2002) with the exception of fixation conditions (3.7% formaldehyde in 100 mM potassium phosphate (pH 6.8), 450 mM KCl, 150 mM NaCl and 20 mM MgCl2) and sectioned using a Leica CM3050S cryostat (Wetzlar, Germany). For comparisons, animals were stained concurrently, imaged and processed identically.

The following antibodies were used: anti-Chaoptin 24B10 1:100 (Developmental Studies Hybridoma Bank, DSHB); rabbit anti-Syt I, 1:4,000 (Mackler et al., 2002); mouse anti-BRP, nc82, 1:100 (Hofbauer et al., 2009a); mouse anti-GluRIIA, 1:100 (DSHB); rabbit anti-GluRIIB, 1:2500 (Marrus et al., 2004); rabbit anti-GluRIIC, 1:2000 (Marrus and DiAntonio, 2004); rabbit anti-pMAD, PS1, 1:2000 (Persson et al., 1998); mouse anti-FasII, 1D4, 1:20 (DSHB); mouse anti-Futsch, 22C10, 1:100 (DSHB); rabbit anti-Tkv, 1:1,250 (McCabe et al., 2004); mouse anti-Wit, 23C7, 1:10 (Aberle et al., 2002; Wang et al., 2007); goat anti-MAD, 1:100 (Santa Cruz Biotechnology, Santa Cruz, CA; Zhu et al., 1999; Li and Li, 2006); Cy5- or FITC-conjugated goat anti-HRP, 1:100 (Jackson Immunoresearch, West Grove, PA); Alexa Fluor 594 conjugated WGA at 5 μM (Invitrogen-Molecular Probes, Carlsbad, CA); FITC or Cy3 conjugated secondary antibodies, 1:200 (Jackson Immunoresearch).

Imaging and analysis parameters

All images were processed using a Zeiss LSM 510 Meta laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) and either a 63×, 1.4 NA or 40×, 1.0 NA oil immersion objective. LSM software and Adobe Photoshop (Adobe Systems, San Jose, CA) were used for image processing. For bouton quantification, larvae were stained with antibodies to HRP and boutons counted on muscles 6 and 7 in segment A2. Muscle surface area was calculated from 10× DIC micrographs of muscle fields; length and width of each muscle was used to calculate a surface area. Statistics were performed as repeated measurement ANOVA tests with a posthoc Dunnett's multiple comparison test between all combinations of genotypes. Bruchpilot (BRP), GluRIIA, GluRIIB and GluRIIC puncta were manually counted at MN4b synapses on muscle 4. Terminal area was calculated using the threshold function in Metamorph for the HRP channel. Statistical analysis was conducted using GraphPad Prism 5 software (Graphpad Software, La Jolla, CA) and significance values calculated using an unpaired student's t-test. All histograms and measurements are shown as mean ± SEM; sample size (n) is described either in the figure legend or in the results section.

Electrophysiology

NMJ recordings were performed as previously described (Dickman et al., 2005). Briefly, recordings were conducted in HL3 (Stewart et al., 1994) saline containing 20 mM Mg2+ and either 1mM Ca2+ or 0.6mM Ca2+ as indicated. All physiology was recorded from muscle 6 in abdominal segments A2 to A4. Recordings conducted on muscles with Vm ≤ -55 were included for analysis. Quantal content was corrected for non-linear summation (Martin, 1955).

ERGs were performed as described (Dickman et al., 2005). HL3 saline solution was used in the stimulating electrode placed in the thorax of the adult fly. The recording electrode was filled with 3 M KCl and placed on the surface on the eye. Light stimuli lasted one second.

Philanthotoxin homeostasis experiments were conducted as described (Goold and Davis, 2007). A final concentration of 4 μM Phtx in 1 mM Ca2+ HL3 saline was applied to a semi-intact larval preparation for 10 minutes. The PhTx was washed out and recordings were made as above.

Statistics were calculated using ANOVA followed by a Bonferroni post-comparison test (GraphPad Prism, La Jolla, CA). All histograms and measurements are shown as mean ± SEM; sample size (n) is described either in the figure legend or in the results section.

Electron Microscopy

Laminas innervated by either control or impβ11 mutant photoreceptors, were prepared for electron microscopy (EM) using previously reported methods (Meinertzhagen, 1996). Single sections containing cartridge profiles cut in fair cross-section were examined and digital montages collected from images obtained with a Philips Tecnai 12 electron microscope operated at 80 kV, using a Kodak Megaview II camera with software (AnalySIS: Soft Imaging System GmbH, Münster, Germany).

We made counts of the following organelle profiles: ~30-nm synaptic vesicles; T-bar presynaptic ribbons at tetrad synapses; and glial invaginations into R1-R6 terminals, called capitate projections. The perimeters and cross-sectional areas of terminal profiles were measured with software (ImageJ: NIH) to determine the packing density of synaptic vesicle profiles. Tests for statistical significance in organelle counts were first made by an unweighted means ANOVA, followed by a Tukey HSD test, using software (Systat 5.2.1). Error bars in figures are mean ± SEM.

Immunoblots and SDS-PAGE Analysis

For immunoblots, extracts were made from timed embryo collections or from intact third-instar larvae by drop-freezing specimens in liquid N2 and homogenizing them directly in 2× Laemmli buffer (Laemmli, 1970). Synaptosome fractions were prepared from adult heads as described (Kelly, 1983) in Synaptosome Buffer (10 mM HEPES, pH 7.5, 1 mM MgCl2, 0.32 M sucrose). Proteins were separated on 8% polyacrylamide gels and transferred to nitrocellulose membranes. Primary antibodies were applied overnight at 4°C and secondary antibodies for 1 hour at 21°C. The following antibodies were used: rabbit anti-importin-β11, 1:7500 (this study); mouse anti-Bruchpilot, 1:100 (Hofbauer et al., 2009b); mouse anti-Lamin C LC28.26 and anti-Lamin Dm0 ADL101, 1:5000 (Riemer et al., 1995), and mouse anti-α-tubulin 1:25000 (Sigma-Aldrich, St. Louis, MO). HRP-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were used at 1:10000 (anti-mouse) or 1:20000 (anti-rabbit). Blots were developed using the SuperSignal West Dura Extended Duration Substrate Kit (Thermo Scientific, Waltham, MA).

Fluorescence Intensity Measurement

Fluorescence from the neuronal marker anti-HRP was used to generate a region of interest (ROI) around the synaptic terminal using the thresholding function. Integrated fluorescent intensity was then measured within the ROI in arbitrary units and divided by the area of the ROI. Metamorph software (Molecular Devices, Downingtown, PA) was used for all measurements and analyzed using ANOVA followed by a Bonferroni post-comparison test or student t-test analysis (GraphPad Prism, La Jolla, CA). All histograms and measurements are shown as mean ± SEM.

Results

Photoreceptor neurotransmission defects in impβ11 mutants

A lethal complementation group consisting of five impβ11 mutant alleles (70, 9, 24, 20 and 202) was isolated in a forward genetic screen that has previously yielded a collection of mutants with defective synaptic development and transmission (Stowers et al., 2002; Dickman et al., 2005; Pack-Chung et al., 2007; Dickman et al., 2008). This screen examined flies whose eyes were completely homozygous for a mutated chromosome arm 2R while the rest of the fly was heterozygous for that chromosome (Stowers and Schwarz, 1999). A primary screen selected flies that failed to perform phototaxis and was followed by a secondary screen for abnormalities in the electroretinogram (ERG). Each allele of the complementation group described here failed to complement the X-ray induced deficiency Df(2R)ΔM22-103 (hereafter referred to as Df; generous gift from W. Boll and M. Noll). With partially overlapping deficiencies (Fig. 1A), the location of the alleles was restricted to an area bordered by the genes vha36 and poxn in the cytological region 52C. Sequencing of candidate genes revealed nonsense mutations in all five alleles in impβ11, whose closest homologs include human importin-β11 and yeast Kap120p / Lph2p sharing 34% and 23% amino acid identity, respectively (Plafker and Macara, 2000; Caesar et al., 2006). The sequence of impβ11 predicts a protein of 1075 amino acids. At the amino-terminus, it possesses a conserved Ran-binding domain characteristic of all importin-βs (Fig. 1B, RBD). This region is followed by a long carboxy terminal region that shares homology only with the β11 class of importins. Three alleles introduced a stop codon before or just at the beginning of the Ran-binding domain (Fig. 1B). Given that the Ran-binding domain is essential for nuclear import and most of the protein is absent, these are likely null alleles. The remaining two alleles, although independently derived, introduced stop codons at the same position, amino acid 853 (Fig. 1B). All alleles when placed over the deficiency (Df) failed to produce a full-length protein (Fig. 1E).

Figure 1
impβ11 mutants have defective photoreceptor neurotransmission but normal projections

When these alleles were made homozygous selectively in the eye (Stowers and Schwarz, 1999), their ERGs lacked ‘on’ and ‘off’ transients in response to brief exposures to light (Fig. 1C, arrows). These transients are reported to arise from the synchronous activation of second order cells in the visual system (Coombe, 1986); their absence indicates a failure of normal transmission by the photoreceptors. In contrast, the persistence of the sustained negative phase of the response indicates that the phototransduction cascade remained intact (Heisenberg, 1971). The 4 additional alleles displayed a similar ERG profile (data not shown; n ≥ 4). Thus, the impβ11 mutants were defective at a step downstream of the depolarization of the photoreceptor but upstream of activating the target neurons. We were able to fully restore the ‘on’ and ‘off’ transients by specifically expressing a UAS-impβ11-eGFP transgene in the photoreceptors by means of ey-GAL4 (Fig. 1C; Brand and Perrimon, 1993; Hazelett et al., 1998; Stowers and Schwarz, 1999). The successful rescue indicates that the ERG defect resulted from loss of importin-β11 and not from potential second-site mutations.

The ERG defect could have arisen from a functional defect in transmission or a developmental defect in the connections made by the photoreceptors. To analyze the photoreceptor axon projections, adult heads with homozygous mutant eyes were sectioned at 10 μm with a cryostat and photoreceptors were immunolabeled with a photoreceptor-specific antibody against Chaoptin. No gross abnormalities were detected in these projections: R1-R6 endings appeared to terminate appropriately in the lamina, and normal terminals appeared in the layers of the medulla that receive the endings of photoreceptors R7 and R8 (Fig. 1D). In electron micrographs of the lamina from animals with homozygous mutant photoreceptors (y,w;FRT42D, impβ1170/FRT42D, GMR-hid, cl;EGUF/+), the ordered structure of the cartridges containing endings of the R1-R6 photoreceptors appeared normal, and synapses were identified with appropriate T-bar ribbons, presynaptic densities and clusters of vesicles. Small clear vesicles were normal in size but more numerous at mutant terminals (Supplemental Fig. S1A and B), a phenotype consistent with a decrease in vesicle release (Hiesinger et al., 2006). The cross-sectional area of the terminal profile was also significantly increased resulting in normal vesicle density (Supplemental Fig. S1C and D). In addition, capitate projections were almost absent from mutant terminals, a phenotype that has previously been interpreted to result from decreased endocytosis, a likely consequence of decreased vesicle release (Supplemental Fig. S1E; Fabian-Fine et al., 2003).

Importin-β11 expression in the larval nervous system

Each impβ11 allele, when placed over the Df, was lethal at the pharate adult stage, indicating that its function was not restricted to photoreceptors. To determine the developmental profile of importin-β11, we generated an antiserum (5157) to amino acids 923 to 1075 (Fig. 1B). On immunoblots of homogenates of third-instar larvae, this antiserum detected a band of the predicted molecular weight, 110 kDa, that was absent from impβ1170/Df (Fig. 1E). Non-specific bands of 70 and 60 kDa were also observed that could not be removed by affinity purification.

Importin-β11 protein was detected throughout the life cycle in wild-type animals, including lysates from embryos aged zero to two hours after egg deposition, a stage prior to initiation of zygotic transcription (Fig. 1F; Anderson and Lengyel, 1979). In addition, lysates of homozygous Df embryos had a detectable band at 110 kDa (Fig. 1F). Because this deficiency removes the entire impβ11 gene, this early expression must result from maternally contributed mRNA or protein. We reasoned that maternally derived importin-β11 might mask the requirements for this protein prior to the pupal stage. Therefore, we used the ovoD method (Chou and Perrimon, 1992, 1996) to generate maternal germline clones homozygous for the impβ1170 and impβ1120 alleles. Mutant females did not lay eggs, and on examination of mutant ovarioles, the ovoD phenotype was evident, consistent with the hypothesis that importin-β11 plays an essential role in the female germline (Chan et al., 2008). Hereafter, unless otherwise notated, late third-instar larvae of genotype y,w;FRT42D, impβ1170/Df were used for experiments and will be referred to as impβ11 null larvae. At this stage, maternally supplied importin-β11 was no longer detectable by Western blot in this genotype (Fig. 1E).

Third-instar larvae offered us the advantage of using homozygous mutant animals to characterize the phenotype at the neuromuscular junction (NMJ), a synapse amenable to both anatomical and electrophysiological analysis (Schwarz, 2006). Identifiable motor neurons exit the larval ventral nerve cord (VNC) and innervate specific muscles in each hemisegment of the body wall, where they form synaptic varicosities or boutons (Keshishian et al., 1996).

To examine importin-β11 immunoreactivity at these NMJs, body wall preparations were labeled with antiserum 5157 (Fig. 2A-F). In control body walls, muscle nuclei were immunoreactive and the protein was enriched in puncta on the nuclear envelope, presumably corresponding to nuclear pores (Fig. 2B). This immunoreactivity was absent in impβ11 mutants (Fig. 2C and D). The antibody also labeled the NMJ (Fig. 2A and E), but this signal was not exclusively due to importin-β11 because it was also present at mutant NMJs (Fig. 2C and F). To circumvent this problem, we created a transgenic line (UAS-impβ11–eGFP) expressing importin-β11 fused to an enhanced green fluorescent protein (eGFP) and placed under the control of a UAS promoter (Brand and Perrimon, 1993). When expressed either ubiquitously (with a da-GAL4 driver; Wodarz et al., 1995), or exclusively in the nervous system (with an elav-GAL4 driver; Luo et al., 1994), but not when expressed in muscle (with a G14-GAL4 driver; Aberle et al., 2002) this construct rescued the lethality of impβ11 null larvae, indicating both that the transgene was functional and that lethality at the pupal stage was due to an essential neuronal function of importin-β11.

Figure 2
Importin-β11 localization in third-instar larvae

In the nervous system, importin-β11-eGFP protein localized to puncta in axons, to the cytoplasm and nuclei of many cells in the VNC, including motor neurons, and in the neuropil, the synaptic region of the VNC (Fig. 2G-I). As expected for a nuclear import factor, at high magnification, importin-β11-eGFP was observed at the nuclear envelope of these neurons where it concentrated at nuclear pores (Fig. 2J). Consistent with the importin-eGFP signal in nerves and the synaptic neuropil, neuronally expressed importin-β11-eGFP was also present at presynaptic neuromuscular terminals (Fig. 2K and L). The fluorescent importin-β11, however, was only prominent in approximately 30% of body wall NMJs in any given larva and filled some but not all boutons within a given synapse (Fig. 2L). This irregular distribution likely reflects the pattern of expression of the GAL4 line elav-GAL4, the insertion site of the UAS-impβ11–eGFP transgene, or a combination of both. A stronger, ubiquitous driver (tubulin-GAL4; Lee and Luo, 1999) caused importin-eGFP to be visible in all boutons within a given NMJ (Fig. 2M). Further, because the NMJ at muscles 6/7 contains the terminals of two axons, we also examined the simpler, singly innervated muscle 4 (Fig. 2N; Lnenicka and Keshishian, 2000; Hoang and Chiba, 2001). Here too, all boutons were positive for eGFP. These data suggest that importin-β11 can localize to synaptic terminals in addition to the synapse-rich region of the VNC, nuclear pores, cell bodies, and muscle nuclei. We also observed importin-β11-eGFP localization to muscle nuclei and trachea when expressed ubiquitously (Fig. 2M and N). To address whether endogenous importin-β11 could also localize to synapses, we performed a subcellular fractionation of adult head lysates (Kelly, 1983). As expected, importin-β11 immunoreactivity was detected in the nuclear fraction, on long exposure, but it also appeared in the synaptosome fraction and in the cytosol (Supplemental Fig. S2). Thus, importin-β11 is likely to mediate import through nuclear pores in both neurons and muscle cells, but may also have a function at synaptic terminals.

Structural and functional defects at the impβ11 mutant NMJ

Because we could detect a synaptic pool of importin-β11, we next examined the NMJ for potential structural and developmental phenotypes by counting the number of boutons present at the NMJ in wild-type and impβ11 mutant larvae. Although the size of their muscle fibers was normal, impβ11 mutant larvae had a 30% reduction in bouton number when compared to either control larvae or heterozygote mutant larvae (Fig. 3A and B). Bouton counts are frequently expressed normalized to muscle surface because bouton number is known to scale with muscle size during development (Lnenicka and Keshishian, 2000; Marques et al., 2002). Thus, normalized to account for variations in larval size, impβ11 null mutants showed a similar 30% decrease in bouton density (Fig. 3B and Table 2). Synaptic footprints, a sign that boutons have retracted (Eaton et al., 2002), were scant in both mutant and control (control = 0 footprints in ~300 boutons; impβ11 = 2 footprints in ~500 boutons). Therefore, the altered bouton number likely reflects a reduction in bouton addition as the terminal developed.

Figure 3
Reduced bouton number and quantal content in impβ11 mutants
Table 2
Bouton number per 1000 μm2 muscle surface area in various heterozygous combinations

Inputs from both type Ib (large) and type Is (small) boutons contribute to the synaptic arbor and electrophysiological profile (Johansen et al., 1989; Lnenicka and Keshishian, 2000). There was no preferential loss of Ib or Is boutons in impβ11 NMJs, but arbor length and bouton diameter were both slightly decreased when compared to control (data not shown). In addition, the geometry of the synapse was altered in impβ11 mutants with axonal branches remaining close to the inter-muscular cleft rather than spreading across the width of the muscle (Fig. 3A).

To determine if the reduction in bouton number was primarily the result of pre- or postsynaptic defects, we expressed UAS-impβ11-eGFP in all tissues (with a da-GAL4 driver), in neurons (with an elav-GAL4 driver) or in muscle (with a G14-GAL4 driver). The reduction in bouton number/muscle surface area was statistically rescued to control values when impβ11-eGFP was expressed in all cells and in neurons alone, but not by expression in muscle alone (Fig. 3B control = 0.88 ± 0.048/1000 μm2, impβ1170/Df = 0.62 ± 0.044/1000 μm2, all cells rescue = 0.93 ± 0.036/1000 μm2, neuron rescue = 0.92 ± 0.034/1000 μm2, muscle rescue = 0.61 ± 0.029/1000 μm2; n ≥ 17 for each genotype). Thus, importin-β11 is primarily required presynaptically for normal bouton formation and arborization.

To determine if these structural defects were accompanied by electrophysiological defects, we stimulated the motor nerve that innervates muscle 6 to evoke excitatory junctional potentials (EJPs). Consistent with the morphological defects, the amplitude of EJPs was reduced by 40% in the mutant larvae as compared with control (Fig. 3C and E; control = 35.6 ± 1.08 mV, impβ11 = 21.1 ± 1.81 mV; n ≥ 12 for both genotypes). Vesicles also fuse spontaneously at the NMJ giving rise to quantal events or miniature excitatory junctional potentials (mEJPs). mEJPs were also altered (Fig. 3D and F,G); impβ11 released vesicles at a lower frequency about 65% less than control (Fig. 3F control = 3.34 ± 0.367 Hz, impβ11 = 1.19 ± 0.171 Hz; n ≥ 11 for both genotypes). This decrease could, at least in part, reflect fewer release sites in the smaller synaptic arbor. There was also a small, but statistically significant, decrease in the amplitude of the mEJPs (Fig. 3G control = 0.95 ± 0.052 mV, impβ11 = 0.77 ± 0.035 mV; n ≥ 11 for both genotypes) suggesting that impβ11 mutants may have a mild reduction in postsynaptic receptor density or sensitivity (Petersen et al., 1997; DiAntonio et al., 1999). As calculated from these data, quantal content, or the number of vesicles released per action potential, was reduced by 30% in impβ11 (Fig. 3H control = 43.3 ± 2.40, impβ11 = 30.7 ± 2.82; n ≥ 10 for both genotypes) when corrected for non-linear summation (Martin, 1955).

Defects in synaptic transmission can result from either pre- or postsynaptic mechanisms. We therefore selectively restored function of importin-β11 using UAS-impβ11-eGFP in either neurons or muscle. The observed defects in quantal content and EJP amplitude in impβ11 were fully rescued with expression of the UAS-impβ11-eGFP transgene in the nervous system with elav-GAL4 (Fig. 3E EJP amplitude - neuron rescue = 32.3 ± 1.71 mV, muscle rescue = 27.1 ± 1.11 mV; Fig 3H quantal content - neuron rescue = 50.1 ± 2.71, muscle rescue = 39.7 ± 1.98; n ≥ 10 for each genotype). Although quantal content was somewhat increased by transgene expression in muscle, it did not correspond to a statistically significant restoration of EJP amplitude. In contrast, neuronal rescue was significant and complete indicating that neuronal expression of importin-β11 was both sufficient and necessary to rescue the electrophysiological defect (P values: compared to control - neuron rescue p ≥ 0.05, muscle rescue p < 0.0001; compared to mutant – neuron rescue p < 0.0001, muscle rescue p ≥ 0.05). Separately, neither neuronal nor muscle expression of importin-β11-eGFP was adequate to restore mEJP frequency or amplitude to control levels, however concurrent expression in both neurons and muscle significantly increased mini frequency (Fig. 3F neuron rescue = 1.64 ± 0.250 Hz, 0.73 ± 0.033 mV; muscle rescue = 1.55 ± 0.314 Hz, 0.76 ± 0.035 mV; all cells rescue = 3.02 ± 0.546 Hz, 0.64 ± 0.025 mV; p ≥ 0.05 when da-GAL4 frequency was compared to control; n ≥ 11 for each genotype). mEJP amplitude was not rescued, and may be particularly sensitive to appropriate expression levels of the importin. Thus, presynaptic importin-β11 was sufficient to restore bouton number and EJP amplitude suggesting that importin-β11 at the presynapse was required for normal NMJ arborization and function.

To characterize further impβ11 abnormalities at the NMJ, we examined proteins of known importance to the development and function of this synapse. Several structural components were investigated that could potentially contribute to the phenotype. However, both the cell-adhesion molecule, Fasciclin II (FasII), and the microtubule-associated protein Futsch (Schuster et al., 1996; Hummel et al., 2000; Roos et al., 2000) localized normally in impβ11 mutant NMJs (Supplemental Fig. S3). The reduction in bouton number was therefore not likely to arise from gross abnormalities in these structural components. Similarly, Synaptotagmin I (Syt I) immunolabeling indicated that synaptic vesicles were concentrated within boutons (Fig 4A; DiAntonio et al., 1993) and contained slightly more Syt I reactivity as measured by fluorescence intensity (Supplemental Fig. S4). NMJs were also labeled with the monoclonal antibody nc82, which recognizes the active zone component Bruchpilot (BRP; Kittel et al., 2006; Wagh et al., 2006; Hofbauer et al., 2009a). The density and number of BRP puncta per bouton in impβ11 mutant NMJs were unchanged (Fig. 4B; control = 0.70 ± 0.029 puncta/μm2, impβ1170/Df = 0.79 ± 0.035 puncta/μm2; n ≥ 13 for both genotypes). However, because there were fewer boutons per NMJ, there was a net decrease in total active zone number and this loss of release sites was likely an important factor in the reduction in synaptic strength.

Figure 4
Synaptic pMAD is reduced in impβ11 mutants but other synaptic proteins appear normal

The impairment in mEJP amplitude suggested that there might have been an alteration in postsynaptic glutamate receptors (Petersen et al., 1997; DiAntonio et al., 1999; Marrus et al., 2004). To examine receptor density and localization, we therefore undertook immunolabeling for GluRIIA, GluRIIB and GluRIIC, but all appeared normal in the impβ11 mutant NMJs. The intensity of the labeling was unchanged and clusters of receptors properly localized beneath active zones (Fig. 4C and Table 1). Thus, within the reduced synaptic arbor of impβ11 mutants, each bouton retained active zones with synaptic vesicles and faced a postsynaptic membrane with properly clustered glutamate receptors.

Table 1
Quantification of glutamate receptors

Reduced pMAD at impβ11 NMJs

The quantitative reduction in bouton number suggested that impβ11 mutants may be defective in one of several signaling pathways known to modulate the development of this synapse (Collins and DiAntonio, 2007). These include the BMP pathway, the Wnt pathway and the mitogen-activated protein kinase (MAPK) pathways (Marqués, 2005; Collins et al., 2006; Speese and Budnik, 2007). In particular, disruption of the BMP pathway reduces bouton number and EJP amplitude, and thus resembles the impβ11 phenotype. At the NMJ, secretion of the ligand, Glass bottom boat (Gbb), activates the BMP pathway by causing the receptor complex composed of Wishful thinking (Wit), Thickveins (Tkv) and Saxophone (Sax) to phosphorylate the signaling molecule MAD (Mothers Against Decapentaplegic; Keshishian and Kim, 2004). The C-terminally phosphorylated form, pMAD, is abundant both at the NMJ and in motor neuron nuclei and therefore may act locally or by retrogradely translocating to the nucleus. (Allan et al., 2003; McCabe et al., 2003; Marqués, 2005). The pathway is likely to function bidirectionally, with some pMAD generated in the muscle as well as in the nerve terminals (Dudu et al., 2006; Collins and DiAntonio, 2007). Recently, several studies have identified proteins that interact with and modulate the BMP pathway at the Drosophila NMJ (Wang et al., 2007; O'Connor-Giles et al., 2008; Merino et al., 2009). We therefore examined the distribution of pMAD as a read-out of the activity of this pathway and its potential interaction with importin-β11.

In control animals, pMAD immunoreactivity (Persson et al., 1998) was present both at the NMJ, overlapping with the neuronal marker HRP, and in motoneuron nuclei (Fig. 4D and F). In impβ11 mutants, pMAD still accumulated in motor neuron nuclei (Fig. 4G), but synaptic pMAD was markedly reduced at the NMJs of all muscles (Fig. 4E and quantified in Supplemental Fig. S5). This phenotype is distinct from that of wit mutants, which lose both nuclear (Marques et al., 2002) and synaptic pMAD (Supplemental Fig. S6).

Like the localization of pMAD itself (Dudu et al., 2006; Collins and DiAntonio, 2007), the reduction in synaptic pMAD in impβ11 appeared to have both pre- and postsynaptic components. Restoration of importin-β11 expression to the nervous system, by means of elav-GAL4 or ubiquitous expression with da-GAL4, was sufficient to restore pMAD immunolabeling to the nerve terminals to control levels (Fig. 4H, I and quantified in Supplemental Fig. S5). Expression of the transgene in muscle with G14-GAL4, however, was also capable of increasing pMAD immunoreactivity to the mutant NMJ, but to a lesser extent than neuronal or ubiquitous rescue (Fig. 4J and quantified in Supplemental Fig. S5). The NMJ phenotypes of MAD mutants and other genes in the BMP signaling pathway are known to arise from presynaptic requirements for this pathway (Aberle et al., 2002; McCabe et al., 2004). Because bouton number, EJP amplitude and pMAD phenotypes of impβ11 were rescued to control values with presynaptic restoration of importin-β11, loss of specifically presynaptic pMAD is also the most likely explanation of the anatomical and electrophysiological defects in impβ11.

BMP pathway components are present at impβ11 mutant NMJs

Importins, by mediating the nuclear import of transcription factors, might be required for the expression of a component of the BMP pathway and thus influence the production of pMAD. We therefore investigated whether the loss of synaptic pMAD resulted from the loss of a component of the pathway. To test whether low Gbb production caused the reduced pMAD, we expressed a UAS-gbb transgene in muscle and determined whether it would restore pMAD to the mutant NMJ. Although expression of the gbb transgene increased nuclear pMAD in muscle, thereby confirming the efficacy of the transgene, pMAD was not restored to impβ11 mutant NMJs (Fig. 5A-E). Thus, loss of Gbb is unlikely to explain the loss of pMAD in impβ11 mutants. Reductions in synaptic pMAD might also have arisen due to the absence of the receptors or MAD itself. We addressed these possibilities by comparing the immuno-localization of MAD, Wit and Tkv. MAD and Wit labeling in control NMJs were detected both within boutons and at their margins; the latter staining likely represented both pre- and postsynaptic pools. Anti-Tkv clearly labeled both the pre- and postsynaptic compartments. Similar patterns of immunolabeling for MAD, Wit, and Tkv were also observed in impβ11 NMJs (Fig. 5F-K), indicating that the loss of pMAD from the NMJ was not due to the absence of the substrate or receptors from the synapse. Because anti-MAD and anti-Wit only dimly labeled some boutons, we used additional methods to examine the presence of each protein in impβ11. For Wit, we examined the VNC where the receptor had previously been localized (Aberle et al., 2002) and found, that anti-Wit immunoreactivity was not markedly altered in impβ11 (Supplemental Fig. S7A and B). As an additional method to test whether a lack of MAD could account for the loss of pMAD we expressed a UAS-GFP-MAD transgene in the nervous system of impβ11 mutant larvae with elav-GAL4. This transgene can functionally replace the endogenous protein in MAD mutants (Dudu et al., 2006). Expression of UAS-GFP-MAD however, did not rescue the pMAD defect in impβ11, further indicating that the mutant defect was not due to a reduction in the substrate MAD (Supplemental Fig. S7C-F). Thus, in impβ11 mutants, the reduction in synaptic pMAD could not be explained by a loss of the ligand, receptors or MAD itself.

Figure 5
BMP pathway components are present at impβ11 NMJs

To determine if other functions that rely on BMP signaling were intact in impβ11 mutants, philanthotoxin-induced synaptic homeostasis was examined. This phenomenon entails a compensatory presynaptic increase in quantal content after the acute blockade of transmission at third-instar NMJs by means of the use-dependent glutamate receptor antagonist, philanthotoxin (PhTx; Frank et al., 2006). This process fails in gbb and wit mutant larvae and the BMP pathway is therefore thought to have a permissive role, although not necessarily be acutely involved in the upregulation of transmission (Goold and Davis, 2007). To examine this phenomenon, both control and impβ11 mutant third-instar larvae were exposed to PhTx for 10 minutes and compared with preparations of the same genotypes mock-treated with saline. In both mutant and control, PhTx decreased mEJP amplitude while EJP amplitude remained unchanged (Supplemental Fig. S8). Thus, the mechanisms governing the homeostatic increase in quantal content remained operational in the impβ11 larvae, despite the lack of pMAD at these NMJs. Therefore, the local pool of pMAD at the NMJ may not be required acutely for PhTx-induced synaptic homeostasis. Because block of retrograde transport of pMAD has been shown to hinder PhTx-induced homeostasis (Goold and Davis, 2007), nuclear pMAD is most likely responsible for permitting this form of plasticity. Thus, although pMAD is generated in the periphery by BMP signaling, it does not need to remain resident in the terminals for homeostasis to be enabled.

Genetic interaction of impβ11 and the BMP pathway

The similarity between published BMP pathway mutants and those associated with loss of importin-β11 function, as well as the observed reduction in pMAD at impβ11 terminals, suggested that importin-β11 and the BMP pathway interacted to regulate the development of the synapse. To test this hypothesis, we examined genetic interactions between impβ11 and the BMP pathway. Although mutations in impβ11 and BMP pathway components each reduce bouton counts, this phenotype is recessive; heterozygotes for these alleles are normal (Fig. 6A, ,3B3B and Table 2). In contrast, larvae made heterozygous for both impβ1170 and either a null allele of MAD (MAD10; Sekelsky et al., 1995), a wit null allele (witA12; Marques et al., 2002) or a hypomorphic allele of wit (witHA1; Aberle et al., 2002) had significantly fewer boutons than either wild-type or each heterozygous allele alone (Fig. 6A and Table 2). These genetic interactions were specific for mutations in the BMP pathway; larvae heterozygous for impβ11 and the synaptic transmission mutant neuronal synaptobrevin (n-syb; Deitcher et al., 1998) had normal synaptic arbors. Neuronal expression of UAS-wit was able to revert the reduction in bouton number in impβ1170/+; witHA1/+ larvae (Fig. 6A and Table 2). The interaction between importin-β11 and BMP components is therefore likely to occur presynaptically and, consistent with a role for importin-β11 in BMP signaling, resembles the genetic interactions of other heterozygous combinations of mutations in the BMP pathway (McCabe et al., 2004).

Figure 6
Genetic interaction of impβ11 and the BMP pathway

Because BMP pathway mutants resemble impβ11 mutant alleles, reducing both bouton number and synaptic strength, loss of pMAD could explain these impβ11 phenotypes. Restoration of synaptic pMAD in impβ11 null larvae would then be predicted to rescue the phenotypes. To this end, we introduced into motor neurons expression of constitutively active receptors with the driver OK6-GAL4 (Aberle et al., 2002) and UAS-tkvact, saxact transgenes (Holley et al., 1996; Hoodless et al., 1996). This expression, but not postsynaptic expression (G14-GAL4), restored pMAD to impβ11 mutant terminals, a finding consistent with our conclusion that an absence of MAD cannot account for the loss of pMAD in impβ11 mutants (Fig. 6B-F). Concomitant with pMAD restoration, expression of the activated receptors in motor neurons also rescued the bouton number and EJP phenotypes in impβ11 to control levels, although mEJP frequency was not rescued (Fig. 6A, G-J and Table 2). Expression of the activated receptors in muscle (G14-GAL4) did not rescue any of the phenotypes (Fig. 6A, G-J and Table 2). In control larvae, expression of the activated receptors in motor neurons did not change pMAD levels or the electrophysiological profile at the NMJ (Fig. 6C and G-J). Thus, restoring the presynaptic pool of pMAD was sufficient for normal bouton number and EJP amplitude. Moreover, the rescue of these phenotypes in the impβ11 mutants cannot be attributed to a simple addition of a UAS-tkvact, saxact-induced increase and an impβ11-induced decrease as there is no gain-of-function phenotype, but rather due to overcoming a particular deficit in pMAD signaling in the impβ11 genotype.

Reduction of retrograde transport restores impβ11 phenotypes

The levels of pMAD in nerve terminals may also be controlled by the rate at which it, or a component of the pathway, is transported away from the terminal to the nucleus or for degradation. Inhibiting the retrograde motor dynein, by expression of p150-Glued blocks retrograde BMP signaling and reduces nuclear pMAD with consequent effects on NMJ development (Eaton et al., 2002; Allan et al., 2003; Marques et al., 2003; McCabe et al., 2003). The dynein motor may transport pMAD itself or another signaling component required for pMAD production or maintenance. Therefore, we hypothesized that the structural and functional phenotypes of impβ11 might also be rescued by inhibiting retrograde transport. Overexpression of dynamitin inhibits transport by destabilizing the dynein motor complex, (Duncan and Warrior, 2002; Melkonian et al., 2007) but to a lesser extent than p150-Glued expression. When UAS-dynamitin was expressed in motor neurons with the OK6-GAL4 driver in a wild-type background, pMAD immunoreactivity at the NMJ was similar to control levels (Fig. 7A and B) and nuclear pMAD showed no obvious reduction (Fig. 7E and F). Bouton number and electrophysiological properties were likewise unchanged (Fig. 7G-K). When UAS-dynamitin was expressed in the impβ11 mutant background, however, the loss of synaptic pMAD was prevented (Fig. 7C and D). These findings confirmed that the elements of the BMP pathway in the terminal were preserved and at least partially active in the impβ11 larvae and suggested that the reduction in synaptic pMAD might arise from an excess of retrograde transport away from the terminal. Coincident with pMAD restoration to the mutant terminal, EJP amplitude, mEJP frequency, quantal content and bouton number were rescued to control values (Fig. 7G-K). No defect in mEJP amplitude was detected in impβ1170/70 larvae (Fig. 7I). These data indicate that restriction of retrograde transport is sufficient to revert impβ11 phenotypes and suggest that importin-β11 promotes or stabilizes pMAD localization to the NMJ which is in turn required for synapse formation and transmission.

Figure 7
Overexpression of dynamitin can revert impβ11 phenotypes

Discussion

Drosophila mutants have allowed us to examine, in vivo, importin-β11 and uncover its synaptic function and relationship with the BMP pathway. Loss of importin-β11 causes specific developmental and functional defects: motor neurons develop fewer boutons and EJP amplitudes are 40% lower than controls.

Importin-βs are structurally diverse; they share a small N-terminal Ran-binding domain but the remaining C-terminus of each is unique and allows for substrate specificity and functional diversity (Mosammaparast and Pemberton, 2004). This diversity extends from interactions with nucleoporins to functions in the cell periphery or neuronal synapses (James et al., 2007; Ting et al., 2007). At these locations they can begin the process of movement to the nucleus but also have additional functions (Harel and Forbes, 2004; Otis et al., 2006; Perry and Fainzilber, 2009).

The functional specificity of importins is illustrated by the impβ11 phenotypes, phenotypes that are not shared by other known importin mutants and arise only in a subset of tissues. For example, mutations in imp13 do not alter NMJ bouton number and enhance rather than decrease neurotransmitter release (Giagtzoglou et al., 2009). Similarly, mutations in impα3 lead to errors in the axonal projections of photoreceptors, a defect not seen in impβ11 (Ting et al., 2007; T. Herman, personal communication). Thus, the loss of this importin does not grossly disrupt all nuclear import, but rather causes a restricted and distinct phenotype. Homozygous null impβ11 clones in the eye primordium undergo cell division and differentiation and give rise to outwardly normal photoreceptors with correct axonal projections, but have defects in synaptic transmission. At the NMJ, the phenotype is similarly restricted to a synaptic defect with no apparent compromise in motor neuron viability, differentiation or axon guidance.

The NMJ defects in impβ11 null larvae are partial reductions in bouton number and synaptic strength rather than a complete failure of development or transmission. The electrophysiological defect in photoreceptors is less understood, but sufficient to prevent phototaxis. In addition to these defects, some neuronal subtypes must be more drastically impaired than motor neurons because neuronal importin-β11 is essential for viability. Additional non-neuronal functions are indicated by the failure of oocyte maturation in mutant germline clones, the expression of importin-β11 in early embryos and importin-β11 immunoreactivity at the nuclear envelope in muscle. Although importin-β11 is expressed in both nerve and muscle, the reduction in bouton number and synaptic strength in impβ11 null larvae results primarily from a loss of neuronal importin-β11 and can be restored by neuronal expression of importin-β11. Other aspects of NMJ development, however, are dependent on importin-β11 in muscle (Mosca and Schwarz, in preparation). Thus, importin-β11 functions are both diverse and highly selective.

Of the many factors contributing to NMJ development and function (Keshishian et al., 1996; Collins and DiAntonio, 2007), importin-β11 action appears particularly connected to the BMP pathway. The evidence for this connection is fourfold. 1) At impβ11 NMJs, pMAD is markedly reduced. Neuronal expression of an impβ11 transgene restores pMAD levels in parallel with restoration of bouton number and EJP amplitude. The decrease in pMAD is not part of a widespread loss of synaptic proteins as FasII, Futsch, Syt I, BRP, GluRIIA, GluRIIB and GluRIIC are not lacking. 2) Mutations in the BMP pathway, including mutations in MAD, result in impβ11-like phenotypes: fewer boutons and smaller EJPs (Marqués, 2005). 3) Mutations in impβ11 interact genetically with mutations in MAD and wit; heterozygous mutations that are recessive independently interact to reduce bouton number when combined. These genetic interactions with impβ11 are comparable to those between established BMP-pathway members (McCabe et al., 2004). 4) Restoration of synaptic pMAD levels in impβ11 mutants either by constitutively activated BMP receptors or impaired retrograde transport also rescues the bouton and EJP phenotypes.

These data suggest that, under normal conditions, importin-β11 permits a synaptic pool of pMAD to exist and that this pool of presynaptic pMAD is required for normal NMJ structure and function. Although SMADs chiefly act as transcription factors, there is growing precedent for alternative post-transcriptional functions (Davis et al., 2008; Hoover and Kubalak, 2008; Tang et al., 2008). A local, synaptic function of pMAD presently provides the simplest explanation of the correlation of the impβ11 null phenotypes and local pMAD levels. The mechanism underlying the reduction in synaptic pMAD, however, remains unknown. We have been unable to detect by immunoprecipitation a direct interaction between importin-β11 and several members of the BMP pathway (data not shown). Moreover, the essential elements of the pathway, Wit, Tkv and MAD itself, remain in the mutant terminals and overexpression of GFP-MAD neither restores pMAD levels nor alleviates the impβ11 phenotype. Therefore, a failure to transcribe or traffic these proteins cannot account for the loss of pMAD. In contrast, over-activation of the receptors by constitutively activated tkv and sax overcomes the impβ11 phenotype. A standard genetic explanation of this rescue by activated receptors would be that the disruption of the pathway is upstream of the receptor, in this case loss of Gbb secretion by the muscle. This is unlikely to explain the impβ11 phenotype, however, because importin-β11 is required in the nervous system, not muscle, for maintaining normal pMAD levels. Further, overexpression of Gbb does not suppress the loss of pMAD at impβ11 synapses. Thus, fundamentals of the BMP pathway, the receptors, their ligand and substrate, are intact at the impβ11 NMJ. Indeed, the continued functioning of the pathway is suggested by the presence of nuclear pMAD in the motor neurons and by the occurrence of homeostatic compensation to PhTx at the synapse, a process that fails in mutations of the BMP pathway (Goold and Davis, 2007). Instead, the loss of synaptic pMAD in impβ11 may likely arise from a modulation of this pathway that causes pMAD to be lost locally from the terminals while keeping intact the pathway for pMAD production and nuclear localization.

Excess retrograde transport of pMAD from the NMJ might plausibly explain pMAD loss in impβ11. Importin-β11 might inhibit, either directly or indirectly, retrograde transport of pMAD from terminals and thereby retain a pool of synaptic pMAD for local functions. This hypothesis is consistent with an intact BMP pathway, persistent nuclear pMAD in motor neurons and selective loss of synaptic pMAD. Synaptic receptors are needed for nuclear pMAD in a pathway that depends on retrograde transport (Marqués, 2005). Although we do not see increased nuclear pMAD in mutant motor neurons, increased transport out of the terminals need not cause a detectable increase in the nuclear pool. pMAD-dependent transcriptional regulation likely promotes synapse development, probably as a permissive signal for NMJ growth (Collins and DiAntonio, 2007; Goold and Davis, 2007). Synaptic pMAD may instead maintain or remodel the NMJ more subtly. This difference can explain why mutations in wit, which reduce pMAD in both terminals and nuclei, cause stronger synaptic defects than impβ11 (Aberle et al., 2002; Marques et al., 2002). Other possible explanations of the pMAD phenotype include a role for importin-β11 in protecting the synaptic pool from dephosphorylation or degradation, or a modulatory effect of the importin on the basal activation of the BMP receptors. In addition, we cannot rule out subtle alterations in nuclear pMAD due to potential redundancy of importin-β11 with other importins (Xu et al., 2007) or effects of maternally contributed importin-β11.

A defect in nuclear import might indirectly, perhaps through transcriptional changes, control synaptic pMAD levels. Such a mechanism would be consistent with importin-β11 localization at neuronal nuclear pores and with the canonical function of importins. However, because importin-β11-eGFP localizes to the NMJ and other importins occur at synapses (Thompson et al., 2004; Ting et al., 2007), importin-β11 might instead act synaptically to modulate pMAD. Whether acting locally, at the nuclear pore or both, our data reveal a potentially novel function for an importin in regulating BMP signaling. Because many other factors and pathways interact with BMP signaling (von Bubnoff and Cho, 2001; Keshishian and Kim, 2004; Collins and DiAntonio, 2007), there are many potential targets for further investigation of importin-β11 mechanisms.

Although importins have primarily been examined at the nuclear pore, they also undergo long distance translocations in the cytoplasm that can be important for signal transduction (Guillemain et al., 2002; James et al., 2007). These functions are of particular interest in the nervous system and include synapse to nucleus shuttling where they are implicated in synaptic plasticity, axonal regeneration and axonal targeting (Otis et al., 2006; Ting et al., 2007; Perry and Fainzilber, 2009). With the characterization of Drosophila impβ11, the regulation of synaptic development and strength can be added to the repertoire of importin-dependent functions, while the phenotype of the mutations serves to highlight the functional selectivity of an individual importin.

Supplementary Material

Supp1

Acknowledgments

This work was supported by National Institutes of Health Grant RO1 NS041062 and MH075058 (T.L.S.) and predoctoral fellowships from NIH/NINDS Ruth L. Kirschstein National Research Service Award (M.E.H.K.), National Defense Science and Engineering Graduate Fellowship (T.J.M.) and the Howard Hughes Medical Institute (D.K.D.). We thank D. Schmucker for assistance in generating transgenic flies, D. Allan for intellectual input and Drosophila stocks, T. Herman for R7 photoreceptor projection analysis, A. Deb and E. Pogoda for technical assistance and L. Bu of the MRDDRC Imaging Core under the support of NICHD P30HD18655 for imaging assistance. We thank A. DiAntonio, N. Reist, P. ten Dijke, M. Noll, W. Boll and M. Gonzales-Gaitan for antibodies and Drosophila stocks. We also thank the Bloomington Stock Center at the University of Indiana for fly stocks and Developmental Studies Hybridoma Bank at the University of Iowa for antibodies.

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