| Description |
The majority of studies of intracellular transport have focused on cargos that move well over some distance. However, a significant percentage of intracellular cargos at any given time remain in a stalled or locally diffusive state. The goal of my graduate work was to study the processes that lead to localized non-motile or weakly motile cargos. In my first work (Chapter 2), I showed that cargos driven by a team of kinesins can spend a significant amount of time at microtubule (MT) intersections, especially when the intersecting MTs are counter-aligned. The work in Chapter 2 was done in two dimensions (2D) with MT attached to the surface of a coverslip. To enable more biologically faithful studies, I contributed to the development of a novel technique (Chapter 3) to manipulate MTs in three dimensions (3D). This work now enables the construction of MT structures in a controlled environment in a fully suspended geometry. The study in Chapter 4 was motivated by the need to understand cold block of MT-based transport seen in some mammalian neurons and how this is overcome in cold adapted organisms. I have explored the differences in how temperature affects the activity of biological motors and helped model how these differences modulate the tug-of-war between the two opposite directed motors. Lastly, it is critical in the study of nearly immotile cargos to distinguish when the cargo is not moving due to opposite-directed motor activity from when it is driven by a thermally-driven random process such as Brownian diffusion. I showed (Chapter 5) that in contrast to diffusion, tug-of-war follows Arrhenius kinetics which reflects motor activation energy. I used a bi-directional kinesin NCD N340K mutant, on a single MT in-vitro. I also showed that this system of tug-of-war displays complex behavior. In contrast to thermal diffusion, this tug-of-war is sub-diffusive at short time intervals and it only becomes effectively diffusive at longer time intervals. In addition, I showed that the tug-of-war leads to an unexpected skewed distribution of diffusion coefficients within experimental sample range and I have helped develop a theoretical understanding of how this could arise in my model system. |
