Article Figures & Data
Figures
- Figure 1.
Western blot reveals Hiw expression profile in wild-type and hiw mutant alleles. a, The gene structure of hiw. Black boxes indicate exons. Alleles ND8, ND9, ND42, and ND69 have nonsense mutation at amino acid 1930, 2001, 3173, and 3812, respectively. Molecular lesions of alleles ND17 and ND36 have not been identified by sequencing; however, they are proposed to be nonsense mutations at amino acid proximately 2700 (indicated by arrows). The locations of two P-element alleles, BG0215 and EP1305, are indicated. These two P-elements were used to generate two deletion mutants, hiwΔN and hiwΔC (the molecular lesions of which are indicated), by imprecise excision. The locations of the antigens that are used to generate the rabbit polyclonal antibody HIW2b and the monoclonal antibody 6H4 are indicated. Two genes, CG5541 and CG32594, are located in the large intron (indicated by a dotted line), which spans ∼33 kb. b, Expression of the endogenous Hiw protein in wild-type and individual hiw alleles was analyzed by immunoblot with antisera HIW2b, as shown in the top panel. Numbers to the left are molecular masses (in kilodaltons). The arrowhead points to the full-length Hiw band at ∼570 kDa. The same blot was probed for DVGLUT as a loading control, as shown in the bottom panel.
- Figure 2.
Presynaptic expression of Hiw rescues morphological defects in hiw mutants. a, Representative confocal images of muscle 4 synapses costained with DVGLUT (green) and FasII (red) in WT (elav Gal4/+), hiwND8, hiwΔN, hiwΔC, and hiwND8-rescue (hiwND8/Y; UAS-Hiw/+; elav Gal4/+) third-instar larvae. All hiw alleles were out-crossed with wild-type flies. b-e, Quantification of bouton number (b), number of branch points (c), synaptic span (d), and bouton size (e) in WT, hiwND8, hiwΔN, hiwΔC, and hiwND8-rescue third-instar larvae. For bouton number, the number of branch points and synaptic span are n = 30, 30, 26, 37, and 51 cells, respectively; for bouton size, n = 15, 15, 21, 20, and 18 cells, respectively. The excision mutants of hiw, hiwΔN, and hiwΔC have essentially identical synaptic phenotypes to the nonsense allele hiwND8 (p > 0.5 among hiwND8, hiwΔN, and hiwΔC). All three hiw alleles are significantly different from WT in all parameters measured (p ≪ 0.001). The hiw morphological defects are rescued by presynaptic expression of Hiw in all parameters measured (p ≪ 0.001). Error bars represent SEM.
- Figure 3.
Expression of Hiw. a, b, synaptic staining with the mouse Hiw antibody (6H4) remains in the hiwΔN deletion mutant, as shown in the representative confocal images of muscle 4 synapses stained with 6H4 alone (a) or costained with 6H4 (red) and anti-HRP (green) (b). c, Expression of the total Hiw protein in the brains of WT, hiwΔN (ΔN), and GFP-Hiw overexpression (GFP-Hiw) larvae was analyzed by immunoblot with antisera HIW2b, as shown in the top panel. Neuronal expression of GFP-Hiw was driven by ELAV-GeneSwitch with RU486 in a wild-type background. The blot in the bottom panel was probed for β-tubulin as a loading control. Numbers to the left are molecular masses (in kilodaltons). d-f, Misexpressed GFP-Hiw in muscle is not recognized by 6H4, as shown in the representative confocal images of muscle 4 synapses double stained with anti-GFP and 6H4. The muscle expression of GFP-Hiw was driven by 24B Gal4 driver. g-o, The localization of neuronally expressed GFP-Hiw at the synapse was analyzed by double staining with anti-GFP and synaptic vesicle marker CSP (g-i), anti-GFP and periactive zone marker FasII (j-l), or anti-GFP and active zone marker NC82 (m-o). Scale bars: (in b) a, b, 25 μm; (in f) d-f, 13 μm; (in o) g-o, 5 μm.
- Figure 4.
The putative E3 ligase RING finger domain is essential for Hiw function. a, Representative confocal images of muscle 4 synapses costained with DVGLUT (green) and FasII (red), in WT (elav Gal4/+), hiwND8, Hiw-rescue (hiwND8/Y; UAS-NT-Hiw/+; elav Gal4/+), and HiwΔRING-rescue (hiwND8/Y; UAS-NT-HiwΔRING/+; elav Gal4/+) third-instar larvae. b, Expression of the transgenic NT-Hiw proteins in the rescue larvae was analyzed by immunoblot with peroxidase-antiperoxidase complex, as shown in the top panel. The arrowhead points to the NT-Hiw band at ∼590 kDa. The blot in the bottom panel was probed for β-tubulin as a loading control. The numbers to the right are molecular masses (in kilodaltons). c-f, Quantification of bouton number (c), number of branch points (d), synaptic span (e), and bouton size (f) in WT, hiwND8, Hiw-rescue (Hiw-res), and HiwΔRING-rescue third-instar larvae. For bouton number, the number of branch points and synaptic span are n = 31, 26, 42, and 50 cells, respectively; for bouton size, n = 15, 16, 16, and 19 cells, respectively. Neuronal expression of HiwΔRING failed to rescue the morphological defects of hiw in all parameters (p > 0.3, HiwΔRING-rescue vs hiwND8). Error bars represent SEM.
- Figure 5.
Impaired neurotransmission at hiw mutant NMJs is rescued by presynaptic expression of wild-type Hiw but not Hiw with a disrupted RING finger domain. a, Representative traces of evoked and spontaneous transmitter release recorded from WT (elav Gal4/+), hiwND8, Hiw-rescue (Hiw-res) (hiwND8/Y; UAS-Hiw/+; elav Gal4/+), and HiwΔRING-rescue (hiwND8/Y; UAS-NT-HiwΔRING/+; elav Gal4/+). Calibration: 20 ms, 5 mV for evoked release; 200 ms, 2 mV for spontaneous release. b-d, The mean mEJP amplitude (b), EJP amplitude (c), and quantal content (d) are plotted for WT, hiwND8, Hiw-rescue, and HiwΔRING-rescue (n = 19, 23, 10, and 11 cells, respectively). The electrophysiological defects in the hiw mutant are rescued in all three parameters by presynaptic expression of Hiw (p ≪ 0.001, Hiw-res vs hiwND8; p > 0.1, Hiw-res vs WT) but not HiwΔRING (p > 0.5, HiwΔRING-res vs hiwND8). Error bars represent SEM.
- Figure 6.
Neuronal expression of HiwΔRING inhibits hiw function. a, Representative confocal images of muscle 4 synapses costained with DVGLUT (green) and FasII (red) in wild-type (ELAV-GeneSwitch/+), Hiw overexpression (+Hiw, UAS-NT-Hiw/+; ELAV-GeneSwitch/+), and HiwΔRING overexpressing (+HiwΔRING, UAS-NT-Hiw/+; ELAV-GeneSwitch/+) third-instar larvae. b-e, Quantification of bouton number (b), number of branch points (c), synaptic span (d), and bouton size (e) in WT, +Hiw, and +HiwΔRING third-instar larvae. For bouton number, the number of branch points and synaptic span are n = 30, 29, and 29 cells, respectively; for bouton size, n = 18, 17, and 17 cells, respectively. Overexpression of Hiw does not significantly change the number of bouton, branch points, and synaptic span (p > 0.1, +Hiw vs WT) but leads to a decrease in bouton size (p < 0.005, +Hiw vs WT). In contrast, larvae overexpressing HiwΔRING show markedly expanded synapses (p ≪ 0.001 for all parameters, +HiwΔRING vs WT). Error bars represent SEM.
- Figure 7.
The timing of hiw function in regulating synaptic morphology and physiology. a, b, The header of the figure describes the genetic background, the presence of rescue transgene, and the manipulation of RU486 in the samples quantified underneath. Synaptic morphology and function were analyzed in four scenarios: (1) baseline hiwND8 phenotype, (2) full rescue with RU486, (3) leaked rescue without RU486, and (4) later rescue with RU486 switch. a, Quantification of bouton numbers of muscle 4 synapses in the four scenarios (n = 36, 33, 35, and 42 cells, respectively). Leaked rescue shows a 27% reduction in bouton number compared with baseline hiwND8 (*p < 0.001). Later rescue shows an additional 25% reduction in bouton number compared with leaked rescue (**p < 0.005). b, Histogram showing the quantal content for the four scenarios recorded from muscle 6 of third-instar larvae (n = 12, 15, 13, and 10 cells, respectively). Leaked rescue shows a twofold increase on the quantal content compared with baseline hiwND8 (***p<0.005). Later rescue shows an additional 52% increase in quantal content compared with leaked rescue (****p < 0.005). Error bars represent SEM.
