Date of Award
Doctor of Philosophy (PhD)
In my thesis work, I explored the organization and dynamics of the nuclear pore complex (NPC) through coarse-grained modelling and polarized-total internal reflection fluorescence (pol-TIRF) microscopy. The NPC is a large (~120 MDa in humans) macromolecular assembly that controls the flow of molecules into and out of the nucleus. The NPC is a key regulator of intracellular trafficking, nuclear organization, and gene expression. The study of this cellular gatekeeper is hampered by its size and complexity, as well as the difficulty of measuring protein dynamics in vivo. Although various models for how cargo translocates the NPC have been proposed, the mechanism remains unclear. However, most models for transport propose a crucial role for the phenylalanine-glycine nucleoporins (FG-Nups). I constructed a coarse-grained model to investigate the dynamics of these proteins. My coarse-grained model was able to recapitulate in vivo experimental results previously measured in our lab. My results also suggest that, at the time-scale of cargo translocation, some of the FG-Nups are highly mobile and capable of translocating back and forth across the NPC in milliseconds. This coarse-grained computational model and its results will be discussed in Chapter 2. The entire NPC and its constituent nucleoporins (Nups) have been subject to a great many structural studies; however, monitoring the dynamics of the NPC in vivo has proven to be highly difficult. In order to overcome this challenge, I have built and validated a series of fluorescent orientational sensors. These sensors can be used in conjunction with a pol-TIRF imaging scheme to resolve the orientations and dynamics of specific Nups inside individual NPCs within living cells. By rigidly conjugating mEGFP to various Nups in the scaffold rings of the NPC, I have been able monitor Nups within the Y-shaped complex, the adaptor ring, and the inner channel ring of the NPC. In Chapter 3, I will describe the tools I have built to test NPC dynamics. I proceeded to measure the orientations and dynamics of the Nups under different transport conditions with these tools. In Chapter 4, I show that Nup54, a member of the inner channel ring, undergoes a shift in conformation under a variety of transport conditions. An orientational shift was not observed in Nup133, a member of the Y-shaped complex. These results suggest that the inner channel ring may reorganize with respect to the NPC in response to cargo translocation. In the final chapter of this thesis, I explore the implications of our findings and outline the immediate future directions of this work. I will close by describing how polarization microscopy can be applied to other biological systems and proposing some basic technological improvements to this technique.
Pulupa, Joan, "Conformational Dynamics of the Nuclear Pore Complex Studied With Coarse-Grained Modelling and Polarized Light Microscopy" (2019). Student Theses and Dissertations. 509.