Date of Award

7-13-2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Michael Atkinson

Abstract

Gap junctions are aggregates of intracellular channels (plaques) that allow the direct exchange of small molecules and ions between cells (≤ 1kD). Previous research has shown gap junction plaques composed of connexin 43 (Cx43) to be dynamic and mobile structures. We are utilizing a normal rat kidney cell line (NRK), which contains a temperature sensitive viral oncogene (v-src), to study the mobility of gap junction channels within a plaque in transformed (v-src active) and non-transformed NRK cells (v-src inactive). Previous research in our lab has shown a correlation between channel mobility and v-src activation. On average, plaque mobility was reduced in NRK cells with a transformed phenotype (v-src active) when compared to NRK cells with a normal fibroblastic phenotype (v-src inactive). The mechanisms that underlie channel mobility are not known. Current literature has shown Cx43 to interact directly with β-tubulin and indirectly actin. The purpose of this study was to investigate the role of α- and β-tubulin, actin, and connexon trafficking on gap junction channel mobility. Immunofluorescence was performed to identify the localization of our proteins of interest in transformed and non-transformed NRK cells. Fluorescence recovery after photobleaching (FRAP) was used to visualize and quantify the mobility of gap junction channels in plaques of treated (nocodazole or cytochalasin B or brefeldin A) and non-treated NRK cells containing an EGFP tagged connexin 43 (Cx43). Immunofluorescence studies indicate a disruption of the actin and microtubule networks and a reduction cadherin staining at the cell-cell interface in transformed cells. Disrupting microfilaments with cytochalasin B or halting connexon trafficking did not significantly affect fluorescence recovery t1/2 (time it takes for the photobleached area to reach half of the non-photobleached area fluorescence equilibrium). However, disrupting microtubules with nocodazole yielded a significant difference in fast complete t1/2 fluorescence recovery. These results suggest that neither actin nor connexon trafficking affects gap junction channel mobility but that the presence of microtubules may increase gap junction channel mobility.

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