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Integrins regulate DLG/FAS2 via a CaM kinase II-dependent pathway to mediate synapse elaboration and stabilization during postembryonic development

Department of Biology, University of Utah, Salt Lake City, UT 84112-0840, USA

View larger version (75K): [in a new window]   Fig. 1. Synaptic bouton quantification in mysolfC alleles. (A) Representative muscle 12 NMJ morphology in mature third instars of wild-type, mysb9, mysts1 and mysolfCX17 alleles. Black arrowheads indicate type I boutons. White arrowheads indicate type II boutons. Scale bar indicates 20 µm. (B) Quantification of boutons for each allele and for mysolfCX17/mysXG43, a null allele. Note that mysts1 is among the least severe of the mys overgrowth alleles, whereas the mysb9 undergrowth allele is clearly very different. Error bars indicate s.e.m. (*P<0.05; **, P<0.01; ***, P<0.001).

View larger version (57K): [in a new window]   Fig. 2. ßPS integrin interacts with DLG. (A) Co-localization of DLG (red) and ßPS integrin (green) in the third instar NMJ. ßPS staining overlaps, but is not restricted to, the domain of DLG staining. (B) Immunoprecipitations of fly head extracts using protein A beads or beads coupled to either anti-kinesin or DLG were examined by western analysis. Note detectable ßPS is found only in the IP with DLG beads validating the DLG IP assay. (C) Anti-DLG immunoprecipitation (IP) experiments from adult heads were performed and western blots of total lysate (LYS), material on beads (IP-resuspended to same volume as lysate) and unbound proteins or flow through (FT) were probed with either anti-DLG or anti-ßPS antibodies. These results indicate that quantitative immunodepletion of DLG leads to a substantial reduction of ßPS from the lysate.

View larger version (74K): [in a new window]   Fig. 3. Rescue of mys mutant phenotypes by genetic manipulation of CaMKII expression. (A) Phenotypic rescue of structural defects at the third instar NMJ on muscle 4. Black arrowheads indicate type Ib boutons; open arrowheads indicate type 1s boutons. Scale bar: 20 µm. (B) Quantification of synaptic bouton number in mys and CaMKII overexpression mutant animals. In all cases, type I boutons on a minimum of 20 muscle 12 NMJs in hemisegment A2 were quantified. Error bars indicate s.e.m. Asterisks indicate significance relative to wild type. Circles indicate significance relative to each mys mutant without manipulated CaMKII levels. (* or °P<0.05; *** or °°°, P<0.001).

View larger version (53K): [in a new window]   Fig. 4. FAS2 is increased in mys mutant NMJ synapses. (A) Anti-FAS2 staining (green) and anti-DLG staining (red) at the third instar NMJ. FAS2 staining is significantly increased in both mys alleles. Arrowhead marks a type Ib bouton; arrow indicates a satellite bouton. Scale bar: 12 µm. High magnification views of FAS2 in NMJ boutons. Scale bar: 3 µm. (B) Quantification of the average intensity of FAS2 staining normalized to HRP (neuronal marker). Each bar represents the average of 80-100 boutons for each genotype. Error bars indicate s.e.m. Asterisks indicate significance. (C) Quantification of FAS2 in animals overexpressing wild type CaMKII. (***, P<0.001) (D) Sample western blots of FAS2 in the same mys mutants and the FAS2 mutants, fas2e76 and fas2e86. (E) Graph quantifying band intensities for western blot experiments. Staining intensities are normalized to wild type.

View larger version (88K): [in a new window]   Fig. 5. Rescue of mys synaptic structure defects by genetic manipulation of FAS2 expression. (A) Phenotypic rescue at the third instar muscle 4 NMJ. mysb9 causes undergrowth, whereas mysts1 causes overgrowth. Other phenotypic characteristics include increased satellite boutons and irregular bouton size. Black arrowheads indicate type Ib boutons. Open arrowheads indicate type 1s boutons. Scale bar: 20µm. (B) Quantification of NMJ synaptic growth in mys and fas mutant animals. In all genotypes, type I boutons on a minimum of 20 muscle-12 NMJs in hemisegment A2 were quantified. Error bars indicate s.e.m. Asterisks indicate significance relative to wild type. Circles indicate significance relative to each genotype without manipulated FAS2 levels. (* or °, P<0.05; ** or °°, P<0.01; *** or °°°, P<0.001).