Evaluating the Time Course of Dye Uptake for Mechanically Stimulated HeLa Cells Expressing Cx43 Hemichannels
The formation of a continuous channel between two cells
uniquely defines gap junctions in comparison to desmosomes and tight junctions.
With over 20 different types in mammals, connexins (Cxs) are the major subunits
of gap junctions in all vertebrates.
Two hemichannels (connexons), each formed from six connexin subunits,
align to form one cell-to-cell channel.
A collection of these channels form a plaque referred to as a gap
junction. Analysis of the on-going
synthesis and degradation of gap junction channels has led to research about
their trafficking and regulation. The gating (opening and closing) of channels provides
an additional layer of regulation. It is now clear that hemichannels have the
ability to open and connect the intra- and extracellular environment. Mechanical stimulation (at low calcium
concentrations) greatly enhances the probability of hemichannels being open in
a population of cultured cells.
This technique, in conjunction with dye uptake and image analysis,
offers a novel approach for quantifying the presence of hemichannels in the plasma
membrane. This capstone project
evaluated the time course of dye uptake for mechanically stimulated HeLa cells
expressing Cx43 hemichannels. The
following hypothesis was proposed: Cellular uptake increases linearly for the entire
time period of dye exposure during a 5 minute treatment. The results provide
evidence of biphasic dye uptake, with a rapid phase followed by a sustained,
linear phase.