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First published online 26 April 2005
doi: 10.1242/jcs.02329

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ZO-1 alters the plasma membrane localization and function of Cx43 in osteoblastic cells

Department of Internal Medicine, Washington University School of Medicine, St Louis, MO 63110, USA

View larger version (30K): [in a new window]   Fig. 1. The ZO-17-444 fusion protein interacts with the carboxyl termini of Cx43, Cx45 and Cx46, but not Cx32. The ZO-17-444 fusion protein was fractionated on agarose beads conjugated to the carboxyl termini of Cx43, Cx45, Cx46 or Cx32. The resins were washed and the bound material was analyzed by SDS-PAGE and fluorography. The position of molecular mass markers is given in kDa.

View larger version (53K): [in a new window]   Fig. 2. Expression of the ZO-17-444 fusion protein in transfected ROS cells. (A) ROS/ZO-1dn cells were harvested and analyzed by immunoblotting with the Xpress tag antibody. A 54 kDa immunoreactive band is seen in the transfected cells. The position of molecular mass markers is given in kDa. (B) Localization of the ZO-17-444 fusion protein in ROS/ZO-1dn cells. ROS and ROS/ZO-1dn cells (clone E) were fixed, permeabilized, and stained with Xpress epitope tag antibody. Scale bar: 25 µm.

View larger version (51K): [in a new window]   Fig. 3. Expression of the ZO-17-444 fusion protein disrupts the Cx43-ZO-1 interaction. (A) Cx43 associates with the ZO-17-444 fusion protein in transfected cells. ROS/ZO-1dn cells were solubilized in lysis buffer and lysates were fractionated on a cobalt chelate affinity resin. Bound material was analyzed by immunoblotting with Cx43 antiserum. Cx43 was detected in precipitates from high expressor ROS/ZO-1dn clones E, J, and K, but not in ROS or low expressor ROS/ZO-1dn clone A cells. (B) ROS/ZO-1dn cells were solubilized, immunoprecipitated with Cx43 antiserum, run on SDS-PAGE, and immunoblotted with an anti-ZO-1 monoclonal antibody. ZO-1 co-precipitated with Cx43 in ROS and in the low expressor ROS/ZO-1dn clone A cells, but not in high expressor ROS ZO-1dn clones E, J, or K. The position of molecular mass markers is given in kDa.

View larger version (13K): [in a new window]   Fig. 4. Disruption of the Cx43/ZO-1 interaction reduces gap junctional communication in transfected ROS cells. Lucifer yellow was injected into single cells in confluent monolayers, and the number of dye filled cells was scored after 3 minutes (N=25). Dye transfer was significantly reduced in ROS/ZO- and clones E, J, and K compared to parental ROS cells. Results are expressed as the mean number of dye filled cells per injected cell ± s.d.

View larger version (41K): [in a new window]   Fig. 5. Expression of the ZO-17-444 fusion protein does not alter Cx43 abundance. (A) Transfected ROS cells were harvested and solubilized in 1% SDS and analyzed by immunoblotting. (B) Cells were incubated for 1 hour in PBS containing 100 µg/ml NHS-LC-biotin and solubilized in lysis buffer, and the biotinylated proteins were isolated with Neutravidin agarose (Pierce). The resins were extensively washed, and the bound material was analyzed by immunoblotting with Cx43 antiserum. The position of molecular mass markers is given in kDa.

View larger version (73K): [in a new window]   Fig. 6. Expression of the ZO-17-444 fusion protein reduces appositional membrane staining for Cx43 in transfected ROS cells. (A) Cells were fixed, permeabilized and simultaneously stained with the Sigma polyclonal antibody directed against Cx43 and a monoclonal antibody directed against ZO-1. (B) Expression of ZO-1 in transfected ROS cells. ROS cells and transfected ROS cells were analyzed by immunoblotting with the ZO-1 monoclonal antibody. Scale bar: 25 µm.

View larger version (68K): [in a new window]   Fig. 7. Expression of the ZO-17-444 fusion protein alters the mobility of Cx43 on sucrose flotation gradients. ROS and ROS/ZO-1dn cells were harvested in 1% Triton X-100, equilibrated in 40% sucrose, overlaid with a discontinuous 5-30% sucrose gradient, and centrifuged. Gradient fractions were collected, separated by SDS-PAGE and Cx43, ZO-1 and caveolin-1 were identified by immunoblotting. Fraction 1 was derived from the bottom of the sucrose gradient (40% sucrose), while fraction 9 was derived from the top of the sucrose gradient (5% sucrose).

View larger version (79K): [in a new window]   Fig. 8. Expression of the ZO-17-444 fusion protein does not alter the expression of N-cadherin in osteoblastic cells. (A) Immunofluorescence analysis of transfected ROS cells with Cx43 polyclonal antibody and the N-cadherin monoclonal antibody. (B) ROS cells and transfected ROS cells were analyzed by immunoblotting with the N-cadherin monoclonal antibody. Scale bar: 25 µm.

View larger version (55K): [in a new window]   Fig. 9. Over-expression of ZO-1 increases gap junctional communication and punctate appositional membrane staining for Cx43. (A) Immunofluorescence localization of Cx43 in ROS cells and ROS/ZO-1myc cells with antibodies directed against Cx43, ZO-1 or the myc epitope. Scale bar: 25 µm. (B) Cells were fixed, permeabilized, and simultaneously stained with the Sigma polyclonal antibody directed against Cx43 and a monoclonal antibody directed against ZO-1. The cells were then probed with a CY-3 donkey anti-rabbit IgG and an Alexa 488 goat anti-mouse IgG, and examined with the appropriate fluorescence filters. Scale bar: 25 µm. (C) Over-expression of the ZO-1 increases gap junctional communication in transfected ROS cells. Lucifer Yellow was injected into single cells in confluent monolayers, and the number of dye filled cells was scored after 3 minutes (N=25). Results are expressed as the mean number of dye filled cells per injected cell ± s.d.

View larger version (15K): [in a new window]   Fig. 10. Altered expression of Cx43 in ROS/ZO-1myc cells. (A) ROS cells and transfected ROS cells were solubilized in 1% SDS. Equal aliquots of protein were analyzed by immunoblotting with a Cx43 antibody. (B) Cx43 associates with ZO-1myc in the ROS/ZO-1myc cells. Cx43-associated proteins were isolated from lysates derived from ROS/ZO-1myc cells using our co-immunoprecipitation protocol. Immunoblotting with the anti-myc monoclonal antibody identified ZO-1myc in the immunoprecipitates derived from ROS/ZO-1myc cells. (C) Cells were incubated 1 ml of PBS containing 100 µg/ml NHS-LC-biotin and solubilized in lysis buffer. The biotinylated proteins were isolated with Neutravidin agarose (Pierce). The resins were extensively washed and the bound material was analyzed by immunoblotting with Cx43 antiserum.

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