This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The pattern of gap junctional coupling between cells is thought to be important for the proper function of many types of tissues. At present, little is known about the molecular mechanisms that control the size and distribution of gap junctions. We addressed this issue by expressing connexin43 (Cx43) constructs in connexin-deficient HeLa cells. HeLa cells expressing exogenously introduced wild-type Cx43 formed small, punctate gap junctions. By contrast, cells expressing Cx43-GFP formed large, sheet-like gap junctions. However, the effect of Cx43-GFP on gap junction size was rescued by co-expression of untagged wild-type Cx43 using an adenoviral vector. GFP-tagging of Cx43 has been shown to abolish ZO-1 binding (Giepmans et al., 2001) These results suggest that the GFP tag, which is fused to the C-terminus of Cx43, alters gap junction size by masking the C-terminal amino acids of Cx43 that comprise a zonula occludins-1 (ZO-1) binding site. We are currently testing this hypothesis using deletion and dominant-negative constructs that directly target the interaction between Cx43 and ZO-1.GoalTo further address whether the loss of ZO-1 interaction is responsible for the observed increase in Cx43-GFP gap junction size, we will extend our studies by using a smaller epitope tag that can be introduced intramolecularly and that is suitable for live cell and high-resolution imaging. The best candidate for this purpose is the tetracysteine motif (developed in the Tsien & Ellisman labs), which binds fluorescent biarsenic compounds with high affinity, and has shown promise as a fully functional intramolecular tag (unpublished data). In addition, in vitro studies performed by Dr. Hunter at MUSC will address the function of microtubule binding to the C-terminal juxtamembrane domain of Cx43. Specifically, we will determine whether the tubulin binding domain of Cx43 interacts preferentially with the ends of microtubules using a bead binding assay. These in vitro studies will complement current work by Dr. Giepmans at NCMIR which focuses on the interaction between Cx43 and microtubules in eukaryotic cells.Materials & MethodsThe turnover rates of tagged and untagged Cx43 will be determined using both biochemical (at MUSC) and fluorescence microscopy (at NCMIR and MUSC) pulse-chase assays. Subsequent to metabolic labeling with [35S]methionine and various periods of cold chase, cells expressing recombinant connexins will be fractionated into detergent soluble and insoluble pools, which will allow the turnover rate of junctional connexin (insoluble pool) to be differentiated from that of total cellular connexin (soluble pool). Alternatively, the turnover rate of gap junctions will be measured using the technique of Gaietta et al. (2002), in which pre-existing tetracysteine-tagged connexins are pulse-labeled with a fluorescent compound (FlAsH), cells are chased for various periods without label, and then connexins newly synthesized during the chase period are labeled with a second fluorescent compound (ReAsH). Determination of the relative turnover rates of gap junctions composed of different recombinant connexins (e.g. tetracysteine fused to the C-terminus of Cx43-GFP or wild-type Cx43, and internally tagged Cx43 with or without the residues essential for ZO-1 binding), combined with the viral rescue experiments described above, will help to elucidate the mechanisms that regulate the size, distribution and turnover of Cx43 gap junctions. For the microtubule bead binding assay, purified polypeptide comprising the tubulin binding domain of Cx43 and a tetracysteine motif will be bound to FlAsH-conjugated beads. To determine if Cx43 binds preferentially to microtubule ends, beads will be mixed with polarity-marked fluorescent microtubules and the location of bead binding will be assayed by fluorescence microscopy.
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