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First published online 2 November 2004
doi: 10.1242/jcs.01512

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Myosin 3A transgene expression produces abnormal actin filament bundles in transgenic Xenopus laevis rod photoreceptors

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA

View larger version (16K): [in a new window]   Fig. 1. Schematic diagram of whole myosin 3A and myosin 3A transgenes. Shown at the top is Myo3A with its kinase (purple), motor (blue), nine IQ motifs (yellow), and tail domains 3THDI (red) and 3THDII (black). Below Myo3A are the various Myo3A transgenes that were fused to the C terminus of GFP (green) and expressed in Xenopus tadpole rods: the entire Myo3A protein (GFP:Myo3A), Myo3A with the motor inactivated by mutation (GFP:Myo3A – motor inactive), Myo3A with the tail beyond the IQ motifs deleted (GFP:Myo3AT), Myo3A with 3THDII domain deleted (GFP:Myo3A3THDII), the Myo3A tail fragment (GFP:Myo3AT), the Myo3A tail with 3THDII deleted (GFP:Myo3AT3THDII), and the Myo3A tail domain 3THDII (GFP:3THDII). The GFP transgene alone was expressed as a control.

View larger version (99K): [in a new window]   Fig. 2. Transgenic Myo3A fusion protein localizes to the distal end of Xenopus rod inner segment actin filament bundles. The entire Myo3A protein fused to the C-terminal of GFP (GFP:Myo3A) was used to make transgenic Xenopus that express the transgene in rod photoreceptors using a modified Xenopus rod opsin promoter. (A) In a section from a GFP:Myo3A transgenic tadpole retina stained with Texas-Red phalloidin to visualize actin filament bundles, GFP:Myo3A (green) accumulates at the distal end of phalloidin-stained actin filament bundles (red). Arrows indicate the presence of the fusion protein at the distal end of actin filament bundles that protrude into the retinal pigmented epithelium (RPE). No fluorescence was detected in the rod nuclei (N). (B) Diagram of the structural features of rod photoreceptors and the overlying RPE layer in retinal sections of the photoreceptor. OS, outer segment; CP, calycal processes; IS, inner segment; CC, connecting cilium; N, nucleus; S, synapse. Actin filament bundles in photoreceptors extend from the inner segment and terminate in the calycal processes surrounding the proximal outer segment in rod photoreceptors. (C) A retinal section from a control GFP transgenic tadpole retina exhibits fluorescence in rod nuclear (labeled with DAPI, blue) and cytosolic (inner segment, synapse and connecting cilium) compartments of rods. The arrow in panel C indicates fluorescence in the connecting cilium. Scale bar: 10 µm.

View larger version (122K): [in a new window]   Fig. 3. Expression of the GFP:Myo3A transgene results in the formation of abnormally large rod calycal processes. Rods from a GFP:Myo3A transgenic retina contain thicker rod calycal processes (A,C, black arrows; E, white arrowheads) than those found in wild-type rods (B,D, arrows). In addition, a calycal process from a GFP:Myo3A rod protrudes into the RPE layer and extends beyond the normal distal boundary seen in wild-type rod calycal processes (C, arrow). Actin filament bundles are visible in a longitudinal section through an enlarged calycal process found in a GFP:Myo3A retina (C, inset). Cross-sections of rod outer segments from wild-type (D) and GFP:Myo3A transgenic tadpoles (E) contain numerous normal-sized calycal processes (white arrows) but two abnormally large calycal processes are observed in the GFP:Myo3A retina (white arrowheads). Actin filament bundles are present in rod calycal processes of a wild-type retina (F) and of the GFP:Myo3A retina (G; boxed region in E), but there are fewer actin filament bundles visualized in the wild-type calycal process compared with the enlarged calycal process in the GFP:Myo3A retina. Scale bars: (A-C) 2 µm; (inset in C) 0.2 µm; (D,E) 1 µm; (F,G) 0.25 µm.

View larger version (24K): [in a new window]   Fig. 4. GFP:Myo3A transgenic retinas contain rod calycal processes with increased cross-sectional area. Cross-sections of rods from two GFP:Myo3A, a wild-type and GFP control retina similar to those shown in Fig. 3D and E were used to measure the cross-sectional area of calycal processes from randomly chosen rods. For each rod, the calycal processes were categorized by size (cross-sectional area), and the average percentage of calycal processes per rod within a cross-sectional area range was plotted for each experimental and control tadpole retina. Note that the first three bins on the x-axis represent 0.01 µm2 and together represent the size of the remaining six bins (0.03 µm2). Rod calycal processes with cross-sectional areas less than 0.03 µm2 were seen in both control and experimental retinas, but only rods from the two GFP:Myo3A transgenic tadpoles contained calycal processes larger than 0.03 µm2. In the wild-type and GFP control retinas most of the calycal processes had cross-sectional areas less than 0.01 µm2 but a few were in the ranges 0.01-0.02 and 0.02-0.03 µm2. Therefore, any calycal processes less than 0.03 µm2 in cross-sectional area are within the normal range, while those greater than 0.03 µm2 are defined as being clubs, since calycal processes of this size are never found in wild-type or control rods.

View larger version (131K): [in a new window]   Fig. 5. Retinas containing the GFP:Myo3A transgene have reduced numbers of rod photoreceptors compared with cones. (A) GFP:Myo3A retina with areas that have reduced numbers of rods (arrows) compared with other regions within the retina and to wild-type retina (B). The other retinal layers in the transgenic eye are unaffected by the transgene. (C) The GFP:Myo3A retina contains predominantly cone photoreceptors (C) that are characterized by lipid droplets in their inner segment and fewer rods. No rod inner segments and only a few rod outer segments (ROS) are observed in the transgenic retina unlike the greater number of intact rod inner and outer segments interspersed with cones found in a wild-type retina (D). N, nucleus. Scale bars: (A,B) 100 µm; (C,D) 1 µm.

View larger version (43K): [in a new window]   Fig. 6. Expression of transgenic GFP:Myo3A causes rod cell death in the retina. Three to four 0.5 µm sections from transgenic and control tadpole retinas were counted to determine the average rod:cone ratio for retinas from GFP:Myo3A (black), GFP (gray) and wild-type (white) tadpoles. The average rod:cone ratio in GFP:Myo3A transgenic retina (1.06±0.020, n=18 tadpole retinas) was reduced compared with control GFP transgenic (1.62±0.020, n=15 tadpoles retinas) and wild type (1.78±0.056, n=14 tadpole retinas) retina. The reduction in the rod:cone ratio from GFP:Myo3A retinas compared with either GFP or wild-type retinas was statistically significant (P<0.001) presumably because of the death of rods expressing the Myo3A transgene. There was no difference in average rod:cone ratios of control transgenic GFP and wild-type retinas. Error bars, standard error of the mean.

View larger version (56K): [in a new window]   Fig. 7. Motor activity and the Myo3A tail are required for Myo3A localization to the distal end of inner segment actin filament bundles. (A) A retinal section from a transgenic tadpole containing the GFP:Myo3A (motor-inactive) protein displayed fluorescence in the inner segment (IS), connecting cilium (arrow) and synapse (arrowhead). (B) Deletion of the Myo3A tail (GFP:Myo3AT) also resulted in fluorescence confined to the cytosol of the rod inner segment, connecting cilium and synapse. (C) Fluorescence from a GFP:Myo3A transgene with a deletion of the 22 amino acid 3THDII domain (GFP:Myo3A3THDII) localized to inner segment actin filament bundles (arrow) but also displayed extensive levels of fluorescence in the inner segment cytosol and synapse. (D) In addition to cytosolic fluorescence, an accumulation of fluorescence from GFP:Myo3A3THDII was also observed in the distal ends of actin filament bundles (arrows). None of the nuclei of transgenic retinas exhibited fluorescence (N, blue DAPI label). RPE, retinal pigmented epithelium; OS, outer segment. Scale bar: 10 µm.

View larger version (62K): [in a new window]   Fig. 8. 3THDII is important for Myo3A tail localization to inner segment actin filament bundles. (A) The Myo3A tail fusion protein (GFP:Myo3AT, green) co-localized with inner segment actin filament bundles (stained red with phalloidin) as well as being localized to the rod inner segment cytosol (IS) and nuclei (N). (B) Elimination of 3THDII from the Myo3A tail fusion protein (GFP:Myo3AT3THDII) resulted in fluorescence being confined to cytosolic compartments of rods and not inner segment actin filament bundles. Instead, fluorescence is located in the rod connecting cilium (arrow), inner segment, and synapse but is excluded from the outer segment (OS) and nucleus (N, labeled in blue with DAPI). (C) Fluorescence from a GFP:3THDII fusion protein in inner segment actin filament bundles, inner segment cytosol and nuclei of rods. RPE, retinal pigmented epithelium. Scale bar: 10 µm.