| dc.description.abstract | Metastasis is the most clinically significant step in cancer progression. Migration and
metastasis are not fully understood, but it is clear that the actin cytoskeleton plays an essential role.
Palladin is specifically involved in metastasis of cancer cells, but also co-localizes with actin stress
fibers in normal cells. The 90 kDa palladin is the only ubiquitously expressed isoform and contains
three Ig domains and one proline-rich region. This proline-rich region has been shown to bind
directly to the actin-regulating protein VASP. In a previous paper, our lab showed that the Ig3
domain of palladin is the minimal binding site for F-actin. In this work we wanted to compare
functions of the 90 kDa palladin to the isolated actin binding domain. Our hypothesis was that the
90 kDa palladin may be autoinhibited and may thereby block the binding site for monomeric actin.
To understand the mechanism of action for how palladin can influence actin assembly, we used
fluorescence spectroscopy to monitor pyrene actin polymerization. By using site-directed
mutagenesis via PCR we were able to mutate the putative VASP binding site within the prolinerich
region of the 90 kDa palladin. We then examined binding between VASP and WT or mutant
palladin using a pulldown assay and far Western blot. Both palladin and VASP proteins are
involved in the regulation of actin filaments and understanding the fundamental mechanism of
these proteins may help us eliminate the progression of cancer invasion and metastasis. In addition,
we sought to determine how palladin and VASP are involved in actin assembly required for cell
motility. A facultative intracellular pathogen, Listeria monocytogenes, has been used to study the
regulation of palladin in the regulation of actin dynamics. Our aim is to test the hypothesis that
palladin promotes the nucleation, elongation and the stabilization of actin-based structure during
cell motility. Understanding the role of palladin in actin cytoskeleton may help us prevent cancer
cells from reaching the metastasis stage of cancer progression. | |
