Francis Cavanna (The University of Texas at Austin)

"In-vitro Active Gels: Quantifying Actin Network Interactions and Morphologies"

Abstract: Actin is a biopolymer found in living cells and used for structural and locomotive work. While this protein is often studied within in-vivo work, it is often difficult to disentangle causality for actin-specific interactions from the broader constellation of cellular processes. For this reason, in-vitro studies purify actin and its associated proteins and reconstitute these networks within a simplified experimental system. In this same tradition, we investigate actin network parameters in four ways. First, we characterize pore size of these networks with respect to four separate actin bundling agents: MgCl_2, PEG, α-actinin, and fascin. We find that fascin and MgCl_2 bundled networks exhibit increasing pore size as bundling agent concentration increases, while α-actinin and PEG bundled networks exhibit positive trends in mesh size as the bundling agent concentration increases. For the second project, we characterize PEG networks more closely. We vary the molecular weights of actin networks with PEG molecules of molar weights 2, 6 and 20 kDa. As PEG molecular weights increase from 6 kDa and PEG 20 kDa, a phase transition is found from filamentous to bundled networks for Actin-PEG solutions with a PEG concentration at 0.1% w/v ratio. We investigate this boundary further with dynamic light scattering and 2D infrared spectroscopy. We find that these networks have lower diffusivity in solution, and that when PEG bundled actin transitions from the filamentous state to the bundled state, β sheets within the protein are decreased in favor of loops. Next, we measure ATP consumption within actomyosin networks through the use of an NADH readout assay and simulate the results. Contractile actomyosin networks are found to consume energy beyond the end of deformation, while the simulation is used to fit different values of myosin activity and diffusion constants. The diffusion constants are found to not have statistical significance between experiment populations, while the myosin activity rates seem to increase as myosin concentration increases. For the final project, we perform exploratory work towards encasing a core of undyed yield stress fluid within a dyed shell. We characterize length, cap length, and core and wall width of these shells as we vary the input parameters for generating these shells. These results help illuminate properties of actin networks, and prepare future work on network parameters