Does overexpression of ZO-1 alter the gap junction permeability in bone cells? Tony Chen, Thomas H. Steinberg, Washington University School of Medicine, Infections Diseases Division Gap junctions are specialized sections of the cell membrane that connect one cell to another. Gap junctions allow cells to communicate with each other. They are made up of organized collections of protein channels that allow ions and small molecules to be transmitted between cells passively. The individual channels are made up of two connexons, one for each cell; they align and form a continuous pathway between the two cells. Each connexon is made up of six connexins. There are several different connexins found in the bone cells used for experimentation; the one being tested on is Connexin 43. There are several different proteins that are known to interact with Connexin 43, the most prominent one being Zonula Occludens-1, or ZO-1. The rat osteoblastic cells, or ROS cells, that were used in lab expressed Cx43, so they were transfected with the ZO-1myc protein in order to observe the effects of overexpression of the protein on gap junction permeability. One method of testing the effects of ZO-1 on gap junction permeability was dye coupling. Cells were injected with the fluorescent dye Lucifer yellow. Dye was allowed to pass from cell to cell for 3 minutes. A picture was taken, and the number of dye filled cells were counted. It was observed that there is more dye coupling in the ROS/ZO-1myc cells than there is in the ROS cells. The other method of testing ZO-1 was intercellular calcium waves. Calcium waves are increases in the intracellular calcium ion concentration that spread from cell to cell, and were elicited by mechanical stimulation of a single cell. In ROS cells calcium waves spread to other cells via gap junctions. The waves are detected and measured using Fura-2, a fluorescent dye whose fluorescence properties change when the dye binds to a calcium ion. When calcium is not present in the system, Fura-2 emits more 510nm light when excited with 380nm light. When calcium is present in the system, Fura-2 emits more 510nm light when excited with 345nm light. The ROS cells were mechanically stimulated with a blunt end micropipette, which flooded the cells with calcium ions. The cells were excited alternately by 345nm and 380nm light, and the emission of 510nm light was captured in paired digital images, the ratio of which (345/380) was related to the concentration of calcium present in the cells. It was observed that the ZO-1myc cells propagate calcium waves to more cells than the ROS wild type does. The conclusion that can be drawn from these results is the following: because expression of the ZO-1myc protein results in better dye coupling and more calcium waves, it can be concluded that overexpression of ZO-1 increases gap junction coupling in cells, and thus regulates gap junction permeability. Scholar Index Next   Natural Sciences Learning Center Washington University - Biology All contents copyright © 2004