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First published online 25 September 2007
doi: 10.1242/jcs.000935

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Promotion of lens epithelial-fiber differentiation by the C-terminus of connexin 45.6 – a role independent of gap junction communication

Department of Biochemistry, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA

View larger version (68K): [in this window] [in a new window]   Fig. 1. Expression and co-distribution of wild-type and mutant Cx45.6. (A) Recombinant retroviruses containing cDNAs encoding wild-type Cx45.6 (lane 3) and mutant Cx45.6(D47A) (lane 1) and Cx45.6(P88S) (lane 2) proteins were used to infect CEF cells. After 6 days of infection, cells were lysed, and crude membranes were prepared and analyzed by SDS-PAGE. Western blots were performed by probing PVDF replicas with affinity-purified antibody recognizing the C-terminus of Cx45.6. The membrane was stripped and re-probed with monoclonal antibody against the control protein, -actin. (B) Eight days after infection of recombinant retroviruses, primary cultured cells expressing exogenous wild-type Cx45.6 (a-c), mutant proteins Cx45.6(D47A) (e-g) and Cx45.6(P88S) (i-k) were fixed, stained with DAPI (a,e,i) and labeled with antibody against FLAG (b,f,j) or against Cx45.6 (c,g,k). The primary antibodies were detected by fluorescein-conjugated anti-mouse IgG for the antibody against FLAG and rhodamine-conjugated anti-rabbit IgG for the antibody against Cx45.6. The immunostaining was visualized by confocal fluorescence microscopy. The corresponding merged images derived from a,b,c, e,f,g and i,j,k are shown in d, h and l, respectively. Bar, 10 µm.

View larger version (29K): [in this window] [in a new window]   Fig. 2. Wild-type, not Cx45.6 mutant, proteins form functional gap junction channels that mediate dye transfer. (A,B) Six days after retroviral infection with recombinant retroviruses RCAS(A)-Cx45.6, RCAS(A)-Cx45.6(D47A) and RCAS(A)-Cx45.6(P88S), the scrape-loading dye-transfer assay was performed using RD as a tracer dye (A,B) and Alexa488 (A) or LY (B) as transferring dyes. Bar, 40 µm. The extent of dye transfer was quantified by measuring the distance from scrape lines to the migrated front of cells stained with Alexa 488 (A, lower panel) or LY (B, lower panel). The data are presented as the mean±s.e.m.; n=5. (*P<0.05; ***P<0.001, in comparison with the non-Cx45.6-overexpressing control). Bar, 10 µm. (C) Six days after retroviral infection with recombinant retroviruses RCAS(A)-Cx45.6, RCAS(A)-Cx45.6(D47A) and RCAS(A)-Cx45.6(P88S), a parachuting dye-transfer assay was performed using Dil as a tracer dye and calcein as a transferring dye. The extent of dye transfer was quantified by measuring the area of calcein-fluorescence-stained cells (NIH image; lower panel). The data are presented as the mean±s.e.m.; n=3. ***P<0.001, in comparison with the non-Cx45.6-overexpressing control. Bar, 10 µm.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Mutant proteins Cx45.6(P88S) and Cx45.6(D47A) inhibit the intercellular coupling mediated by wild-type Cx45.6. CEF cells were co-infected with recombinant retroviruses RCAS-Cx45.6 and mutants RCAS(A)-Cx45.6(P88S) and RCAS(A)-Cx45.6(D47A). The extent of dye transfer was quantified by the scrape-loading dye-transfer assay using LY-RD (A) and parachuting dye-transfer assay using calcein-Dil (B). The data are presented as mean±s.e.m.; n=3. ***P<0.001, in comparison with the non-Cx45.6-overexpressing control.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Loss-of-gap-junction-function Cx45.6 mutants stimulate the expression of MIP(AQP0) to levels comparable to those of wild-type Cx45.6. Lens primary cultures were infected with recombinant retroviruses RCAS(A), RCAS(A)-Cx45.6, RCAS(A)-Cx45.6(D47A) or RCAS(A)-Cx45.6(P88S) for 8 days. (A) Crude membrane preparation from cells infected with retroviruses RCAS(A)-Cx45.6(D47A) (lane 1), RCAS(A)-Cx45.6(P88S) (lane 2), RCAS(A)-Cx45.6-WT (lane 3) and RCAS(A) vehicle (lane 4) were loaded on SDS-PAGE gels and immunoblotted with antibodies against Cx45.6, -actin (A) or MIP(AQP0) (B). The Cx45.6 (A, right panel) and MIP(AQP0) (B, lower panel) bands from three separate western blot analyses were quantified by densitometry. The data are presented as the mean±s.e.m.; n=3. *P<0.05, in comparison with the non-Cx45.6-overexpressing control. (C) Primary cells were immunolabeled with monoclonal antibody against MIP(AQP0). The primary antibody was detected by fluorescein-conjugated anti-mouse IgG. The phase-contrast (upper panel) and immunostaining (lower panel) images were captured by a fluorescence microscope. The size of the MIP(AQP0)-stained area was quantified (UTHSCSA ImageTool Software) and presented as a percentage in the x-axis (C, right panel). The data are presented as the mean±s.e.m.; n=3. *P<0.05, in comparison with the non-Cx45.6-overexpressing control. Bar, 10 µm.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Stimulation of lentoid formation and levels of lens differentiation markers filensin and CP49 by wild-type and loss-of-gap-junction mutants of Cx45.6. (A) Lens primary cultures were infected with recombinant retroviruses RCAS(A), RCAS(A)-Cx45.6, RCAS(A)-Cx45.6(D47A) or RCAS(A)-Cx45.6(P88S) for 8 days and the total numbers of lentoids were quantified. (B) Lysates from cells infected with retroviruses RCAS(A) vehicle (lane 1), RCAS(A)-Cx45.6-WT (lane 2), RCAS(A)-Cx45.6(D47A) (lane 3), and RCAS(A)-Cx45.6(P88S) (lane 4) were loaded on SDS-PAGE gels and immunoblotted with antibodies against filensin, CP49 or -actin. (C) The CP49 protein bands from three separate western blot analyses were quantified by densitometry. *P<0.05, in comparison with the non-Cx45.6-overexpressing control.

View larger version (20K): [in this window] [in a new window]   Fig. 6. Stimulation of lens differentiation by Cx56*45.6C but not by Cx45.6*56C. (A) Cx45.6*56C was generated by fusing Cx45.6 lacking its C-terminus with the C-terminus from Cx56. Cx56*45.6C was made by fusing Cx56 lacking its C-terminus with the C-terminus from Cx45.6. (B) Recombinant retroviruses containing cDNAs encoding wild-type Cx45.6 (lane 3), Cx45.6*56C (lane 1) and Cx56*45.6C (lane 2) or RCAS(A) vehicle control (lane 4) were infected into primary lens cultures. After 6 days of infection, lens cell lysates were analyzed by SDS-PAGE and immunoblotted with antibodies against FLAG or -actin. (C) RCAS(A) retrovirus containing Cx45.6, Cx56, Cx45*56C and Cx56*45.6C sequences were infected into CEF cells and intercellular coupling was determined by scrape-loading dye transfer using LY-RD; n=3. (D) 6 days after infection, the total lentoid numbers were quantified at various time points; n=3. (E) 6 days after injection, primary lens cells were labeled with antibody against MIP and detected by fluorescein-conjugated anti-mouse IgG. The percentage of areas with MIP expression versus whole-image areas was determined; n=5. The data are presented as the mean±s.e.m.

View larger version (17K): [in this window] [in a new window]   Fig. 7. Stimulation of lens differentiation by the C-terminus of Cx45.6. (A) Cx45.6C contains the entire C-terminus of Cx45.6. Cx45.6(–C) is a truncated mutant without its C-terminus. (B) Recombinant retroviruses containing wild-type Cx45.6 (lane 1), Cx45.6C (lane 2) and Cx45.6(–C) (lane 3) were infected into primary lens cultures. After 6 days of infection, lens cell lysates were analyzed by SDS-PAGE and immunoblotted with antibodies against FLAG or -actin. (C) 6 days after infection, the total numbers of lentoids formed were quantified at various time points.