Fluorescent Labeling of Connexin with As Complex and X-Y Coordinate Registration of Target Single Cells Based on a Trian
Gap junction (GJ) research has entered a new stage focusing the concerted dynamic behavior of multiple isoforms of connexin (Cx) in the cell membrane, cytosolic vesicles, and space between them. To proceed with this research, imaging technologies are impo
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Fluorescent Labeling of Connexin with As Complex and X-Y Coordinate Registration of Target Single Cells Based on a Triangle Standard Chip for the Image Analysis of Gap Junctional Communication Mikako Saito Abstract Gap junction (GJ) research has entered a new stage focusing the concerted dynamic behavior of multiple isoforms of connexin (Cx) in the cell membrane, cytosolic vesicles, and space between them. To proceed with this research, imaging technologies are important. Here we describe two novel protocols for this purpose. At first, the adoption of a small motif of Cys-Cys-X-X-Cys-Cys as a visualization tag is described. An As complex, FlAsH, can bind to this tetra-Cys (TC) tag to form a fluorescent conjugate. Its introduction into the C-terminal of Cx43 is demonstrated. Next, a novel triangle chip for the accurate x-y registration is described. Target single cells of HeLa marked with a fluorescent dye can be easily recognized by electron microscopy based on this chip. Key words Connexin, Tetra-Cys tag, Target single cells, Triangle standard chip
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Introduction Gap junctional communication is well understood to be relevant to various growth stages, metabolic conditions, and diseases by facilitating direct intercellular movement of small molecules [1, 2]. A gap junction (GJ) comprises six connexin (Cx) molecules. There are 21 Cx isoforms in humans and 20 isoforms in mice [3]. Cx proteins are synthesized in the endoplasmic reticulum and transported to the Golgi apparatus [4]. During this transportation, a Cx molecule is folded into three-dimensional structure and then oligomerized into a hexameric hemichannel, a connexon. Connexons in the Golgi apparatus are packaged into vesicles and transported to the cell membrane. An individual connexon from one cell associates with a corresponding connexon on a neighboring cell to form a GJ channel. Usually multiple channels aggregate in the cell membrane to form GJ plaques [5]. Conversely, the process of decomposition into single Cx molecules is also understood to progress in the
Mikako Saito
cytoplasm [6]. To date, such a general view of the dynamic behavior of Cx has been analyzed mostly by molecular biological methods with image data provided by fluorescent microscopy. Recently, special attention has been focused on the potential role of Cx as tumor suppressors [7]. The specific roles of several isoforms such as Cx43 and Cx26 have been well investigated because they are more ubiquitously expressed than other isoforms. Until now, however, it is still unclear whether they are suppressive [8, 9] or progressive [10, 11]. A breakthrough to find a solution to this problem should be given by the analysis of the spatiotemporal behavior of Cx and its associated clusters with as high resolution as possible. In this new research stage, it has been recognized to be important to pay special attention to such isoforms that might be formerly ignored because of low expression and short lifetime [12]. Consequently, we have focused our efforts on the improvement of live imaging applica
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