Dynamics of Protein-Mediated Polymer Coupling and their Implications in Antibody Production and Emergent Patterning

Citation

Hirokawa, Soichi (2023) Dynamics of Protein-Mediated Polymer Coupling and their Implications in Antibody Production and Emergent Patterning. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/fpmm-a552. https://resolver.caltech.edu/CaltechTHESIS:08252022-153300158

Abstract

Proteins serve a wide range of functions in and out of the cell, from signaling and gene regulation to transport and structural reinforcement. These functions are usually carried out from interactions with other molecules in the surrounding medium such as other proteins, small molecules, or DNA. One such class of proteins are what I will call polymer-coupling proteins: these proteins intentionally link identical polymers or two regions of the same polymer together so that their coupled interactions critically affect the state of the biological system. A vast array of such proteins exist in nature with roles such as the looping of DNA to physically inhibit the expression of a gene or the formation of the cytoskeleton which provides a cell with its shape. In this thesis, I use in vitro experimental methods to explore two cases of coupling proteins and understand their roles not only in reorganizing their complementary polymers but influencing the final state of their respective systems.

In Chapter 2, I examine the starting process for the assembly of an antibody-encoding gene in developing immune cells. Motivated by data suggesting that some antibodies are less likely to be made than others, I explore how the early steps of constructing an antibody-encoding gene affect this uneven frequency of assembly. To initiate recombination, the recombination-activating gene (RAG) protein complex simultaneously binds and cuts two well-recognized sequences neighboring two antibody-encoding gene segments in order to allow other proteins to combine these exposed segments together. The sequences to which the RAG protein performs its binding and cutting functions have certain identifiable sequence patterns but can still vary. Through a single-molecule experimental method known as tethered particle motion (TPM) I show how changes to the binding site sequence can enhance or diminish the propensity of the RAG protein to bind and cut the DNA and thus explore the consequences of these altered interactions in the unequal selection for certain antibody gene segments over others.

In Chapter 3, I turn to questions of the emergence of order from self-organization in biological systems. From the molecular to the population scale, biology constantly demonstrates that with an injection of energy, systems can be driven out of equilibrium and allow for the organization of its constituents. A case of such organization in cells is the coupling of microtubules by motor proteins to create and maintain the mitotic spindle, a critical biological architecture for ensuring that each cell obtains a copy of the genome during division. In vitro experiments that exploit similar motor-microtubule interactions have become a convenient way to identify the effects of perturbing a key player such as motor properties or boundary conditions of the system on the spatiotemporal extent of organization. However, in many instances, the dynamics under which such cytoskeletal systems reduce their entropy over the course of creating order have not been carefully examined in experimental systems. Here, I use engineered light-dimerizable motors that can give rise to the formation of a highly connected network that compacts to form a dense, organized structure, and through the use of a noninvasive imaging technique observe how the polymers that make up the network continually reorganize in the bulk during a global contraction of the network.

Thesis Files