Single -molecule biophysical assays with electrokinetics

Molecular motors and actin filaments play critical roles in disease processes and, more generally, in cell motility. The thesis developed new techniques to study the mechanical and electrical properties of actin filaments and the motion of motor proteins unhindered by nearby surfaces. Devices consisting of a pair of electrodes separated with a small gap were patterned on a glass wafer. With the application of AC electric fields, rhodamine-phalloidin-labeled actin filaments were polarized, attracted to the gap, and became suspended across the two electrodes. The thermal fluctuations of the suspended actin filaments were imaged, and the filament’s tension was estimated by comparing the experimental observations with theoretical predictions of a linear, Brownian dynamics model for filament thermal vibrations. The filament’s tension increased linearly with the electric field intensity-squared. The Brownian dynamics theory was verified by carrying out a set of experiments in which forces were controllably applied to the filaments with optical traps. The theoretically-estimated forces were compared and agreed with the applied forces. The optical trap experiments highlighted the importance of selecting appropriate exposure times to avoid biasing the experimental position data. Furthermore, optical tweezers were used to bring a motor protein-coated bead into close proximity with a pre-selected, suspended actin filament, facilitating the myosin-mediated bead attachment to the filament. The clearance beneath the filament allowed the bead to move freely along and around its filamentous track. The bead\u27s three-dimensional position was tracked as a function of time to obtain its trajectory. The combined use of electrical and optical tweezers provides a new and convenient means to study motor motility. Variants of this technique would enable studies of motor motility in the presence of filaments\u27 networks such as found in cells. Finally, the persistence lengths of free-floating filaments were measured as a function of their solution’s ion concentration. Preliminary observations were made of free floating filaments orienting and straightening in spatially uniform, AC electric fields. The thesis contributes to biophysics by providing new means to manipulate filaments and motor proteins and by contributing to our understanding of the effects of electric fields on actin filaments