(447a) Investigating the Spatiotemporal Dynamics of Paxillin Isoform Switching in Epithelial-Mesenchymal Transition

Authors: 
Reddick, M., Stanford University
Ghosh, R., Stanford University
Liphardt, J., Stanford University

The ability of cells to crawl in their microenvironment is
the dominant means of motility in eukaryotic cells. In mammals, cells that
normally are connected via cell-cell junctions must break their connections in
order to transform to a motile phenotype and crawl independently, a progression
known as epithelial-mesenchymal transition (EMT). EMT is necessary for proper
organization of organs and tissues throughout development and for wound healing
in response to lesions. However when EMT is not properly regulated, it may lead
to life threatening consequences. For example, pathogenic fibrosis can occur when
chronic inflammation leads to excessive accumulation of extracellular matrix
(ECM) material deposited by too many EMT produced mesenchymal
cells. Another example is that of cancer cells that highjack the EMT regulatory
pathway, allowing cells to metastasize by separating from the primary tumor
site and migrate to new regions of the body. Once the metastatic stage is
reached, complete removal of the cancer from the patient is highly improbable.

Interestingly, in 2005 Tumbarello et. al. showed
that a translationally truncated isoform of the focal
adhesion protein paxillin (termed paxillin
d)
is exclusively expressed in epithelial cells and not in cells of mesencyhmal origin, suggesting that paxillin
isoform switching plays a role in EMT. We have developed a means to study paxillin isoform switching dynamics within many individual
focal adhesions across different cell types and under various chemical and
physical perturbations. Briefly, we have utilized CRISPR-Cas9 genome editing to
tag both the paxillin N and C termini, each respectively
with a fluorescent protein in the human mammary epithelial cell line MCF10A and
the cancer derived SUM159 cell line (see figure). We then use confocal
fluorescent microscopy and Total Internal Flourescence
Microcsopy (TIRM) to visualize the isoform
distribution within focal adhesions of single cells, and observe their
spatiotemporal dynamics. The advantage of our CRISPR-Cas9 generated cell
lines are both the maintenance of the native regulation of paxillin
expression and the ability to capture paxillin protein
dynamics in living cells, both of which are not achievable with previous
methods alone (e.g. exogenous protein expression or immunofluorescence).

Here we will discuss the relationship between paxillin isoform distributions and their respective
lifetimes within focal adhesions, spatial location within cells (e.g. leading
edge of lamellipodia), across epithelial and mesenchymal cell types, and EMT. Furthermore, we explore
how biasing focal adhesions to be predominantly populated by one isoform versus
the other affects cell phenotype and motility, allowing us to construct a
simple systems biology description of how paxillin
isoform switching influences large scale changes in the cell.