Striped bass, Morone saxatilis, were purchased from Chico Game Fish Farm, Chico, CA, and were kept in circular constant-flow indoor tanks containing chloramine-free tapwater at a constant 14:10 light/dark cycle. At 1–2 h before handling, artificial sea salt was added to the tank at a 1:10 dilution, and 15–30 min before handling, bass were treated with 3-aminobenzoic acid ethyl ester (methanesulfonate salt, MS222; Sigma, St. Louis, MO) at 9.4 g/100 gal.
Dark-adapted bass (1 h total darkness) were caught and killed in total darkness and immediately dissected under low light. Light-adapted bass were subjected to bright light (1 h), killed, and dissected in light. Eyes were enucleated, hemisected, and immersed in HBSS (Life Technologies/BRL, Gaithersburg, MD) supplemented with 1 μM calpeptin (Calbiochem-Novobiochem, La
Jolla, CA). Similarly, the sacculus was dissected into HBSS/calpeptin. The retina, RPE (dark-adapted) or retina/RPE combined (light-adapted), and sacculus were removed and transferred into either RNA-STAT for preparation of RNA, homogenization buffer, or fresh HBSS/calpeptin for shake-off preparation.
RNA Isolation and cDNA synthesis
RNA was collected from retina and RPE of dark-adapted bass. Total RNA was extracted using RNA-STAT (TEL-TEST, Inc., Friendswood, TX), and m-RNA was purified using the PolyATract mRNA isolation system (Promega, Madison, WI). Rapid amplification of cDNA ends (RACE)–ready cDNA was synthesized from purified mRNA using the Advantage cDNA synthesis kit (Clontech, Palo Alto, CA).
Myo3A and 3B Cloning
A myosin PCR screen was performed on bass retina/RPE cDNA using the degenerate myosin primers ATP-3 and EAF-A (Bement et al., 1994
) to amplify the myosins expressed in these tissues. The resultant ~150–base pair PCR products were subcloned, sequenced, and analyzed against GenBank, EMBL, DDBJ, and PDB sequences using the BLAST Network Service of the National Center for Biotechnology Information. Two closely related clones displayed very little sequence similarity to previously identified myosins. The first of these two clones, now called Myo3A, was amplified as four RACE fragments using nested primers designed to amplify in both the 5′ and 3′ directions originating from within the initial 150 base pairs cloned (Hillman et al., 1996
A 567–base pair fragment spanning the kinase/myosin junction of the second clone, now called Myo3B, was amplified in a separate experiment using two degenerate primers. The upstream primer was directed against a conserved kinase motif GITAIE (GGNATHACNGCNATHGA), and the downstream primer was directed against a conserved motor domain motif NPPHIFAV (CNACNGCRAANAYRTGNGGNGGRTT). It was recognized by sequence comparison as one of the novel clones seen in the original PCR screen described above. Six RACE reactions (one in the 5′ direction and five in the 3′ direction) were performed to clone the cDNA (abstract, Wong et al., 1998
Northern Blot Analysis
To compare mRNA levels in retina and RPE, 1.7 μg each of RPE polyA RNA and retina polyA RNA were electrophoresed in 1% agarose/formaldehyde gels (Sambrook et al., 1989
). Multitissue Northern blots were prepared with 4 μg of polyA RNA from dark-adapted retina, light adapted retina/RPE, brain, heart, intestine, kidney, liver, muscle, and testis. The polyA RNA was transferred to Hybond-N (Amersham Pharmacia Biotech, Piscataway, NJ) by capillary action, and the membrane was prehybridized in 50% formamide, 2× Denhardt's solution, 5× SSC, and 0.1 mg/ml salmon sperm DNA overnight at 42°C and hybridized in the same buffer with the 32
P-labeled probes overnight at 42°C. Radioactive labeling of probes was done using Prime-It RmT dCTP labeling reactions (Stratagene, La
Jolla, CA), and probes were added at 2 × 106
cpm/ml. Membranes were washed in 0.1% SDS/0.1× SSC at 42°C and autoradiographed using x-ray film with intensifying screens at −80°C for up to 1 wk.
Tissue Homogenization and Sample Preparation
Dissected tissue was homogenized in a Teflon/glass motor-driven homogenizer (Eberbach Corp., Ann Arbor, MI) at 180–200 rpm, 40 strokes in one of several buffers: buffer A, 50 mM Mops, pH 7.2, 1 mM EDTA, 1 mM EGTA, 160 mM KCl; buffer B, buffer A + 1 mM dithiothreitol (DTT); buffer C, buffer A + 1 mM DTT and 10 mM ATP; and buffer D, buffer A + 10 mM ATP in the presence of a complex protease inhibitor cocktail (PI): 2 μg/μl aprotinin, 100 μM leupeptin, 1 μM pepstatin, 1 μM calpeptin, 5 μM calpain inhibitor III, 400 μM PMSF, 1 mM benzamidine, 2 mM phenanthroline, 10 μg/μl TAME, 200× protease and phosphatase inhibitor cocktail (P8340, Sigma). The homogenate was subjected to centrifugation (100,000 × g); the final supernatant is referred to as high-speed supernatant.
Shake-offs (isolated photoreceptor inner/outer segments) were prepared by agitating dissected retinas in HBS + calpeptin, breaking off rods and cones at the myoid region. These photoreceptor inner/outer segments are collected by a low-speed centrifugation (1000 × g
, 20 min, 4°C) and can be either homogenized or further purified on a Percoll step gradient as described (Pagh-Roehl and Burnside, 1995
). All gradient purification steps were done in the presence of the protease inhibitor cocktail described above.
Generation of Myosin IIIA Antibodies
Three antibodies were generated against bass Myo3A: a “head” antibody raised against the motor domain (aa 260–669), a “tail” antibody raised against the tail region beyond the last IQ motif (aa 1631–1832), and the “3THDII” (used throughout this study) antibody raised against the extreme C-terminal 22 amino acids. The head and tail domains were expressed as histidine-tagged fusion proteins and injected into rabbits for the production of polyclonal antisera (antibody production was performed at Office of Laboratory Animal Care, University of California, Berkeley). The tail-tip antibody was raised against and affinity-purified on the last 22 amino acids of the bass MYO3A (performed by Bethyl Laboratories, Inc., Montgomery, TX). The epitope chosen for antibody production is at the extreme C terminus (NPYDFRHLLRKTSQRRKLIKQY) in a region almost identical to the human MYO3A tail tip (see Figure for sequence comparison). Currently, an antibody raised against Myo3B suggests that it is not expressed in the photoreceptors.
Figure 3 Amino acid sequence alignment of class III myosins. Boxshade output of a clustal W alignment (Young, 1976 ) of fish MyoIIIA (designated FMIIIA) and Myo3B (FMIIIB), human MYO3A (HMIIIA), human MYO3B (HMIIIB), Drosophila NINAC (DMIII), and Limulus MyoIII (more ...)
Protein samples were subjected to electrophoresis on NuPAGE Tris acetate gels (7%) (Invitrogen, Carlsbad, CA) and transferred to Hybond N (Amersham Pharmacia Biotech) in NuPAGE transfer buffer + 10% methanol and 0.05% SDS. The membrane was blocked with 5% milk in PBS and incubated with 1:80,000 α-3THDII O/N in 3% BSA in PBS at 4°C or 1:5000 immunoaffinity-purified anti-tail antibody. Membrane was washed for 1 h with six changes in PBS and incubated with goat anti-rabbit antibody conjugated to horseradish peroxidase (Amersham Pharmacia) for 1 h at room temperature in 3% BSA in PBS. The membrane was washed again for 1 h with six changes of PBS and developed using enhanced chemiluminescence (Amersham Pharmacia).
Photoreceptor inner/outer segments were allowed to settle for 5 min on poly-l
-lysine–subbed slides and coverslips. They were methanol-fixed in −20°C methanol for 2 min, or paraformaldehyde-fixed in 4% paraformaldehyde/PBS and incubated 40 min at room temperature. After fixation, the slides and coverslips were rinsed twice in PBS. Before immunolocalization, paraformaldehyde-fixed cells were treated with 2% glycine/PBS for 2 min, PBS twice for 2 min, five changes of fresh 0.1 mg/ml sodium borohydride for 5 min, twice with PBS for 2 min, 0.1% Triton-X-100 for 3 min, and twice with PBS for 5 min. Immunolocalization was carried out according to Hoang et al. (1999)
. Secondary antibodies were Alexa 488 (Myo3A) and Cy3 (tubulin) (Molecular Probes, Eugene, Oregon). Phalloidin staining was performed by adding a 1:50 dilution of Texas Red or Alexa 488 phalloidin (Molecular Probes) to the secondary antibody incubation.
Cells were observed with an inverted microscope (Carl Zeiss, Inc., Thornwood, NY) using a 63×/NA 1.4 objective. Images were acquired with a cooled CCD camera (Hamamatsu Corp., Bridgewater, NJ) controlled by Open Lab software (Improvision Inc., Lexington, MA). To obtain images of photoreceptor immunofluorescence and phalloidin staining, section series of 15–25 0.5-μm optical sections were taken. The section series was deconvolved with Open Lab software and merged. Stained photoreceptors were also visualized with a Zeiss 510 Confocal microscope. The Myo3A staining pattern was similar in both methanol- and paraformaldehyde-fixed cells; however, the phalloidin did not label actin filaments in methanol-fixed tissue.
Grids were coated with a thin film of Formvar layered on water. Then, 12–15 coated grids were collected on a coverslip and allowed to air-dry. The grids were then coated with 0.01% poly-l-lysine for 5 min at room temperature and left to dry overnight at room temperature. Freshly prepared ice-cold photoreceptor inner/outer segments were allowed to settle on grids for 5–10 min. Cells were permeabilized for 5–10 min with PHEM buffer (6 mM PIPES, 25 mM HEPES, 8 mM EDTA, 2 mM MgCl2, 1 mM DTT, 20 μM taxol, 25 μg/ml phalloidin) containing 1% Triton X-100. Samples were incubated for 1 hr in blocking buffer: PHEM buffer with 2% BSA and 30% normal goat serum (NGS), followed by a 1-hr incubation in primary antibody diluted (1:200, Myo3A; 1:50, anti-actin) in working buffer (PHEM buffer supplemented with 2% BSA and 10% NGS). The grids were washed three times with working buffer and incubated for 30 min in 1:10 secondary goat anti-rabbit antibody conjugated to 18-mm gold spheres (Jackson Immuno Research, West Grove, PA). Finally, specimens were washed two times in working buffer and once in distilled water, allowed to air-dry, and negative-stained using 1% uranyl acetate.
Rod Inner/Outer Segment Cytoskeleton Extractions
Gradient-purified rod inner/outer segments (RIS/ROS) were collected by centrifugation at 1000 × g for 20 min at 4°C and resuspended in buffer B with 1% Triton-X-100 and either 1 mM MgCl2, 1 mM MgATP, or 10 mM MgATP. After 10–20 min on ice, the samples were centrifuged at 100,000 × g for 30 min at 4°C. The supernatant (cytosolic, peripheral, and membrane proteins) was collected, and the insoluble pellet (cytoskeleton and associated proteins) was resuspended in an equal volume of lithium dodecyl sulfate (LDS) sample buffer (Invitrogen). Samples were electrophoresed and analyzed by Western blotting as described above.
Calmodulin Affinity Column
Cell extract was prepared from homogenized whole retinas as described above. The high-speed supernatant containing the soluble fraction from homogenized retinas was diluted to twice its original volume in HBSS/PI and subjected to centrifugation in a Centricon YM-30 (Millipore Corp., Bedford, MA.) with a 30,000 MW cutoff (as described by Battelle et al., 1998
) Once the retinal extract had been reduced to its original volume, it was incubated for 1 h with gentle rocking at 4°C with calmodulin-Sepharose 4B previously washed three times in buffer D/PI (with or without Ca2+
). The calcium-containing buffer was generated by adding 2 mM CaCl2
to achieve a calculated free Ca2+
concentration of 17 μM (Bers et al., 1994
). After the binding step, beads were washed three times in buffer D/PI and incubated with buffer D/PI (with or without Ca2+
) for 1–4 h. The bead supernatant was removed, and electrophoresis sample buffer was added directly to the beads to remove any bound Myo3A. The beads were sonicated and pelleted by centrifugation, and the solubilized sample was examined by SDS-PAGE. As a negative control, the same incubations were done with Sepharose beads alone.