Article Figures & Data
Figures
- Fig. 1.
Distribution of Shaker channels in wild-typeDrosophila larval neuromuscular junctions and colocalization with DLG. A, Anti-Shaker immunoreactivity at Type I boutons in a wild-type third instar larva. Muscle number designations are indicated. Arrows indicate Type I boutons. B, Absence of immunoreactive signal in a deficiency of Shaker larva, demonstrating the specificity of the staining. C–E, Synaptic colocalization of DLG and Shaker visualized in a high magnification view of Type I synaptic boutons double-labeled with anti-Shaker (C, green channel) and anti-DLG (D, red channel). E, Merged red and green channels. At this magnification, intense punctuate Shaker immunoreactivity is revealed within the area of the synaptic bouton that is stained with anti-Shaker antibodies. Scale bars: A, B, 80 μm; C–E, 4 μm.
- Fig. 2.
Clustering of Shaker-type K+ channels by DLG in heterologous cells. A, COS7 cell singly transfected with Kv1.4 and stained with anti-Kv1.4 antibodies.B, COS7 cell singly transfected with DLG and stained with anti-DLG antibodies. C, COS7 cell singly transfected with Shaker and stained with anti-Shaker antibodies. D, E, COS7 cells cotransfected with Kv1.4 and DLG and stained with anti-Kv1.4 (D) or with anti-DLG (E) antibodies. F, COS7 cells cotransfected with Shaker and dlg and stained with anti-Shaker antibodies. When expressed alone, Kv1.4, DLG, and Shaker are distributed diffusely in the cell with some perinuclear accumulation. On coexpression of DLG and Kv1.4, or DLG and Shaker, both proteins are redistributed into plaque-like clusters (D–F) that are essentially identical to those seen with PSD-95 and Kv1.4 (Kim et al., 1995). Scale bar, 10 μm.
- Fig. 3.
Lack of Shaker clustering inSh102 mutants. A, Anti-Shaker immunoreactivity in a third instar Sh102 larva, showing the absence of immunoreactivity at synaptic regions.B, Anti-DLG immunoreactivity in a differentSh102 preparation, showing normal DLG distribution at type I synapses. Scale bar, A, B, 20 μm. C, Western blot analysis of CS andSh102 cytosol (c) and membrane (m) fractions. Molecular weights (kDa) are indicated to the right of the blot. Multiple bands in the immunoblots are attributable to different Shaker isoforms produced by alternative splicing, which are detected by the antiserum (Rogero and Tejedor, 1995). Note the absence of proteolysis products and the very low levels of Sh102 protein in the cytosolic fraction, suggesting normal insertion of the truncated Sh protein in the plasma membrane.
- Fig. 4.
Genetic analysis of Shaker channel clustering and localization by DLG. A, Schematic diagram of the domain organization of the DLG protein. Bars in the bottom ofA indicate defects in several dlg mutant alleles (Woods and Bryant, 1991; Woods et al., 1996).Black, Intact protein region; gray, deleted protein region; asterisk, amino acid substitution. The vertical black bar between SH3 and GUK represents localization of the putative band 4.1 binding site.B, Western blot of body-wall muscle proteins stained with anti-DLG antibodies. Two bands of 97 and 108 kDa are observed in both wild-type and mutants, but the levels indlgX1-2 are decreased to <5% of wild-type levels. Each lane was loaded with 97 μg of body-wall muscle protein. C, Anti-Shaker immunoreactivity in dlgX1-2 body wall muscles showing lack of clustering at synaptic boutons.D, dlgv59 mutant body wall muscles labeled with anti-Shaker antibodies showing normal clustering of Shaker channels at Type I boutons. Scale bar, C, D, 40 μm.
- Fig. 5.
Targeting of Shaker clusters to Type I synapses is altered in dlg mutants containing only PDZ1–2 but can be rescued by postsynaptic dlg targeting. A, Anti-Shaker immunoreactivity indlgm52 . In these mutants Shaker clusters (arrow) are formed at ectopic muscle regions.B, High-magnification view of ectopic clusters in muscles 12 and 13 of a dlgm52 sample. Arrows indicate the localization of synaptic boutons determined by double labeling with anti-HRP antibodies (data not shown). C, Anti-Shaker immunoreactivity in adlgm52 strain carrying the P[Gal-4] element BG487, which expresses Gal-4 in a subset of muscles, and UAS-dlg. Note that in this strain clustering of Shaker channels around Type I synapses is normal and that only small ectopic clusters are observed (arrow). Scale bars: A, 90 μm; B, 25 μm;C, 20 μm.
- Fig. 6.
Model of Shaker clustering and Shaker synapse targeting by DLG. A, In wild-type, Shaker channels are clustered at the muscle junctional region by the interaction of their carboxyl–ETDV motif and PDZ1–2 of DLG. B, In the absence of DLG, Shaker channels fail to aggregate at the junction and remain diffusely distributed along the muscle membrane.C, When only PDZ1–2 domains are intact in DLG, Shaker channels are clustered, but at extrajunctional regions of the muscle membrane. D, Lack of ET/SXV motif in Shaker channels prevents their clustering and their localization at the junction even when DLG is normally localized. E, Abnormal SH3 or GUK domains do not prevent Shaker clustering at the junction.
Tables
- Table 1.
Shaker (ETDV) Kv1.4 (ETDV) Kv1.4 mut (ETDE) β-Gal HIS3 β-Gal HIS3 β-Gal HIS3 dlg PDZ1–PDZ2 +++ +++ +++ +++ − − dlg PDZ3 − − − − − − PSD-95 PDZ1–PDZ2 +++ +++ +++ +++ − − PSD-95 PDZ3 − − − − − − pGAD10 − − − − − − Binding of Shaker-type K+ channels by DLG as determined by semiquantitative yeast two-hybrid assay, based on induction of yeast reporter genes HIS3 and β-gal. HIS3 activity was measured by the percentage of colonies growing on histidine-lacking medium [+++ (>60%); ++ (30–60%); + (10–30%); − (no significant growth)], and β-gal activity by determining the time taken for colonies to turn blue in X-gal filter lift assays at room temperature [+++ (<45 min); ++ (45–90 min); + (90–240 min); − (no significant β-gal activity)].
