PMC

(A) Schematic of the Anabaena variabilis ATCC 29413 CAST I-B subtype 1 (I-B1) locus and ‘Peltigera membranacea cyanobiont’ 210A CAST I-B subtype 2 (I-B2) locus containing both Cascade proteins, Tn7-like proteins including TnsD and TniQ target selectors (, . ). (B) A dendrogram showing the similarity of TniQ/TnsD proteins of Tn7 and CAST I-B1 and I-B2, CAST I-F, and CAST V-K systems (, ). Dark purple indicates a similar N-terminal region (TniQ/TnsD core). Light purple indicates similarity between the C-terminal regions of Tn7-TnsD, CAST I-B1 TnsD, and remote similarity with CAST I-B2 TnsD (see , S12). Dashed lines indicate boundaries of TniQ/TnsD core. Light and dark yellow colors indicate the protein domains annotated from the CAST I-F TniQ structure (see ). (C) Phylogenetic tree built with FastTree (WAG model) () from the core region () of TniQ/TnsD proteins from CAST I-B (TniQ in red; TnsD in purple), Tn7-TnsD (in green), CAST V-K TniQ (in yellow), and CAST I-F TniQ (in cyan). The tree indicates proximity between CAST I-B1 TnsD and Tn7-TnsD and the distinction between CAST I-B1 and CAST I-B2 (, ).

Above elbow cast (long arm cast) on the left side and below-elbow cast (short arm cast) on the right side. A and C: Long arm cast; B and D: Short arm cast.

Contrast sensitivity and visual acuity of CAST^−/− and CAST^+/+ mice measured with the virtual-reality optomotor system. Average and SEM values are plotted for both genotypes. A, Contrast sensitivity was measured at six different spatial frequencies. Contrast sensitivity of CAST^−/− mice was reduced by a factor of 1.3 to 2.3 compared with their CAST^+/+ littermates. Maximal contrast sensitivity was achieved at a spatial frequency of 0.064 cyc/ ° and was 7.4 ± 0.1 in CAST^−/− compared with 16.7 ± 0.7 in CAST^+/+ mice. At all six spatial frequencies, contrast sensitivity was significantly lower in CAST^−/− compared with CAST^+/+ mice (p < 0.001 for all comparisons, t test with Bonferroni's correction). B, Visual acuity of CAST^+/+ mice was 0.38 ± 0.004 cyc/ ° compared with 0.33 ± 0.002 cyc/ ° in CAST^−/− mice. The difference in visual acuity was statistically significant (p < 0.001, t test).

Synaptic ribbons are anchored in photoreceptors of CAST^−/− mice. A, Maps of the WT CAST gene, targeting vector, and the resulting mutant gene. Position of first exon (black box), restriction enzyme sites, and position of 5′ and 3′ probes (white box) are indicated. Neo, Neomycin; DT-A, diphtheria toxin-A; Sph, SphI; Xb, XbaI. B, Southern blot analysis of targeted mice. C, PCR analysis of targeted mice. D, WB of protein homogenate (20 μg/lane except for Tubulin, 2 μg/lane) isolated from retina of CAST^+/+ and CAST^−/− and stained with indicated antibodies showed absence of CAST and significant increase of ELKS immunoreactive band in the retina of CAST^−/− mice. No significant changes were found in the expression of the ribbon synapse marker RIBEYE (stained by CtBP2 antibody) and Tubulin examined here. ELKS expression level was upregulated 1.74 ± 0.29-fold in CAST^−/− mice. *p < 0.05, n = 3, unpaired Student's t test; error bars represent the SD. Filled and open bars represent the data of CAST^−/− and CAST^+/+, respectively. The value was normalized to that of CAST^+/+ mice for each protein. E, F, Micrograph of vertical sections of CAST^+/+ (E) and CAST^−/− (F) retina with anti-CAST antibody. The prominent staining of CAST seen in the CAST^+/+ ribbon synapses in the OPL is absent in sections of CAST^−/− mice. G–I, Electron micrographs of cross-sections of rod ribbon synapses from CAST^+/+ (G) and CAST^−/− (H) retina show no obvious difference in ribbon synapse architecture. The scheme (I) shows the general configuration of this complex synapse: rod spherules have ribbons anchored to the active zone. The electron-dense arciform density is clearly visible, and several postsynaptic elements from horizontal (H) and bipolar (B) cells invaginate into the presynaptic terminal. ad, Arciform density. Scale bars: E, F, 20 μm; G, H, 200 nm.