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First published online 17 August 2004
doi: 10.1242/jcs.01316

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Myosin Va is required for normal photoreceptor synaptic activity

Richard T. Libby^1,*, Concepcion Lillo², Junko Kitamoto², David S. Williams² and Karen P. Steel^1,,

¹ MRC Institute of Hearing Research, University Park, Nottingham, NG7 2RD, UK
² Departments of Pharmacology and Neurosciences, UCSD School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093-0983, USA

View larger version (43K): [in a new window]   Fig. 1. Mutants in dilute lethal have severely disrupted electroretinographic responses. (A) Waveforms of electroretinograms from a dilute mouse (Myo5ad/Myo5ad) on the left and a dilute lethal mutant (Myo5ad-l/Myo5ad-l) on the right, at the maximum light intensity. The a-wave is the first negative (downward) deflection and the b-wave is the later positive deflection, with oscillatory potentials caused by retinal interneuron activity superimposed. The dilute lethal waveforms show reduced amplitudes of response and an anomalous shape caused by disproportionate reduction of the b-wave. Scale bar for both waveforms shown on left. (B) Response amplitudes ± standard error plotted as a funct ion of light intensity: (left) a-waves; (right) b-waves. There is a significant difference between dilute and dilute lethal amplitudes for a-waves from 4.2 log units of attenuation (4.2-3.6, P<0.01; 3.3-0, P<0.001) and from 7.2 log units of attenuation for the b-waves (7.2-4.8, P<0.005; 4.2-0, P<0.001; see inset). At maximum flash intensity, a-wave amplitudes are reduced by 38% and b-wave amplitudes by 51% in Myo5ad-l/Myo5ad-l (n=12) compared with Myo5ad/Myo5ad or d-l (n=12; includes six Myo5ad/Myo5ad and six Myo5ad/Myo5d-l; there was no significant difference between these two genotypes for any electroretinographic parameter analysed, P>0.25 for all). All mice were between postnatal days 19 and 25 (both groups averaged 21 days of age), ages when the Myo5ad-l/Myo5ad-l are clearly afflicted with the neurological phenotype. (C) Light microscopy of retinas from a Myo5ad/Myo5ad dilute control (left) and a Myo5ad-l/Myo5ad-l dilute lethal mutant (right), at 20 days of age. No obvious differences were seen in outer segment length or any other feature of gross structure. Scale bar, 10 µm.

View larger version (19K): [in a new window]   Fig. 2. Electroretinographic responses suggest abnormal synaptic transmission between photoreceptors and retinal interneurons. (A) Implicit times of a-wave (left) and b-wave (right) peaks, with standard errors, plotted as a function of flash intensity. Times are significantly longer in the Myo5ad-l/Myo5ad-l (n=10) group compared with the controls [Myo5ad/Myo5ad or d-l (n=12; no difference was observed between the two control genotypes, P>0.25); a-wave P<0.01 from 3 log units of attenuation; b-wave, 7.2-2.4, P<0.05, 1.8-0 P<0.001]. (B) Ratios of b- to a-wave amplitudes are significantly smaller in dilute lethal mutants than the control dilute group (P0.001 from 2.4 log units of attenuation).

View larger version (208K): [in a new window]   Fig. 3. Dilute lethal mutants have abnormal photoreceptor synapses. Transmission electron micrographs of synaptic regions. Ribbon synapses (arrows) look normal in the control mice (A). In Myo5ad-l/Myo5ad-l mice, the ribbons of photoreceptor synapses appear club-like (B,E), although the ribbons of bipolar cell synapses appear normal (G). Photoreceptor synapses of Myo5ad-l/Myo5ad-l mutants also possess clusters of presumed synaptic vesicles that are ectopically located (C,D,F, arrows). At least three sections were examined from each of four Myo5ad-l/Myo5ad-l mice (abnormalities were consistently observed in the mutant mice) and two control mice (Myo5ad/Myo5ad or d-l; the synaptic morphology of these mice is similar to all wild-type mice we have previously examined, this dilute genotype does not affect synaptic morphology, which would be expect from their ERGs). Scale bars, 100 nm (F), 300 nm (others).

View larger version (25K): [in a new window]   Fig. 4. Myosin Va and Myosin VIIa have no additive impact on visual function. Response amplitudes ± standard error plotted as a function of light intensity, from mice from the intercross between the Myo5a and Myo7a mutants (a presumptive null allele of myosin VIIa, Myo7a4626SB, was used and is referred to as –): (A) a-waves; (B) b-waves. Four groups of mice are shown: the control group of mice that were heterozygous or wild type at both loci (Myo5a+/+,d or d-l, Myo7a+/?; all had normal pigmentation); mice homozygous for the Myo7a mutation and carrying a non-lethal genotype for Myo5a (Myo5+ or d/+,d or d-l, Myo7a–/–); mice homozygous for dilute lethal and heterozygous or wild type at the Myo7a locus (Myo5ad-l/d-l, Myo7a+/?); and mice homozygous for both dilute lethal and Myo7a mutations (Myo5ad-l/Myo5ad-l, Myo7a–/–). There is no evidence of additivity of the of the 50% reduction in amplitude in dilute lethals and the 20% reduction with Myo7a mutants. Number of mice analyzed for each genotype: Myo5a+/+,d or d-l, Myo7a+/?, 9; Myo5+ or d/+,d or d-l, Myo7/–/–, 8; Myo5ad-l/d-l, Myo7a+/?, 13; Myo5ad-l/d-l, Myo7a–/–, 7.