pH sensitivity in human plastin 3 (PLS3) affects bundling activity and interactions with filamentous actin

Biological Sciences: 1st Place (The Ohio State University Denman Undergraduate Research Forum)In the cell, monomeric actin (G-actin) is polymerized into filaments (F-actin), creating the dynamic cytoskeleton, which provides the network for critical cellular functions such as endo- and exocytosis, signaling, motility, and cytokinesis. Plastin 3 (PLS3) is a protein that binds and bundles F-actin, and thus participates in each of these processes. PLS3 bundling activity is inhibited by Ca2+, however other modes of regulation are likely to exist. We have investigated the activity of PLS3 by monitoring actin bundling with light scattering and differential centrifugation. In doing so, we have recently discovered that PLS3 bundling activity changes dramatically between pH 7 and 8, suggesting that pH may play an important role in regulation of bundling activity. As cysteines and histidines are known to be responsive to physiological pH fluctuations, we are exploring their possible involvement in modulation of PLS3 bundling activity. Cysteine modification with excess n-ethylmaleimide or tetra-methyl rhodamine did not affect bundling activity of PLS3, suggesting that surface exposed cysteines are unlikely to be involved in PLS3 regulation. We are currently performing multi site-directed mutagenesis to create a library of PLS3 constructs with His and Cys residues mutated to Lys and Ala residues, respectively. To analyze properties of multiple PLS3 mutants efficiently, we have optimized a small scale PLS3 expression, purification, and characterization. Bundling assays were conducted under various pH conditions and optimized using differential centrifugation and gel electrophoresis. We will assess the actin bundling activity of the PLS3 constructs from mutagenesis to elucidate which residues are involved in pH-dependent regulation of PLS3 activity. PLS3 and two other isoforms of human plastin are implicated not only in many actin-related cellular processes, but also in unique pathologies such as spinal muscular atrophy and cancer metastasis. The primary objective of our research is to elucidate the role of PLS3 in these processes and pathologies.A three-year embargo was granted for this item.Academic Major: Biolog