Development of HeLa Cell Lines Expressing Specific Connexins
Authors: Saito Mikako , Hiratoko Shoya , Tokunaga Naruwa , Ogawa Yoshihide 
Published: February 2020

Introduction

In a biological system, the cell-cell communication is essential to maintain the homeostasis of the system under stress conditions as well as during drastic transitions of differentiation. Gap junctions (GJs) are thought to be facilitating the intercellular communication by forming channels for ions, small molecules, and second messengers to pass through to the neighbor cells. The GJ comprises various isoforms of connexin (Cx), a functional protein. Actually the GJ molecular movement occurs via GJ plaque, a cluster of a number of GJ.
To date a large number of studies have been reported about the relevance of Cx to various growth stages, metabolic conditions, tissues and organs, and diseases. Special attention has been paid to the potential role as tumor suppressors. Those studies have been concentrated on the specific roles of several isoforms such as Cx31, Cx43, and Cx45, because they are more ubiquitously expressed than other isoforms. However the specificity of their respective roles is still unclear. Moreover it should be necessary to consider the simultaneous actions of multiple isoforms rather than the specific action of a particular single isoform alone.
The open-close properties of GJ were well analyzed formerly. The GJ comprising Cx43, for example, was analyzed by whole cell voltage clamp method. The open state, however, did not imply the single state of complete opening. Rather, there were multiple open states with different opening rates. The transition manners from one state to another were different depending upon chemical conditions in the cytosol and/or membrane potential conditions.
Once GJ plaque is formed between the neighbor cells of interest, we can understand the molecular movement via the GJ plaque occurs. However, we cannot understand why GJ plaques were not formed between every neighbor cell, though there seemed to be no difference between those neighbor cells. In order to solve this problem, we intended to develop an experimental platform to analyze the role of each Cx isoform in the regulation of GJ plaque localization and in the intercellular molecular movement.
Here we have selected several Cxs such as Cx26 and Cx30.3 as a demonstrative isoform and developed the cell lines expressing Cx26 and/or Cx30.3 for the dynamic analysis of the role of respective Cxs in the gap junctional molecular movement. The functional analysis needs the quantitative introduction of a diffusion marker dye into only a target single-cell. Such an experiment could be successfully conducted by means of a femtoinjection system.

Material and Methods

HeLa cells were cultured in GMEM supplemented with 10% FBS for 24 h until a confluent condition. Then pCAG-Cx26-IP-EGFP of pCAG-Cx30.3-IP-EGFP was transfected with Gene Pulser II. The cells were transferred to a selection medium containing 3 µg/µl puromycin.
The molecular size cut off by GJs composed of Cx26 or Cx30.3 is estimated as 1000-1500. Therefore we have selected Lucifer yellow (LY, MW: 443 Da) as a permeable dye. A 5 µg/µl LY solution was filled in a glass capillary. By means of a single-cell manipulation supporting robot, the capillary was injected into a target HeLa single-cell that expressed Cx26 and/or Cx30.3 and the dye solution was introduced into the cell under a condition of 0.7 kg·cm-2 for 10 ms. Then the cells were observed with a fluorescent microscope.

Results and Discussions

A pair of HeLa cells expressing Cx26, which could be confirmed by the expression of EGFP reporter protein, was selected to investigate the intercellular molecular movement via GJ. LY was introduced into one of the pair cells and the fluorescent intensities in both cells were measured. When EGFP fluorescent intensities in both cells were sufficiently high, the LY movement occurred. In the same way, the LY movement was studied using a pair of HeLa cells expressing Cx30.3. However no LY movement was observed. The intracellular localization of Cx30.3-EGFP was mostly in cytosol. In order to facilitate the incorporation of Cx30.3 in the cell membrane, Cx26 or Cx31 was co-expressed in the Cx30.3 expressing HeLa cells. Consequently only the Cx30.3-Cx31 co-expressing cell line could be successfully developed. The diffusion of LY injected at a pinpoint of the cytosol proceeded throughout the cytosol and nucleus within 1.6 s. On the other hand, intercellular diffusion of LY proceeded and became a steady level within 50 s. The dynamic properties of LY movement seemed not to be linear versus the Cx expression intensity as well as versus the time. The potential role of gap junction composed of Cx26 and/or Cx30.3 will be discussed from the viewpoint of regulation of gap junctional molecular movement.