Insights into the Role of Cell–Cell Junctions in Physiology and Disease - International Review of Cell and Molecular Biology - page 202

2005; Kaz et al., 2012; Kurzen et al., 2003; Maynadier et al., 2012; Oshiro
et al., 2005; Yang et al., 2012; Yashiro et al., 2006 ). Whether these alter-
ations act via the release of plakoglobin from desmosomes and subsequently
engage the plakoglobin-dependent pathway, or an alternative pathway, is
still an open question.
5. STUDIES OF JUNCTIONS
5.1. Techniques for studying junctions
Many conventional molecular biology methods were deployed to examine
junctional regulation of cell behavior. These include transfection, immuno-
blotting (western blotting), cell tracking for migration, wound healing,
cell-substrate adhesion assays, and immunofluorescence staining. For studies
focusing on junctions in particular, however, investigators have had to develop
some new approaches to more precisely characterize cellular properties.
A measure of how junctional proteins may respond to external stimuli can
be acquired by quantifying the fluorescence signal for an immunofluorescently
stained sample. In theory, the majority of the junctional proteins reside at cel-
lular junctions; thus by selecting appropriate image intensity thresholds, one
canmeasure the pixel density of the immunoreactive signal and thus gain some
idea about the increase or decrease of the protein at the junction. This mea-
surement was used to demonstrate increases in gap junction and desmosomal
proteins in various cell types in response to applied cyclic stretch ( Huang et al.,
2008; Yamada et al., 2005a ).
Measurement of forces generated across cell junctions required an adap-
tation of classical traction-force microscopy by including a constraint regard-
ing the total force in a region of cells (i.e., no net acceleration in any
particular direction). Using this constraint, Trepat et al. (2009) demonstrated
that cell-cell forces are not uniform in confluent patches of cells; rather there
are focal locations of high cell-cell forces, apparently near the edges of cell
“islands,” with a mostly low force interior.
Micropatterning, expanding from its ability to shape individual cells, is
now being used to shape small groups of contacting cells, whether to pre-
cisely control the contacting geometry or to arrange the type of contacting
cells, such as for heterotypic interactions ( Tseng et al., 2012 ). Recently, the
use of micropillars has given rise to precise studies of cell-cell forces in cell
pairs ( Liu et al., 2010 ) or in cells that are exposed to external mechanical
stimuli ( Ting et al., 2012 ).