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. All higher, multicellular organisms require intercellular communication for normal tissue and organ function. This process typically involves the transport of ions, small metabolites, and messengers from one cell to its neighbors. One kind of cell-cell junction, gap junctions, form between neighboring cells and serve an essential role in the passage of molecules from the cytoplasm of one cell to its neighbor in both ionic, hormonal and homeostatic capabilities. The functions of gap junctions include coordinating action potentials in the heart, synchronizing neuronal firing and switching, sharing of metabolites, embryonic development, facilitation of hormonal secretion, and transmission of signal transduction by passage of signal molecules. Recent intense investigations have been pursuing the effects that defects in gap junction proteins have in hereditary diseases. Gap junctions play an active role in signal transduction pathways using classic signaling molecules such cAMP and other nucleotides, calcium ions, and inositol triphosphate. These small molecules move through gap junctions from cell to cell, however, details of these signaling pathways and their effects are under intense investigation. The assembly and degradation of gap junctions are dynamic processes and their half-lives are typically ~3-6 hours. Published work has shown that there are considerable active pools of connexins within the cell and at the cell surface that are involved in the connexin trafficking pathways, however, these studies have been limited to biochemical analysis and confocal microscopy. Our goal is to characterize the transport, assembly and molecular interactions of selected heart connexins (Cx43, Cx45 and Cx37) along the secretory pathway and during gap junction internalization. We hypothesize that multiple pathways exist for the transport and assembly of heart connexins and these connexins are internalized by clathrin-dependent and/or independent-mechanisms. Previous technologies to investigate connexin export to the plasma membrane and internalization for degradation have been limited. We plan to use the new and novel tetracysteine (TC)-tagging of connexins as fluorescence probes for examining the lifecycle of heart connexins. TC-tagging and labeling with FlAsH/ReAsH provides several advantages which include: 1) the same TC-tagged connexin can be green or red fluorescence, 2) ReAsH fluorescence can easily be separated optically from GFP-tagged connexins developed for, 3) FlAsH and ReAsH binding is permanent allowing for pulse labeled connexins to be followed thought both the secretory and endocytic pathways, 4) the same ReAsH labeled cells that were observed by light microscopy can be fixed with strong cross-linking reagents for high resolution EM, 5) ReAsH can selectively be localized by EM even though the cells are pre-labeled with FlAsH, and 6) the tetracysteine containing motif is small (2 kD) and less likely to interfere with connexin function in comparison to the much larger GFP (27 kD) tag. We are developing heart connexins tagged with the tetracysteine (TC) motif will exhibit wild-type connexin characteristics and allow for high resolution light and EM analysis of the life-cycle of heart connexins. Gap junctions are dynamic with an in vivo half-life of ~5 hours in liver hepatocyte, 1.3 hours in the heart and 2 hours in cultured cardiac myocytes. Consistent with other polytopic membrane proteins, the gap junction proteins, connexins, are co-translationally inserted into the endoplasmic reticulum (ER). Cx43 and possibly Cx46 do not appear to oligomerize into connexons while in the ER but most likely in the trans Golgi Network (TGN). However, substantial evidence indicates that at least Cx32 and Cx26 can oligomerize within ER membrane. Using antibody microinjection studies and GFP-tagged Cx43, we have recently shown that gap junction internalization can occur via the formation of annular gap junctions where an entire, gap junction or a fragment of a junction is removed into one of the two contacting cells. An alternate pathway of gap junction removal has not been ruled-out where gap junctions disassemble into small aggregates and connexons subsequently internalize into endosomes. Although little evidence exists to date, it is plausible that internalized connexons may recycle to form new gap junction channels. Our initial study with TC-tagged Cx43 (Cx43-TC) has produced exciting new insights into Cx43 transport and turnover. We are characterize TC-tagged Cx37 and Cx45 in parallel with Cx43-TC and comparing these to their FP-tagged and wild-type connexin counterparts. To examine if both Cx45-TC and Cx37-TC are functional, wild-type and Cx37-TC or Cx45-TC expressing HeLa cells will be microinjected with Lucifer yellow or neurobiotin and spreading of these small molecules via gap junctions will be determined. We currently have at disposal the cDNA s for all the heart connexins and these connexins have been FP-tagged and characterized when expressed in a number of cell systems. We are currently generating TC-tagged Cx37 and Cx45 and anticipate that these TC-tagged connexins will exhibit wild type connexin characteristics. With Cx43-TC in hand we have begun engineering TC-tagged Cx45 and Cx37 as described. Briefly, DNA encoding the TC peptide or linker variations of this sequence will be fused in frame to the 3 -end of the connexin cDNA and sequenced to ensure fidelity. Selected cell lines are either transfected or infected with cDNAs encoding wild-type, FP-tagged or TC-tagged heart connexins using standard transfection protocols. We developed an antibody against the TC 777 tag as an additional tool for tracking and verifying TC-constructs. We have four cell lines to examine the transport and assembly of heart connexins. 1) Mouse HL-1 cells retain phenotypic characteristics of adult cardiomyocytes expressing -cardiac myosin heavy chain and -cardiac actin, allowing them to actively contract in culture. 2) We are using well- characterized human coronary artery endothelial cells (HCAEC; Clonetics) which mimic the endothelial lining of major vessels. 3) Finally, we use communication-competent Normal Rat Kidney (NRK) cells and communication-deficient human carcinoma (HeLa) cell lines that have been routinely used for several previous studies.
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