Article Figures & Data
Figures
- Figure 3.
The IAV channel is gated in response to hypo-osmotic stress. A, IAV-expressing cells exhibit single Ca2+ peaks (gray) or oscillations (black) during prolonged hypo-osmotic stimulation (214 mOsm/kg, 2 mm Ca2+). U, Arbitrary unit of Ca2+ signals quantified by confocal microscope. B, Dependence of Ca2+ spike amplitude on osmolality. The mean amplitudes of the Ca2+ peak in response to hypotonic solutions (214, 227, 239, 252 mOsm/kg) are normalized by the Ca2+ signals in isotonic solution (298 mOsm/kg). Error bars represent SEM. Data were recorded from >100 cells in five independent experiments. C, D, Ionic currents and I-V relationship stimulated by hypotonic solutions (221 mOsm/kg) in IAV-expressing cells in a whole-cell patch configuration. Step and ramp voltage clamp was applied to IAV channel-transfected HEK293T cells, and the resulting currents were recorded. IAV channel showed delayed activation after hypotonic solution treatment, and the reversal potential was ∼0 mV.
- Figure 1.
Mapping the iav locus. A, Deficiency-duplication mapping. The indicated deficiencies and duplication were crossed to iav1 and scored for ability to complement the locomotor inactivity phenotype. The right edge of the candidate region is defined by the gene ogre, which complements iav and is to the left of Df(1)3196 but is uncovered by Df(1)947 (Curtin et al., 1999). The left edge is defined by the lethal P element insertion l(1)G0254 (Schaefer et al., 1999), which is in the predicted gene CG4094 in 6C10-11 (data not shown). This insertion is not rescued by Dp(1;3)sn[sn13a1], indicating that it is left relative to the duplication. B, The iav candidate region at higher resolution assembled from Flybase (The FlyBase Consortium, 2003). Predicted genes are shown above and below the line representing the genetic region. The circles show P-element insertions, and inverted triangles a-d represent sequence polymorphisms used for recombination mapping (see Materials and Methods). The left extent of the candidate region is defined by the lethal P insertion l(1)G0254 in cytogenetic region 6C5 and the right by the gene ogre in 6E4, because Df (1)3196 (Df(1)Sxl-bt) removes ogre yet complements iav (Curtin et al., 1999). C, CG4536 gene structure and lesions in the iav alleles. Predicted structure of the iav protein and the iav mutant alleles. Gene structure prediction is from Flybase release 3.1 (The FlyBase Consortium, 2003) and from PCR 5′ and 3′ RACE analyses and sequencing.
- Figure 2.
A hearing defect in iav mutants. Recordings of antennal sound-evoked potentials from control (iav+), iav mutant, and transgenic iav; P[iav+] flies in response to repeated pulse trains. Records shown are averaged responses in single antennae to 10 or (where noted) 100 pulse stimuli. Mutants hemizygous for either iav allele show no trace of a response, whereas a single wild-type transgene P[iav+ 2.2] restores a normal response to the iav1 background.
- Figure 4.
Expression, localization, and interdependence of IAV and NAN in chordotonal cilia. A, In situ hybridization of an iav antisense RNA probe to embryos at late stage 16. Lateral (lch) and ventral (vch) chordotonal organs are indicated and enlarged in the inset. B-G, Immunostaining of the second antennal segment of wild-type (B, E), iav1 (C, F), and nan36a (D, G) with anti-IAV or anti-NAN serum (red). The anti-IAV serum was directed against the N-terminal cytoplasmic region (anti-IAV-N; see Materials and Methods); similar results were seen with an anti-IAV C-terminal antiserum (data not shown). The mAb 22C10 (Zipursky et al., 1984), which labels a neural cytoskeletal protein in the cell bodies and inner segments but not the ciliary outer segments, is shown in green. The arrowheads denote the position of the outer segments.
- Figure 5.
GFP-IAV requires NAN for normal localization. Expression of GFP-IAV (green) in Johnston's organ of wild-type (A-C) and nan mutant (D-F) flies. Neurons (except for cilia) were also stained with the mAb 22C10 (B, E); the merged images are also shown (C, F). No ciliary GFP signal is detected in the nan mutants. Scale bars, 5 μm.
- Figure 6.
Restricted localization of IAV in chordotonal cilia. Expression of a functional GFP-IAV (green) or GFP-NOMPA (cyan) transgene in larval chordotonal organs (lch5) showing proximal versus distal intracellular localization. Neurons are counter labeled with RFP (magenta) expressed under the control of the neural-specific elav-GAL4 driver. A, Elav-driven RFP; B, GFP-IAV; C, merge of A and B; D, GFP-NOMPA (cyan) and RFP; E, schematic of a single larval scolopidium (Chung et al., 2001) showing the position of the ciliary dilation and dendritic cap. GFP-IAV labeling is restricted to the region proximal to the ciliary dilation, whereas NOMPA-RFP is distal to the dilation. Scale bars, 5 μm.
Additional Files
Supplemental data
Files in this Data Supplement:
- supplemental material - Supp 1 Clustal Alignment of Iav, CeOSM9, Nan, and hTRPV4. Invariant amino acides are shown in black, similar amino acids in gray. Gray bars above the sequence represent the five ankyrin repeats, and the six black bars represent the transmembrane domains. Ion channel pore domain is shown in gray between the fifth and sixth transmembrane domains.
- supplemental material - Supp 2 Clustal Alignment of Iav, CeOSM9, Nan, and hTRPV4. Invariant amino acides are shown in black, similar amino acids in gray. Gray bars above the sequence represent the five ankyrin repeats, and the six black bars represent the transmembrane domains. Ion channel pore domain is shown in gray between the fifth and sixth transmembrane domains.
- supplemental material - Supp3 PCR primers used for amplification of the polymorphic regions used in iav mapping.
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