rho-type guanine exchange factor
Message rtGEF is found throughout oogenesis and embryogenesis. Of particular interest, message is most abundant in furrows and folds of the embryo where cell shapes are changing and the cytoskeleton is likely to be undergoing reorganization (Werner, 1997).
An antibody was prepared against the SH3 domain of dPix/rtGEF. In accordance with the RNA expression pattern (Werner, 1997), dPix is maternally contributed and, during early embryonic development, is distributed more or less ubiquitously. In late stage 16 embryos dPix is localized to muscle attachment sites, with the highest levels in attachment sites of the ventral muscles. dPix is also concentrated in the axon scaffold of the ventral midline and is uniformly distributed at lower levels throughout the CNS. In larval stages, dPix is expressed in muscles, the brain, the motorneurons (at low levels), and in the imaginal discs (Parnas, 2001).
Most significant, however, is the concentration of dPix at the larval NMJ. Here it is expressed in subsynaptic patches reminiscent of the localization pattern of the GluR and of Pak kinase. GluRII and Pak kinase colocalize in subsynaptic patches opposite of active zones. In order to see whether dPix would also colocalize with Pak kinase and GluRII, larval NMJs were double labelled for dPix and Pak kinase. Indeed, dPix colocalizes with Pak kinase and, hence, GluRII. In contrast, dPix does not colocalize with the presynaptic protein synaptotagmin. This indicated that dPix is mainly postsynaptic (Parnas, 2001).
The protein expression pattern and levels of dPix were examined in the dpix mutants. In the EMS alleles, there is no discernable change in protein expression in embryos; however, in larvae, the protein is missing from the synapse. Protein expression in other tissues appears to be intact. In contrast, in late stage 16 embryos of dpixp1036, the protein is undetectable, although at early stage 17 embryos, faint staining in muscle attachment sites could be discerned. In larvae, the protein is absent from the NMJ, similar to the EMS alleles. In all of the following experiments no differences at the NMJ could be detected between the EMS and the P element-induced alleles. Since dPix is expressed in significant levels in the CNS and in the axon scaffold, whether there are any CNS phenotypes in dpixp1036 was examined. No defects could be detected when CNS markers ID4 and BP102 were used (Parnas, 2001).
To identify proteins involved in synapse formation, maintenance, and growth, a large-scale genetic screen was undertaken in fly larvae. Flies were used that expressed the CD8-GFP-Sh chimera, which is targeted to the PSD via interaction of the Shaker cytoplasmic tail with Dlg. When the chimera was expressed in larval muscles using the myosin heavy chain (MHC) promoter, it enabled visualization of muscles and NMJs in whole-mount larvae. This visualization avoided the need to dissect and stain with antibodies a large number of larvae and enabled a large-scale screen. An EMS screen of 3000 lines on the second chromosome was completed. Three mutant alleles in one gene identified in the screen, dpix, cause a reduction in expression of the CD8-GFP-Sh chimera so that its levels at the PSD are reduced by 79%. This is the only gene on the second chromosome that was identified with this phenotype. Since the CD8-GFP-Sh chimera is targeted to the synapse via interactions with Dlg, the clustering of Dlg at the synapse was examined. The levels of Dlg in dpix mutants were lowered by 74%. The pattern of Dlg localization to periactive zones is also disrupted, which suggests that dPix not only controls the levels of postsynaptic Dlg, but also, to some extent, its targeting. Three alleles of this gene, dpix1,2,3, were examined which were subsequently shown to be mutations in a Rho-type GEF -- rtGEF (Parnas, 2001).
Given the strong expression of dPix/rtGEF at the NMJ, the phenotype of dpix mutants at the synapse was examined directly. dpix mutants were stained with various other synaptic markers, namely Synaptotagmin (Syt), which is a synaptic vesicle protein labeling both type I and type II synapses. In dpix mutants, Syt staining intensity is equivalent to wild-type synapses, and both type I and II synapses are present. However, the boutons in dpix mutants seem to be less defined than wild-type, as seen when stained with Dlg. Other presynaptic markers, such as Dap160 and Still life, are also localized correctly. It was of interest to see whether the expression of other postsynaptic markers in addition to Dlg were also effected in dpix mutants. Fas II has been shown to interact with Dlg; still, levels of Fas II were reduced by only 19.8% in synapses of dpix mutants (Parnas, 2001).
Another prominent postsynaptic protein is the GluR. The Drosophila NMJ contains at least two major types of ionotropic GluRs, GluRIIA and GluRIIB. Antibody directed against GluRIIA was used. Similar to Dlg and the CD8-GFP-Sh chimera, the levels of GluRIIA were reduced by 92%, although low levels of receptor could still be seen. In contrast to Dlg, the localization pattern of the receptor is normal. The localization pattern of myc-tagged GluRIIB expressed using the MHC promoter was examined. Unlike GluRIIA, synaptic levels of the GluRIIB transgene are similar in dpix and wild-type larvae. This is in contrast to the CD8-GFP-Sh chimera, which is also transgenically expressed by the same MHC promoter and is reduced in dpix synapses. Similar to GluRIIA, GluRIIB is still localized opposite active zones in dpix mutants, and targeting of the transgene does not seem to be affected. It is concluded that the localization and levels of GluRIIB seemed to be normal, although it was still possible that endogenous levels of GluRIIB are somewhat reduced. The overall morphology of the synapse in dpix mutants seems more compromised when stained for Syt and Dlg rather than for GluRIIB. This probably stems from the fact that Dlg and Syt have a more diffuse pattern of staining than GluRII and therefore give a better view of synaptic structure (Parnas, 2001).
dPix colocalizes with Pak kinase. In mammals, Pix serves to localize Pak to focal complexes. Therefore, Pak kinase localization was examined in dpix mutants. Pak kinase is completely absent from the synapses of all dpix alleles, demonstrating that dPix is necessary for Pak kinase localization at the NMJ (Parnas, 2001).
One possibility is that Dlg and Pak kinase are not concentrated in the synapse because their levels of expression are reduced in dpix mutants. In order to test this possibility, Western blot analysis was performed on dpix and wild-type larval muscle protein preparations. The expression levels of Dlg and Pak kinase are normal in muscles of dpix1 larvae; thus their elimination from the synapse is not due to reduced expression levels (Parnas, 2001).
To conclude, dPix is important for the targeting or synaptic stabilization of at least three major synaptic proteins: Pak kinase, Dlg, and GluRIIA. To a lesser extent, dPix is also important for the synaptic stabilization of Fas II. Since the dpix mutation eliminates Pak kinase from the synapse, and since Pak is a downstream target of the Rac/Cdc42 pathway, which includes Pix, it was reasoned that the elimination of Pak kinase from the synapse may be responsible for the inefficient clustering of Dlg. Thus, dpak mutants were stained with antibodies for Dlg and dPix. Several combinations of dpak alleles were used. dpak11 is a protein null, and dpak6 has a stop codon at position 113; however, there is still some synaptic expression in this allele. dpak4 has a missense mutation in the Dock binding domain, and Pak kinase protein levels are normal. Finally, dpak7 has not been characterized molecularly, but behaves genetically as a null allele. In all allelic combinations of dpak mutants, dPix levels and localization are normal. In the allelic combination dpak11/dpak4, Dlg levels at the synapse are also normal. However, in dpak11/dpak6, Dlg levels are somewhat lower than wild-type (reduction of 57%), and Pak kinase levels are reduced by 66%. In dpak11/dpak7, Dlg levels are reduced to the same extent as in dpix mutants (75%), and Pak kinase is absent. Fas II levels are also reduced to the same extent as in dpix mutants (a reduction of 19.3%). Levels of GluRIIA are also reduced, although less than levels in dpix mutants (reduction of 56%). These results are consistent with Pak kinase acting as a downstream effector of dPix. Nevertheless, there are differences between dpak and dpix mutants. In dpix mutants, the synapse looks abnormal and irregular; whereas in dpak mutants, even when Dlg levels are lowered, the synapse looks normal. Also, in dpak mutants, the muscles are thin and degenerated, and the muscle nuclei are mislocalized. In dpix larvae, the muscle activity appears weaker (as assessed by larval motility) than in wild-type larvae, but they are not as affected as in dpak mutants, and muscle nuclei are localized normally. It should be noted that the ultrastructure of dpix and dpak muscles are completely normal, and muscle differentiation per se does not seem to be affected. No structural correlate could be found that would explain the weaker muscles of dpix larvae (Parnas, 2001).
The muscle membrane at the fly NMJ is folded into a specialized structure called the subsynaptic reticulum (SSR). In Dlg mutants the SSR is reduced in size. Since the levels of Dlg are reduced in dpix synapses, SSR was examined. The SSR in dpix mutants is almost completely absent. This phenotype is seen in all allelic combinations. In dpix1/dpix2 the presynaptic terminal looked normal, with the characteristic active zones, T bars, and synaptic vesicles. However, when dpix1 or dpixp1036 were crossed to the deficiency Df(2)PJ19, there were presynaptic defects. In these genotypes, the synaptic vesicle size is not homogenous, with numerous larger vesicular structures in the terminal. Still, these terminals contain T bars with docked vesicles (Parnas, 2001).
Six different boutons of the genotype dpixp1036/Df(2)PJ19 were examined and compared with eighteen boutons of wild-type larvae. The only parameter that differs between dpix and wild-type is the number of T bars per active zone. Other parameters, such as surface area per active zone and surface area per T bar were not statistically different. However, it was noticed that the boutons look flatter than in wild-type (Parnas, 2001).
Ultrastructure of several allelic combinations of dpak mutants was also examined. The ultrastructure of the synapse in dpak11/dpak4 larvae is normal. The SSR of dpak11/dpak6 larvae is reduced in size to an intermediate level between that of wild-type and dpix mutants. Finally, in dpak11/dpak7 mutants, the SSR is reduced almost to the same extent as seen in dpix mutants. In this allelic combination, the levels of Dlg are also reduced to similar levels as in dpix mutants. No presynaptic defects were seen in dpak mutant larvae (Parnas, 2001).
Since the SSR is missing in dpix and dpak mutants, it is possible that the reduced levels of CD8-GFP-Sh, Dlg, Fas II, and GluRIIA at the synapse result not from targeting or stabilizing defects, but rather from the lack of SSR. In late stage embryos and first instar larvae, the SSR has not yet developed. Thus, if the SSR controls the localization of these postsynaptic components, no difference in their levels would be expected between wild-type and dpix early first instar larvae. The CD8-GFP-Sh chimera, as well as Dlg, GluRIIA, and Pak kinase, were examined. The dpix phenotype is already fully evident in early first instar larvae just prior to hatching. Given that the area and complexity of the SSR is reduced by at least 50% due to the absence of Dlg, it is most likely that the SSR defect is a secondary defect generated by the lack of several postsynaptic components regulated by dPix (Parnas, 2001).
Synaptic function was examined in dpix mutants. Unexpectedly, the electrophysiological differences between mutant and control larvae are relatively mild. Evoked excitatory junctional potentials (EJPs) were reduced by up to 18% and a greater variation in the EJP amplitude per muscle was noticeable compared to wild-type larvae. The amplitude of spontaneous miniature excitatory junctional potentials (mEJPs) was also reduced by 13% to 17% in dpix mutants. The small reduction in the size of mEJPs can be attributed to the reduction in GluRIIA levels since at the Drosophila NMJ, GluRIIA levels directly correlate with quantal size. The quantal content of dpix mutants differs only slightly from that of wild-type. The most dramatic defect is a 40% reduction of mEJP frequency in dpix mutants. The strong reduction in mEJP frequency is likely due to a presynaptic defect. Short-term facilitation was examined in dpix mutant background. No significant differences between wild-type larvae ) and dpixp1036/Df(2)PJ19 mutants were observed (Parnas, 2001).
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date revised: 25 February 2009
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