Monitoring Palladin's Effect on Actin Dynamics and Organization with TIRF Microscopy

Abstract

The actin-associated human protein palladin plays a critical role in cytoskeletal organization in both normal and cancerous cells. In breast and pancreatic cancer cell lines, palladin expression levels have been shown to correlate with metastatic potential. We have recently established that palladin contributes to actin dynamics in three distinct ways: nucleation of actin, crosslink formation, and filament stabilization. We hypothesize that palladin directly influences cell motility through simultaneous regulation of actin polymerization and organization. In this work we build upon our kinetic assays of actin polymerization to directly visualize actin assembly and protein dynamics with total internal reflection fluorescence microscopy (TIRFM). Bulk co-sedimentation assays can only monitor an increase in the total amount of actin polymerized, whereas the TIRF assay allows one to follow polymerization dynamics and filament topology simultaneously. To gain insight into the role of palladin in the assembly process and in the dynamics of supramolecular actin structures, we imaged the polymerization of monomeric actin and palladin in real time with TIRFM. Our data show that palladin mediates the formation of junctions between filaments. Analysis of filament morphology reveals that palladin promotes side branching in addition to bundles. Through these methods, we hope to understand how palladin contributes to actin-based cell motility in metastatic cells, which will be important for developing new therapies to specifically target this step in cancer progression.