Department of Biology, University of Utah, Salt Lake City, UT 84112-0840, USA
*Author for correspondence (e-mail: broadie{at}biology.utah.edu)
Accepted 11 April 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Drosophila, CaMKII, FAS2, Neuromuscular junction, Synaptic proteins
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Synaptic localization of FAS2 is regulated by Discs Large (DLG) through a C-terminal PDZ consensus binding site (Thomas et al., 1997; Zito et al., 1997
). Drosophila DLG, like its mammalian homologue PSD95, is a PDZ-domain scaffolding protein with multiple binding sites that are capable of mediating the formation of large molecular complexes. PSD95 binds NMDA receptors, K+ channels and semaphorins at central glutamatergic synapses (Chung et al., 2000
; Inagaki et al., 2001
; Kim et al., 1996
; Kim et al., 1995
; Kornau et al., 1995
; Niethammer et al., 1996
; Xia et al., 2000
). Similarly, DLG binds both FAS2 and Shaker K+ channels, and is responsible for their synaptic localization at the Drosophila glutamatergic NMJ (Tejedor et al., 1997
; Thomas et al., 1997
; Zito et al., 1997
).
DLG-dependent localization of FAS2 to the Drosophila NMJ is negatively regulated by Ca2+/calmodulin dependent kinase II (CaMKII). Phosphorylated DLG disassociates from the synaptic protein complex, leading to the synaptic loss of FAS2 (Koh et al., 1999b). NMJs in animals expressing a CaMKII inhibiting peptide (ala) display impaired synaptic modulation and altered synaptic morphology (Griffith et al., 1994
; Wang et al., 1994
). Thus, the current model suggests that synaptic activity leads to CaMKII activation via Ca2+-dependent auto-phosphorylation. CaMKII, in turn, phosphorylates DLG to release FAS2 from the synaptic complex, allowing developmental growth in response to increased synaptic activity (Koh et al., 1999b
).
An independent line of investigation has shown that integrins also regulate activity-dependent development of Drosophila NMJ architecture (Beumer et al., 1999; Rohrbough et al., 2000
). Integrins are heterodimeric transmembrane receptors for the extracellular matrix with both adhesion and bi-directional transmembrane signaling functions. During embryogenesis, integrins play roles in neuronal pathfinding in vertebrates, C. elegans and Drosophila (Baum and Garriga, 1997
; Hoang and Chiba, 1998
; Ivins et al., 2000
). Moreover, at least ten different integrin receptors are found in postembryonic vertebrate synapses, and at least three in larval Drosophila NMJs (Beumer et al., 1999
; Burkin et al., 1998
; Cohen et al., 2000
; Fernandes et al., 1996
; Martin et al., 1996
; Pinkstaff et al., 1999
; Rodriguez et al., 2000
; Rohrbough et al., 2000
), suggesting a persistent synaptic function. Drosophila integrin mutants display disrupted short-term memory and a loss of functional synaptic plasticity (Grotewiel et al., 1998
; Rohrbough et al., 2000
). Mutations of the Drosophila ß integrin (ßPS) subunit, or one
integrin partner (
Volado), cause multiple alterations in synaptic architecture, indicating a role for integrins in synaptic morphological development (Beumer et al., 1999
; Rohrbough et al., 2000
).
Do integrins affect synaptic morphological development via CaMKII or via an independent, parallel pathway? We propose the hypothesis that integrins may act through CaMKII at the Drosophila NMJ to modulate synaptic structure. Integrins regulate CaMKII activation in cultured cells (Bilato et al., 1997; Blystone et al., 1999
), and this regulation has been proposed to mediate communication between heterologous integrin receptors expressed together in single tissues, a condition seen at the Drosophila NMJ (Beumer et al., 1999
; Rohrbough et al., 2000
). We show here that transgenic overexpression of CaMKII completely rescues synaptic structural defects in integrin mutants, indicating that integrins act through alteration of CaMKII expression or activity. We also show that synaptic FAS2, whose regulation is downstream of CaMKII activity, is misregulated in integrin mutants and that genetic compensation for this misregulation rescues synaptic structural defects in integrin mutants. We therefore propose that synaptic integrin receptors act upstream of CaMKII to regulate NMJ morphological development, largely via regulation of FAS2 expression, and that this integrin function is required for synaptic structural alterations.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CaMKII protein levels were manipulated by crossing P[w+, UASCAMKII+] R3, located on chromosome 3 (V. Budnik) (Koh et al., 1999b) into mys mutant backgrounds. To drive the inducible UAS constructs in muscle and nerve, we used previously described GAL4 constructs (FlyBase, 1998
). One GAL4, elavGAL4 (Lin and Goodman, 1994
) is located on the tip of the X chromosome, and was crossed onto each of the mys mutant chromosomes and the stock tested for proper genotype by testing for viability and wing blister phenotypes over mysxg43. To inhibit CaMKII, we used the heat-shock inducible construct P[hsp70-ala]2, which expresses the CaMKII inhibitor peptide (ala) at a low level without heat shock induction (Griffith et al., 1993
; Wang et al., 1994
). For descriptions of the marker mutations used in this study see Lindsley and Zimm (Lindsley and Zimm, 1992
).
To genetically reduce FAS2 levels in integrin mutant backgrounds, we recombined mys chromosomes with the P-element alleles fas2e76, which reduces wild-type FAS2 to 10%, and fas2e86, which reduces FAS2 protein
50% (C. Goodman) (Grenningloh et al., 1991
). Recombinants were tested for proper expression of the FAS2 protein by western blot analysis of flies putatively homozygous for the appropriate fas2 mutant allele but heterozygous for the integrin allele (i.e., fas2e76 sn mysb9 v / fas2e76). The continued presence of the mys allele was reconfirmed in each new stock by testing for viability and wing blister phenotypes over mysxg43.
Immunocytochemistry and imaging
Wandering third instar larvae were dissected and immunologically stained as previously reported (Beumer et al., 1999; Broadie and Bate, 1993
). To examine NMJ morphology, preparations were probed with a mouse monoclonal anti-cysteine string protein (CSP) antibody (1:200) (Zinsmaier et al., 1990
). Staining was visualized using a Vectastain ABC Elite kit with NiCl2 and CoCl2 enhancement (Broadie and Bate, 1993
). Images were captured digitally. For clarity, different focal planes were occasionally combined in one picture using Adobe PhotoShop. In confocal preparations, either rabbit anti-DLG (1:1000) (Woods and Bryant, 1991
), rabbit anti-synaptotagmin (1:500) (Littleton et al., 1993
), mouse anti-CSP (1:500) (Zinsmaier et al., 1990
), rabbit anti-HRP (1:500; Cappel/Oreon Teknica Corp.) or Texas-Red-conjugated goat anti-HRP (1:500; Jackson Laboratories) was used to mark synaptic arbors.
Integrin expression was examined with the following antibodies: mouse monoclonal anti-ßPS integrin antibody CF6G11 (1:500) or CF6G11 ascites (1:300) (Brower et al., 1984); rabbit polyclonal 185E (1:1000; R. Hynes) (Zusman et al., 1993
), rat polyclonal anti-
PS2, PS2hc2 (1:5) (Bogaert et al., 1987
) and rabbit rabPS21 (1:1000) (Bloor and Brown, 1998
). All anti-integrin antibodies were independently tested for their well-characterized embryonic staining patterns prior to probing the larval preparations (Bloor and Brown, 1998
; Brower et al., 1984
). FAS2 was visualized using mouse monoclonal anti-FAS2 1D4G9 (1:10; C. Goodman) (Zito et al., 1999
). CaMKII was visualized with rabbit anti-CaMKII (1:4,000; V. Budnik) (Koh et al., 1999b
). Fluorescent secondaries used were Alexa-red 594- and Alexa-green 488-conjugated goat anti-mouse and goat anti-rabbit (1:500; Molecular Probes). Confocal microscopy was performed on either a BioRad MRC-600 or a Zeiss LSM 510. ImageQuant software from Molecular Dynamics was used to quantify staining intensity. Statistical significance of all measurements was determined with the ANOVA test using InStat software.
Western blot analysis
To prepare proteins for western blot quantification, 10 third instar larvae were homogenized in 100 µl NP40 buffer (150 mM NaCl, 1% IGEPAL CA630, 50 mM Tris (pH 8), 1x CompleteTM Protease inhibitor (Boehringer Mannheim), incubated on ice for 45 minutes and centrifuged at high speed for 20 minutes at 4°C. The supernatant was removed to a fresh tube, and the pellet was resuspended in an additional 100 µl NP40 and spun again for 20 minutes. The supernatants were combined and 25 µl of non-reducing sample buffer was added to 100 µl of the supernatant. The sample was incubated at 85°C for 10 minutes and separated on a 10% SDS-PAGE gel, then transferred onto PVDF membrane. Immunoblotting was performed with anti-FAS2 monoclonal 34B3C2 (1:100; C. Goodman) (G. Helt and C. Goodman, unpublished) and visualized with chemiluminescence reagents (Amersham). Anti-actin was used as a loading control (developed by J. J.-C. Lin and obtained from the Developmental Studies Hybridoma Bank, University of Iowa, Department of Biological Sciences, Iowa City, IA 52242 under contract NO1-HD-7-3263 from the NICHD). Bands were quantified using NIH Image.
Immunoprecipitation
Immunoprecipitation assays were performed essentially as described by Thomas et al. (Thomas et al., 1997); however, antibodies were coupled as described by Zhang et al. (Zhang et al., 2001
) to recombinant Protein A sepharose beads. Briefly, beads were washed in phosphate-buffered saline (PBS) 0.2% NP40, 5% bovine serum albumin (BSA) and incubated for 2 hours with rabbit anti-DLG (Woods and Bryant, 1991
). Beads were washed, coupled with disuccinimidyl suberate, washed again and finally blocked with PBS 0.2% NP40 and 5% BSA. These beads were gently rocked with Drosophila head extract prepared in the following manner. OR flies were frozen in liquid N2, agitated and heads isolated by sieving. Heads were homogenized in 50 mM Tris (pH 7.2) 150 mM NaCl 2 mM EGTA 0.5% Triton X-100 and 2x complete protease inhibitors (Roche). The supernatants were pre-cleared for 45 minutes with protein A Sepharose at 4°C. The cleared supernatant was incubated with Protein A beads coupled to either anti-kinesin (SUK4), anti-DLG (Woods and Bryant, 1991
) or naïve beads for 1 hour at 4°C. The beads were washed six times in 800 µl PBS-NP40 buffer once with 5 ml PBS and resuspended in 2x SDS-sample buffer. The proteins were separated on a 4-20% SDS-PAGE gel and transferred to PVDF membranes. Membranes were blocked (5% powdered milk), and probed with either rabbit anti-DLG (1:1000; D. Woods) (Woods and Bryant, 1991
) or rabbit anti-ßPS (1:1500; R. Hynes) (Zusman et al., 1993
). Bands were visualized with alkaline phosphate using NBT/BCIP as a substrate.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
To test this idea further, we used tissue-specific GAL4 drivers (Brand and Dormand, 1995) to overexpress ßPS integrin specifically either in muscle [myosin heavy chain (MHC)-GAL4] or neurons [embryonic lethal, abnormal vision (elav)-GAL4], and assayed NMJ structure in mys mutants. The undergrowth phenotype of the mysb9 NMJ can be completely rescued by postsynaptic muscle expression, which increases the amount of integrin localized to the synapse, of either ßPS alone or with its alpha partners (Table 1). This observation provides good evidence that the mysb9 synaptic undergrowth is due to postsynaptic misregulation of ßPS. By contrast, expression of ßPS integrin in muscle alone provided no rescue of the mysts1 phenotype (Table 1). We also attempted to rescue the mysts1 phenotype by overexpressing ßPS integrin in the presynaptic neuron, but were unable to do so because overexpression of either ßPS alone or with its
partners in the nervous system caused extensive NMJ overgrowth, even in wild-type animals (data not shown). The observation that both presynaptic overexpression (elavGAL4; UAS-ßPS) and underexpression (mysts1) causes structural overgrowth suggests that ßPS integrins are involved in a regulatory mechanism sensitive to both over- and underactivation and pre- and postsynaptic balance.
|
Integrins and DLG are associated in a neuromuscular junction complex
ßPS integrin is a transmembrane receptor present in both pre- and postsynaptic membranes at the larval NMJ (Beumer et al., 1999). DLG is a synaptic scaffolding protein associated with both pre-and postsynaptic membranes and is also involved in the regulation of synaptic morphology, through the localization of diverse transmembrane proteins (including FAS2) (Guan et al., 1996
; Lahey et al., 1994
). We wished to determine whether ßPS integrin associates with the DLG complex to tie together these disparate receptor components in a common molecular machine.
In confocal analyses, ßPS integrin and DLG co-localize at the larval NMJ (Fig. 2A). DLG clearly has less extensive expression, more tightly localized at NMJ boutons, whereas ßPS is more extensive through the subsynaptic reticulum (SSR) and also localized at extrasynaptic sites in the muscle, including the muscle sarcomere (Volk et al., 1990) and attachment sites (Leptin et al., 1989
). However, both proteins are most intensively expressed at the NMJ pre-/postsynaptic interface, where they co-localize (Fig. 2A). Therefore, we tested to determine if ßPS integrins form part of the synaptic complex linked by DLG. We performed protein immunoprecipitation assays using rabbit anti-DLG to probe Oregon R head extracts. DLG antibodies clearly co-immunoprecipitate DLG and ßPS integrin protein (Fig. 2C), consistent with co-localization observed in confocal analysis. Inspection of the ratio of both bound proteins and proteins not bound to beads (immunoprecipitation versus flow through lanes, Fig. 2C) indicates that a large portion of the ßPS integrin protein associates with a complex containing DLG. Control experiments with either naïve protein beads or beads coupled to anti-kinesin antibodies failed to immunoprecipitate sufficient levels of ßPS integrin protein to be detected (Fig. 2B), demonstrating the specificity of the co-immunoprecipitation. The fact that ßPS was found to co-immunoprecipitate with the complex mediated by DLG provides support for integrins existing in a synaptic complex with FAS2 and CaMKII at the synapse.
|
We first inhibited synaptic CaMKII activity in mys mutants with constructs that constitutively expressed the inhibitory peptide ala (Griffith et al., 1993; Wang et al., 1994
). In wild-type animals, expression of the ala peptide results in a
40% increase in NMJ bouton number (Wang et al., 1994
) (data not shown). Expression of ala in integrin mutants resulted in similar, though smaller, increases in bouton number, with 20% and 16% increases in mysb9 and mysts1 mutants animals, respectively (data not shown). Thus, integrin mutant phenotypes cannot be rescued by inhibition of CaMKII activity. Indeed, inhibition of CaMKII has less effect in altering NMJ structure in mys mutants than in controls.
We next overexpressed CaMKII+ through introduction of an inducible UAS-CaMKII+ construct (Koh et al., 1999b). We again used a muscle driver (MHC-GAL4) and neuronal driver (elav-GAL4), and also expressed CaMKII ubiquitously (GAL4-e22c) (Koh et al., 1999a
). Overexpression of CaMKII ubiquitously, or either pre- or postsynaptically, in wild-type animals produced no significant alteration in synaptic architecture (Fig. 3). Similarly, presynaptic CaMKII over-expression in mysb9 mutants did not detectably alter the NMJ undergrowth phenotype. By contrast, transgenic expression of CaMKII in postsynaptic muscle completely rescued the mysb9 undergrowth phenotype (Fig. 3A). The mysb9 NMJ is spatially contracted and contains 35% fewer type 1 boutons than normal, but CaMKII expression in muscle rescued both the abnormal growth and the reduced number of type I boutons to normal levels (Fig. 3B). This finding reinforces our conclusion that the mysb9 defect of NMJ undergrowth is due entirely to a postsynaptic impairment.
In the mysts1 mutant, expressing CaMKII in the presynaptic terminal caused no detectable alteration in the overgrowth phenotype (Fig. 3A). Expressing CaMKII in postsynaptic muscle caused a slight, but insignificant, decrease in the overgrowth phenotype, to partially rescue the mysts1 mutant overproduction of type I boutons (Fig. 3A,B). By contrast, transgenically expressing CaMKII ubiquitously completely rescued the mysts1 overgrowth phenotype (Fig. 3A). The mysts1 NMJ is overly expansive and contains 25% more type 1 boutons than normal, but combined pre/postsynaptic CaMKII expression rescued the abnormal growth and reduced the number of type I boutons to normal (Fig. 3B). Ubiquitous CaMKII overexpression in wild-type animals had no effect on synapse morphology, but also rescued mysb9, as expected (Koh et al., 1999a) (Fig. 3B).
We conclude that mysb9 synaptic undergrowth can be rescued by genetically increasing CaMKII only postsynaptically, whereas CaMKII must be increased on both sides of the synapse to completely rescue mysts1 synaptic overgrowth. These results are consistent with integrins regulating synaptic growth through a CaMKII-dependent pathway, and with mysts1 having defects in both pre- and postsynaptic CaMKII regulation, whereas mysb9 is specifically defective in postsynaptic CaMKII regulation.
Fasciclin 2 expression is increased in myospheroid integrin mutants
Although CaMKII is known to have multiple synaptic targets, one of the most clearly understood is DLG. The ultimate result of CaMKII phosphorylation of DLG is to limit the localization of the homophilic adhesion receptor FAS2, which dictates the directionality and degree of synaptic elaboration. As we have demonstrated that ßPS integrin lies upstream of CaMKII, we hypothesized an alteration in FAS2 localization. We therefore assayed FAS2 expression in mysb9 and mysts1 alleles in two ways: (1) quantitative confocal immunocytochemistry to assay FAS2 localization at the NMJ (Fig. 4A,B), and (2) western blot analysis to assay FAS2 levels in whole animal protein extracts (Fig. 4C,D).
|
We next assayed total FAS2 protein levels using quantified western blot analysis of staged third instar larva (Fig. 4D,E). As a control and verification test for subsequent experiments (see below), we quantified FAS2 levels in two fas2 mutants, fas2e76 and fas2e86. We observed a 90% and
50% reduction of FAS2, respectively, in these alleles which is consistent with published levels of expression (Grenningloh et al., 1991
). In both mysb9 and mysts1, quantification of the FAS2 band relative to an actin internal control band supported the increased FAS2 expression observed at the synapse in confocal immunocytochemistry experiments (Fig. 4E). We measured a 35% increase in FAS2 in mysts1 animals and a 17% increase in mysb9 animals. Note that the relative change of FAS2 levels in mysb9 and mysts1 mutants is the same using both techniques. Thus, at least part of the increased synaptic FAS2 levels could be explained by more abundant FAS2 protein in the mys mutants.
Taken together, these data are consistent with the hypothesis that the ßPS integrin receptor, together with its partners, functions via CaMKII to regulate both FAS2 expression and its localization at the NMJ synapse. By contrast, FAS2 has no detectable effect on the expression or localization of ßPS integrin.
Genetically reducing FAS2 expression in mys mutants rescues synaptic structural defects
As the synaptic structural defects of mysb9 and mysts1 are accompanied by a striking increase in FAS2 expression at the synapse, we next determined if genetically reducing FAS2 expression would rescue these developmental phenotypes. We recombined two fas2 regulatory alleles, fas2e76 (90% loss of FAS2) and fas2e86 (
50% loss of FAS2) (Fig. 4E) (Grenningloh et al., 1991
) onto chromosomes carrying the ßPS integrin mutations, generating four stocks: fas2e76 mysb9, fas2e76 mysts1, fas2e86 mysb9 and fas2e86 mysts1. Each genotype was then tested for alterations in the synaptic architecture phenotype at the third instar NMJ.
Animals doubly mutant for fas2e76 and either mys allele reduced FAS2 expression to very low levels, below 20% of wild type (Fig. 4E), and showed reduced NMJ growth phenotypes, consistent with those published for fas2e76 mutants alone (data not shown) (Schuster et al., 1996a; Schuster et al., 1996b
; Stewart et al., 1996
). By contrast, fas2e86 in conjunction with either mysb9 or mysts1, produced FAS2 protein at near normal levels, as measured by western blot analysis (Fig. 4E). Most strikingly, NMJ synaptic architecture, including bouton size, neurite branch length and number, and synaptic area, is altered towards normal by genetically reducing FAS2 levels in both mysb9 and mysts1 animals (Fig. 5A). In mysts1 mutants, the reduced FAS2 level resulted in a highly significant (P=0.0002) decrease in bouton number to near normal numbers (Fig. 5B), resulting in no significant difference between wild-type and mysts1 fas2e86 bouton numbers. This result is particularly striking because either mysts1 or fas2e86 (Stewart et al., 1996
) alone causes excessive NMJ overgrowth. By contrast, in mysb9 mutants, the reduced FAS2 level resulted in an increase in bouton number towards normal, although the rescue was not complete (Fig. 5B).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To assay the mys requirement in synaptic development comprehensively, we compared and contrasted two regulatory alleles with opposing structural phenotypes: mysb9, which causes NMJ undergrowth, and mysts1, which causes NMJ overgrowth (Beumer et al., 1999). In both mutants, we have observed a correlation between the morphological alterations and synaptic function (Beumer et al., 1999
), in contrast to the homeostasis seen in fas2 mutant synapses (Davis et al., 1997
; Stewart et al., 1996
) but consistent with the parallel alterations seen in CaMKII-inhibited synapses (Wang et al., 1994
). However, the correlation between synaptic structural and functional alterations in mys mutants is not striking, and it is clear that integrins primarily mediate architectural regulation. Therefore, our focus in this study was exclusively on the mechanism(s) by which integrins modulate NMJ structural development. This article presents molecular and genetic evidence that strongly support the hypothesis that synaptic integrin receptors containing ßPS modulate CaMKII activation on both sides of the synaptic cleft and, through CaMKII, control both the expression and the synaptic localization of FAS2 at the synapse. The primary experimental results supporting these conclusions are detailed below.
First, transgenic expression of CaMKII is sufficient to completely rescue all synaptic structure defects in mys mutants. Genetically increasing CaMKII in postsynaptic muscle, but not presynaptic neuron, completely rescues the structural undergrowth of the mysb9 integrin mutant, whereas it is necessary to elevate CaMKII both pre- and postsynaptically to rescue structural overgrowth in the mysts1 mutant. These results are consistent with the postsynaptic mislocalization of ßPS integrin in the mysb9 mutant, as opposed to the global loss of synaptic integrin in the mysts1 mutant (Beumer et al., 1999). These data indicate that coordinate regulation of CaMKII in the muscle and motoneuron is necessary for proper development of synaptic architecture.
Second, at the distal end of the cascade, both the expression and synaptic localization of FAS2 are increased in mys mutants, although the extent of FAS2 misregulation was significantly different between the two regulatory mutants. Importantly, both the NMJ overgrowth (mysts1) and undergrowth (mysb9) phenotypes are rescued towards wild-type structure by genetically reducing the amount of FAS2 available at the synapse to near normal levels. In mysts1 mutants, correcting for FAS2 level suppresses the synaptic overgrowth, while in mysb9 mutants, correcting the FAS2 level suppresses the synaptic undergrowth. These results support the conclusion that FAS2 is centrally involved in mediating synaptic growth, but suggest that the FAS2-mediated mechanism is more complex than previously thought.
Do integrins do more than regulate FAS2 at the synapse?
Our results demonstrate that integrins regulate morphological growth at the postembryonic NMJ through activation of CaMKII in both pre- and postsynaptic compartments. One important target of CaMKII is FAS2 and it is clear that regulation of FAS2 expression and localization is an important component of integrin regulation. However, the modulation of FAS2 levels alone is unlikely to account fully for the alterations in synaptic architecture in integrin mutants. In particular, both mysts1 and mysb9 upregulate synaptic FAS2 levels, albeit to different degrees, yet show opposite alteration in synaptic growth. Moreover, reducing FAS2 levels rescues both under- and overgrowth defects, but the rescue is not perfect.
Precise control of FAS2 levels finely tunes morphological development at the NMJ. Different degrees of reduced FAS2 expression can either facilitate or inhibit the growth/maintenance of the NMJ, and reduced FAS2 expression has been demonstrated in other overgrowth mutants (Schuster et al., 1996b). However, overexpression of FAS2 in specific muscles drives increased NMJ elaboration/bouton differentiation and selective preference for a high-expressing muscle over a low-expressing muscle (Davis and Goodman, 1998
). How can this complexity be interpreted? One likely explanation is interaction between FAS2 and other developmental pathways regulated by CaMKII. To date, the only known FAS2-independent regulation of synaptic morphology involves a deubiquitinating protease encoded by fat facets (faf), and a putative ubiquitin ligase encoded by highwire (hiw), which have been shown to work together to modulate the degree of NMJ growth (DiAntonio et al., 2001
; Wan et al., 2000
). Loss-of-function hiw mutants display NMJ structural overgrowth, importantly without a concomitant decrease in FAS2. Indeed, overexpression of FAS2 cannot suppress the overgrowth seen in hiw mutants (Wan et al., 2000
). These observations are consistent with the overgrowth combined with overexpression of FAS2 observed in mysts1 mutants in this study. However, any putative interaction between FAS2-dependent and -independent mechanisms of morphological growth regulation at the Drosophila NMJ synapse remain to be elucidated. We expect that further study will reveal an additional structural control mechanism regulated by CaMKII, acting in parallel to and interacting with FAS2. The ubiquination mechanism discussed here is one candidate mechanism, but as CaMKII is known to have many synaptic targets, it is clearly not the only candidate.
Presynaptic CaMKII has previously been shown to be important for behavioral change, including learning in mice, and habituation in Drosophila (Griffith et al., 1993; Soderling, 2000
). In mice, animals null for
CaMKII are unable to learn, and do not manifest experience-dependent plasticity (Frankland et al., 2001
). Animals missing just one copy of
CaMKII learn normally, but are unable to form long-term memories, which is accompanied by an inability to maintain LTP in the cortex. In Drosophila, presynaptic CaMKII has been shown to bidirectionally regulate habituation of a simple motor response (Jin et al., 1998
). Inhibition of CaMKII activation by expression of the ala peptide reduces the initial response, which is then followed by facilitation instead of habituation. Moreover, presynaptically targeted expression of a constitutively active form of CaMKII eliminated habituation. Thus, appropriate regulation of CaMKII appears necessary for many aspects of developmental plasticity.
In summary, we conclude that architectural developmental defects observed in NMJ synapses mutant for ßPS integrin are due to the loss of the ability to regulate synaptic CaMKII properly. One function of CaMKII is to phosphorylate the scaffolding protein DLG, and thus regulate the proteins synaptically localized by this scaffold. FAS2 is the central known output of this regulatory cascade. Loss of this regulation is central to the mys mutant defects in the postembryonic elaboration of NMJ structure. It is clear from this study, however, that regulation of FAS2 localization via CaMKII is only one facet of how integrins function at the synapse.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ashburner, M. (1989). Drosophila. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Ayyub, C., Rodrigues, V., Hasan, G. and Siddiqi, O. (2000). Genetic analysis of olfC demonstrates a role for the position-specific integrins in the olfactory system of Drosophila melanogaster. Mol. Gen. Genet. 263, 498-504.[Medline]
Bailey, C. H. and Chen, M. (1989). Structural plasticity at identified synapses during long-term memory in Aplysia. J. Neurobiol. 20, 356-372.[Medline]
Bastiani, M. J., Harrelson, A. L., Snow, P. M. and Goodman, C. S. (1987). Expression of fasciclin I and II glycoproteins on subsets of axon pathways during neuronal development in the grasshopper. Cell 48, 745-755.[Medline]
Baum, P. D. and Garriga, G. (1997). Neuronal migrations and axon fasciculation are disrupted in ina-1 integrin mutants. Neuron 19, 51-62.[Medline]
Beumer, K. J., Rohrbough, J., Prokop, A. and Broadie, K. (1999). A role for PS integrins in morphological growth and synaptic function at the postembryonic neuromuscular junction of Drosophila. Development 126, 5833-5846.
Bilato, C., Curto, K. A., Monticone, R. E., Pauly, R. R., White, A. J. and Crow, M. T. (1997). The inhibition of vascular smooth muscle cell migration by peptide and antibody antagonists of the alphavbeta3 integrin complex is reversed by activated calcium/calmodulin-dependent protein kinase II. J. Clin. Invest. 100, 693-704.
Bloor, J. W. and Brown, N. H. (1998). Genetic analysis of the Drosophila alphaPS2-integrin subunit reveals discrete adhesive, morphogenetic and sarcomeric functions. Genetics 148, 1127-1142.
Blystone, S. D., Slater, S. E., Williams, M. P., Crow, M. T. and Brown, E. J. (1999). A molecular mechanism of integrin crosstalk: alphavbeta3 suppression of calcium/calmodulin-dependent protein kinase II regulates alpha5beta1 function. J. Cell Biol. 145, 889-897.
Bogaert, T., Brown, N. and Wilcox, M. (1987). The Drosophila PS2 antigen is an invertebrate integrin that, like the fibronectin receptor, becomes localized to muscle attachments. Cell 51, 929-940.[Medline]
Brand, A. H. and Dormand, E. L. (1995). The GAL4 system as a tool for unravelling the mysteries of the Drosophila nervous system. Curr. Opin. Neurobiol. 5, 572-578.[Medline]
Broadie, K. S. and Bate, M. (1993). Development of the embryonic neuromuscular synapse of Drosophila melanogaster. J. Neurosci. 13, 144-166.[Abstract]
Brower, D. L., Wilcox, M., Piovant, M., Smith, R. J. and Reger, L. A. (1984). Related cell-surface antigens expressed with positional specificity in Drosophila imaginal discs. Proc. Natl. Acad. Sci. USA 81, 7485-7489.[Abstract]
Budnik, V., Koh, Y. H., Guan, B., Hartmann, B., Hough, C., Woods, D. and Gorczyca, M. (1996). Regulation of synapse structure and function by the Drosophila tumor suppressor gene dlg. Neuron 17, 627-640.[Medline]
Bunch, T. A., Salatino, R., Engelsgjerd, M. C., Mukai, L., West, R. F. and Brower, D. L. (1992). Characterization of mutant alleles of myospheroid, the gene encoding the ß-subunit of the Drosophila PS integrins. Genetics 132, 519-528.
Burkin, D. J., Gu, M., Hodges, B. L., Campanelli, J. T. and Kaufman, S. J. (1998). A functional role for specific spliced variants of the alpha7beta1 integrin in acetylcholine receptor clustering. J. Cell Biol. 143, 1067-1075.
Chung, H. J., Xia, J., Scannevin, R. H., Zhang, X. and Huganir, R. L. (2000). Phosphorylation of the AMPA receptor subunit GluR2 differentially regulates its interaction with PDZ domain-containing proteins. J. Neurosci. 20, 7258-7267.
Cohen, M. W., Hoffstrom, B. G. and DeSimone, D. W. (2000). Active zones on motor nerve terminals contain alpha 3beta 1 integrin. J. Neurosci. 20, 4912-4921.
Dahme, M., Bartsch, U., Martini, R., Anliker, B., Schachner, M. and Mantei, N. (1997). Disruption of the mouse L1 gene leads to malformations of the nervous system. Nat. Genet. 17, 346-349.[Medline]
Davis, G. W. and Goodman, C. S. (1998). Genetic analysis of synaptic development and plasticity: homeostatic regulation of synaptic efficacy. Curr. Opin. Neurobiol. 8, 149-156.[Medline]
Davis, G. W., Schuster, C. M. and Goodman, C. S. (1997). Genetic analysis of the mechanisms controlling target selection: target- derived Fasciclin II regulates the pattern of synapse formation. Neuron 19, 561-573.[Medline]
DiAntonio, A., Haghighi, A. P., Portman, S. L., Lee, J. D., Amaranto, A. M. and Goodman, C. S. (2001). Ubiquitination-dependent mechanisms regulate synaptic growth and function. Nature 412, 449-452.[Medline]
Fernandes, J. J., Celniker, S. E. and VijayRaghavan, K. (1996). Development of the indirect flight muscle attachment sites in Drosophila: Role of the PS-integrins and the stripe gene. Dev. Biol. 176, 166-184.[Medline]
FlyBase (1998). Flybase: A Drosophila database. Nucleic Acids Res. 26, 85-88.
Frankland, P. W., OBrien, C., Ohno, M., Kirkwood, A. and Silva, A. J. (2001). Alpha-CaMKII-dependent plasticity in the cortex is required for permanent memory. Nature 411, 309-313.[Medline]
Grenningloh, G., Rehm, E. J. and Goodman, C. S. (1991). Genetic analysis of growth cone guidance in Drosophila: fasciclin II functions as a neuronal recognition molecule. Cell 67, 45-57.[Medline]
Griffith, L. C., Verselis, L. M., Aitken, K. M., Kyriacou, C. P., Danho, W. and Greenspan, R. J. (1993). Inhibition of calcium/calmodulin-dependent protein kinase in Drosophila disrupts behavioral plasticity. Neuron 10, 501-509.[Medline]
Griffith, L. C., Wang, J., Zhong, Y., Wu, C. F. and Greenspan, R. J. (1994). Calcium/calmodulin-dependent protein kinase II and potassium channel subunit Eag similarly affect plasticity in Drosophila. Proc. Natl. Acad. Sci. USA 91, 10044-10048.
Grotewiel, M. S., Beck, C. D., Wu, K. H., Zhu, X. R. and Davis, R. L. (1998). Integrin-mediated short-term memory in Drosophila. Nature 391, 455-460.[Medline]
Guan, B., Hartmann, B., Kho, Y. H., Gorczyca, M. and Budnik, V. (1996). The Drosophila tumor suppressor gene, dlg, is involved in structural plasticity at a glutamatergic synapse. Curr. Biol. 6, 695-706.[Medline]
Hoang, B. and Chiba, A. (1998). Genetic analysis of the role of integrin during axon guidance in Drosophila. J. Neurosci. 18, 7847-7855.
Inagaki, S., Ohoka, Y., Sugimoto, H., Fujioka, S., Amazaki, M., Kurinami, H., Miyazaki, N., Tohyama, M. and Furuyama, T. (2001). Sema4c, a transmembrane semaphorin, interacts with a post-synaptic density protein, psd-95. J. Biol. Chem. 276, 9174-9181.
Ivins, J. K., Yurchenco, P. D. and Lander, A. D. (2000). Regulation of neurite outgrowth by integrin activation. J. Neurosci. 20, 6551-6560.
Jin, P., Griffith, L. C. and Murphey, R. K. (1998). Presynaptic calcium/calmodulin-dependent protein kinase II regulates habituation of a simple reflex in adult Drosophila. J. Neurosci. 18, 8955-8964.
Kim, E., Niethammer, M., Rothschild, A., Jan, Y. N. and Sheng, M. (1995). Clustering of Shaker-type K+ channels by interaction with a family of membrane-associated guanylate kinases. Nature 378, 85-88.[Medline]
Kim, E., Cho, K. O., Rothschild, A. and Sheng, M. (1996). Heteromultimerization and NMDA receptor-clustering activity of Chapsyn- 110, a member of the PSD-95 family of proteins. Neuron 17, 103-113.[Medline]
Koh, Y., Popova, E., Thomas, U., Griffith, U. and Budnick, V. (1999a). Role of CaMKII-dependent phosphorylation on the selective localization of DLG to synaptic junctions (abstract). 40th Annual Drosophila Research Conference Programs and Abstracts 1, 204.
Koh, Y. H., Popova, E., Thomas, U., Griffith, L. C. and Budnik, V. (1999b). Regulation of DLG localization at synapses by CaMKII-dependent phosphorylation. Cell 98, 353-363.[Medline]
Kornau, H. C., Schenker, L. T., Kennedy, M. B. and Seeburg, P. H. (1995). Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95. Science 269, 1737-1740.[Medline]
Lahey, T., Gorczyca, M., Jia, X. X. and Budnik, V. (1994). The Drosophila tumor suppressor gene dlg is required for normal synaptic bouton structure. Neuron 13, 823-835.[Medline]
Leptin, M., Bogaert, T., Lehmann, R. and Wilcox, M. (1989). The function of PS-integrins during Drosophila embryogenesis. Cell 56, 401-408.[Medline]
Lin, D. M. and Goodman, C. S. (1994). Ectopic and increased expression of Fasciclin II alters motoneuron growth cone guidance. Neuron 13, 507-523.[Medline]
Lin, D. M., Fetter, R. D., Kopczynski, C., Grenningloh, G. and Goodman, C. S. (1994). Genetic analysis of Fasciclin II in Drosophila: defasciculation, refasciculation, and altered fasciculation. Neuron 13, 1055-1069.[Medline]
Lindsley, D. L. and Zimm, G. (1992). The Genome of Drosophila melanogaster. San Diego: Academic Press.
Littleton, J. T., Bellen, H. J. and Perin, M. S. (1993). Expression of synaptotagmin in Drosophila reveals transport and localization of synaptic vesicles to the synapse. Development 118, 1077-1088.
Luthl, A., Laurent, J. P., Figurov, A., Muller, D. and Schachner, M. (1994). Hippocampal long-term potentiation and neural cell adhesion molecules L1 and NCAM. Nature 372, 777-779.[Medline]
Martin, P. T., Kaufman, S. J., Kramer, R. H. and Sanes, J. R. (1996). Synaptic integrins in developing, adult, and mutant muscle: selective association of alpha1, alpha7A, and alpha7B integrins with the neuromuscular junction. Dev. Biol. 174, 125-139.[Medline]
Mayford, M., Barzilai, A., Keller, F., Schacher, S. and Kandel, E. R. (1992). Modulation of an NCAM-related adhesion molecule with long-term synaptic plasticity in Aplysia. Science 256, 638-644.[Medline]
Niethammer, M., Kim, E. and Sheng, M. (1996). Interaction between the C terminus of NMDA receptor subunits and multiple members of the PSD-95 family of membrane-associated guanylate kinases. J. Neurosci. 16, 2157-2163.[Abstract]
Pinkstaff, J. K., Detterich, J., Lynch, G. and Gall, C. (1999). Integrin-subunit gene expression is regionally differentiated in adult brain. J. Neurosci. 19, 1541-1556.
Rodrigues, V. and Siddiqi, O. (1978). Genetic analysis of chemosensory pathway. Proc. Indian Acad. Sci. USA(B) 87B, 147-160.
Rodriguez, M. A., Pesold, C., Liu, W. S., Kriho, V., Guidotti, A., Pappas, G. D. and Costa, E. (2000). Colocalization of integrin receptors and reelin in dendritic spine postsynaptic densities of adult nonhuman primate cortex. Proc. Natl. Acad. Sci. USA 97, 3550-3555.
Rohrbough, J., Grotewiel, M. S., Davis, R. L. and Broadie, K. (2000). Integrin-mediated regulation of synaptic morphology, transmission, and plasticity. J. Neurosci. 20, 6868-6878.
Schuster, C. M., Davis, G. W., Fetter, R. D. and Goodman, C. S. (1996a). Genetic dissection of structural and functional components of synaptic plasticity. I. Fasciclin II controls synaptic stabilization and growth. Neuron 17, 641-654.[Medline]
Schuster, C. M., Davis, G. W., Fetter, R. D. and Goodman, C. S. (1996b). Genetic dissection of structural and functional components of synaptic plasticity. II. Fasciclin II controls presynaptic structural plasticity. Neuron 17, 655-667.[Medline]
Soderling, T. R. (2000). CaM-kinases: modulators of synaptic plasticity. Curr. Opin. Neurobiol. 10, 375-380.[Medline]
Stewart, B. A., Schuster, C. M., Goodman, C. S. and Atwood, H. L. (1996). Homeostasis of synaptic transmission in Drosophila with genetically altered nerve terminal morphology. J. Neurosci. 16, 3877-3886.
Tejedor, F. J., Bokhari, A., Rogero, O., Gorczyca, M., Zhang, J., Kim, E., Sheng, M. and Budnik, V. (1997). Essential role for dlg in synaptic clustering of Shaker K+ channels in vivo. J. Neurosci. 17, 152-159.
Thomas, U., Kim, E., Kuhlendahl, S., Koh, Y. H., Gundelfinger, E. D., Sheng, M., Garner, C. C. and Budnik, V. (1997). Synaptic clustering of the cell adhesion molecule fasciclin II by discs-large and its role in the regulation of presynaptic structure. Neuron 19, 787-799.[Medline]
Volk, T., Fessler, L. I. and Fessler, J. H. (1990). A role for integrin in the formation of sarcomeric cytoarchitecture. Cell 63, 525-536.[Medline]
Wan, H. I., DiAntonio, A., Fetter, R. D., Bergstrom, K., Strauss, R. and Goodman, C. S. (2000). Highwire regulates synaptic growth in Drosophila. Neuron 26, 313-329.[Medline]
Wang, J., Renger, J. J., Griffith, L. C., Greenspan, R. J. and Wu, C. F. (1994). Concomitant alterations of physiological and developmental plasticity in Drosophila CaM kinase II-inhibited synapses. Neuron 13, 1373-1384.[Medline]
Woods, D. F. and Bryant, P. J. (1991). The discs-large tumor suppressor gene of Drosophila encodes a guanylate kinase homolog localized at septate junctions. Cell 66, 451-464.[Medline]
Xia, J., Chung, H. J., Wihler, C., Huganir, R. L. and Linden, D. J. (2000). Cerebellar long-term depression requires PKC-regulated interactions between GluR2/3 and PDZ domain-containing proteins. Neuron 28, 499-510.[Medline]
Zhang, Y. Q., Bailey, A. M., Matthies, H. J., Renden, R. B., Smith, M. A., Speese, S. D., Rubin, G. M. and Broadie, K. (2001). Drosophila fragile X-related gene regulates the MAP1B homolog futsch to control synaptic structure and function. Cell 107, 591-603.[Medline]
Zinsmaier, K. E., Hofbauer, A., Heimbeck, G., Pflugfelder, G. O., Buchner, S. and Buchner, E. (1990). A cysteine-string protein is expressed in retina and brain of Drosophila. J. Neurogenet. 7, 15-29.[Medline]
Zito, K., Fetter, R. D., Goodman, C. S. and Isacoff, E. Y. (1997). Synaptic clustering of Fascilin II and Shaker: essential targeting sequences and role of Dlg. Neuron 19, 1007-1016.[Medline]
Zito, K., Parnas, D., Fetter, R. D., Isacoff, E. Y. and Goodman, C. S. (1999). Watching a synapse grow: noninvasive confocal imaging of synaptic growth in Drosophila. Neuron 22, 719-729.[Medline]
Zusman, S., Grinblat, Y., Yee, G., Kafatos, F. C. and Hynes, R. O. (1993). Analyses of PS-integrin functions during Drosophila development. Development 118, 737-750.