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First published online 3 February 2004
doi: 10.1242/jcs.00945

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Interaction of major intrinsic protein (aquaporin-0) with fiber connexins in lens development

Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA

View larger version (68K): [in a new window]   Fig. 1. Amino acid sequence of chick lens MIP(AQP0). (A) Sequence of cDNA clone of MIP(AQP0). A single uninterrupted open reading frame starting at nucleotide 67 and ending at nucleotide 885 (bold, underline), encoding a protein with a predicted molecular mass of 28.1 kDa. The derived amino acid sequence of MIP(AQP0) is shown in lower line. This sequence is registered in the GenBank database under the accession number AY078179. (B) The deduced amino acid sequence of chick MIP(AQP0) in one-letter code is aligned to the bovine lens MIP(AQP0) (K02818). Identical residues are shaded. Putative membrane-spanning domains (TM) are labeled and underlined.

View larger version (49K): [in a new window]   Fig. 2. Co-immunoprecipitation of MIP(AQP0) with embryonic lens fiber connexins. [35S]-Methionine-labeled gap-junction-rich membranes isolated from embryonic-day-9 lenses were immunoprecipitated with affinity-purified anti-Cx45.6 (lanes 1 and 2) and anti-Cx56 (lanes 3 and 4) antibodies in the absence (lanes 2 and 4) or presence (lanes 1 and 3) of 0.6% SDS. Lens lysate was immunoblotted by anti-MIP(AQP0) antiserum (lane 5).

View larger version (98K): [in a new window]   Fig. 3. Expression and colocalization of MIP(AQP0) and Cx45.6 detected by dual-immunostaining of sagittal sections of embryonic-day-10 and -20 lens. (A) Sagittal sections from bow (A-C) and core (D-F) regions of embryonic-day-10 chick lens were prepared and co-immunostained with antibodies specific for MIP(AQP0) and Cx45.6, and subsequently stained with fluorescein-conjugated goat anti-mouse IgG for MIP(AQP0) (A,D) and followed by rhodamine-conjugated goat anti-rabbit IgG for Cx45.6 (B,E). The corresponding images from the same regions were merged together to demonstrate the overlapping patterns between these two proteins (C,F). Scale bar, 20 µm. (B) Sagittal sections from embryonic-day-20 chick lens were similarly prepared and co-immunostained with anti-MIP(AQP0) monoclonal antibody (A,D) and affinity-purified anti-Cx45.6 (B,E) antibodies. The merged images were shown in (C,F). Scale bar, 20 µm.

View larger version (118K): [in a new window]   Fig. 4. Expression and colocalization of MIP(AQP0) and Cx45.6 detected by dual-immunostaining of coronal sections of embryonic-day-10 and -20 lens. (A) Coronal sections from bow (A-C) and core (D-F) regions of embryonic-day-10 chick lens were prepared and co-immunostained with antibodies specific for MIP(AQP0) and Cx45.6, and subsequently stained with fluorescein-conjugated goat anti-mouse IgG for MIP(AQP0) (A,D) and followed by rhodamine-conjugated goat anti-rabbit IgG for Cx45.6 (B,E). The corresponding images from the same regions were merged together to demonstrate the overlapping patterns between these two proteins (C,F). (B) Coronal sections from embryonic-day-20 chick lens were similarly prepared and co-immunostained with anti-MIP(AQP0) (A,D) and Cx45.6 (B,E) antibodies. The merged images were shown in (C,F). Scale bar, 20 µm.

View larger version (28K): [in a new window]   Fig. 5. Co-existence of MIP(AQP0) in the complex formed by Cx45.6/Cx56. Gap-junction-rich embryonic lens membranes were isolated from embryonic-day-11 lenses and immunoprecipitated with immobilized affinity-purified anti-Cx45.6 (A-C, lanes 1 and 2), anti-Cx56 antibodies (A-C, lanes 3 and 4) or anti-MIP(AQP0) antiserum (A-C, lanes 5 and 6) in the absence (A-C, lanes 1, 3 and 5) or presence (A-C, lanes 2, 4 and 6) of SDS. The resulting immunoprecipitates were immunoblotted with affinity-purified anti-Cx45.6 (A) or anti-Cx56 (B) antibodies, or with anti-MIP(AQP0) antiserum (C). The SDS-boiled precipitates of Cx45.6-antibody-conjugated beads alone were blotted by anti-MIP(AQP0) antiserum (C, lane 7). The cross-reacting immunoglobulin light chain was shown as (*). (D) Silver staining of the samples from membrane preparation of embryonic-day-10 lens (lane 1), supernatant of immunoprecipitation of lens membranes with anti-Cx45.6 antibody (lane 2), immunoprecipitates of lens membrane with anti-Cx45.6 antibody (lane 3), fivefold overloaded sample (lane 4), SDS-boiled precipitates with Cx45.6-antibody-conjugated beads in the absence of lysates (lane 5) and precipitates with non-Cx45.6-antibody-conjugated beads (lane 6) in the presence of lysates. The immunoprecipitates of lens membranes with anti-Cx45.6 antibody were immunoblotted by anti-MIP(AQP0) monoclonal antibody (lane 7).

View larger version (12K): [in a new window]   Fig. 6. C-terminus of MIP(AQP0) pulled down lens-fiber connexins from chick embryonic-day-10 lens lysates. Ni-NTA beads conjugated with six-histidine-tagged C-terminus of MIP(AQP0) were used to pull down proteins from lens lysates. Total lens lysates (lane 1) and retained fractions of lysate by Ni-NTA beads in the absence (lane 2) or presence (lane 3) of fusion protein were visualized by Coomassie Blue staining. The retained fraction of lysate by fusion-protein-conjugated-beads (lanes 4 and 7), the flow-through factions after extraction by fusion-protein/Ni-NTA beads (lanes 5 and 8) and total lens lysate samples (lanes 6 and 9) were immunoblotted with affinity-purified anti-Cx45.6 antibody (lanes 4-6) and anti-Cx56 antibody (lanes 7-9).

View larger version (124K): [in a new window]   Fig. 7. Transient interactions of MIP(AQP0) with lens-fiber connexins at bow regions of adult lenses. Lens sections prepared from 1-month-old chicken were double labeled with monoclonal anti-MIP(AQP0) antibody and affinity-purified anti-Cx45.6 antibody, and subsequently labeled with fluorescein-conjugated goat anti-mouse IgG (A,D,G,J) for MIP(AQP0) and followed by rhodamine-conjugated goat anti-rabbit IgG for Cx45.6 (B,E,H,K). The corresponding images from the same regions were merged together to demonstrate the overlapping patterns between these two proteins (C,F,I,L). (A-C) A region at the lens bow regions; (D-F) a region at the anterior part of the lens fibers; (G-I) a region at the posterior part of the lens fibers; (J-L) a region at the center core of the lens fibers. Scale bar, 20 µm.

View larger version (43K): [in a new window]   Fig. 8. MIP(AQP0) did not form a complex with lens-fiber connexins in adult chick lens. Gap-junction-rich lens membranes from postnatal 1-month-old chicken were prepared and immunoprecipitated with immobilized affinity-purified anti-Cx45.6 (lanes 1 and 2) or anti-Cx56 antibodies (lanes 3 and 4) antibody, or anti-MIP(AQP0) antiserum (lanes 5 and 6) in the absence (lanes 1, 3 and 5) or presence (lanes 2, 4 and 6) of SDS. The resulting immunoprecipitates were immunoblotted with anti-Cx45.6 (A) or anti-Cx56 (B) antibodies, or anti-MIP(AQP0) antiserum (C). The cross-reacting immunoglobulin light chain is shown as (*).

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