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Theses Doctoral

Emergent Properties of Biomolecular Organization

Tsitkov, Stanislav

The organization of molecules within a cell is central to cellular processes ranging from metabolism and damage repair to migration and replication. Uncovering the emergent properties of this biomolecular organization can improve our understanding of how organisms function and reveal ways to repurpose their components outside of the cell. This dissertation focuses on the role of organization in two widely studied systems: enzyme cascades and active cytoskeletal filaments.Part I of this dissertation studies the emergent properties of the spatial organization of enzyme cascades. Enzyme cascades consist of a series of enzymes that catalyze sequential reactions: the product of one enzyme is the substrate of a subsequent enzyme. Enzyme cascades are a fundamental component of cellular reaction pathways, and spatial organization of the cascading enzymes is often essential to their function. For example, cascading enzymes assembled into multi-enzyme complexes can protect unstable cascade intermediates from the environment by forming tunnels between active sites.

We use mathematical modeling to investigate the role of spatial organization in three specific systems. First, we examine enzyme cascade reactions occurring in multi-enzyme complexes where active sites are connected by tunnels. Using stochastic simulations and theoretical results from queueing theory, we demonstrate that the fluctuations arising from the small number of molecules involved can cause non-negligible disruptions to cascade throughput. Second, we develop a set of design principles for a compartmentalized cascade reaction with an unstable intermediate and show that there exists a critical kinetics-dependent threshold at which compartments become useful. Third, we investigate enzyme cascades immobilized on a synthetic DNA origami scaffold and show that the scaffold can create a favorable microenvironment for catalysis.

Part II of this dissertation focuses on the organization of active cytoskeletal filaments. Many mechanical processes of a cell, such as cell division, cell migration, and intracellular transport, are driven by the ATP-fueled motion of motor proteins (kinesin, dynein, or myosin) along cytoskeletal filaments (microtubules or actin filaments). Over the past two decades, researchers have been repurposing motor protein-driven propulsion outside of the cell to create systems where cytoskeletal filaments glide on surfaces coated with motor proteins. The study of these systems not only elucidates the mechanisms of force production within the cell, but also opens new avenues for applications ranging from molecular detection to computation.

We examine how microtubules gliding on surfaces coated with kinesin motor proteins can generate collective behavior in response to mutualistic interactions between the filaments and motors, thereby maximizing the utilization of system components and production. To this end, we used a microtubule-kinesin system where motors reversibly bind to the surface. In experiments, microtubules gliding on these reversibly bound motors were unable to cross each other and at high enough densities began to align and form long, dense bundles. The kinesin motors accumulated in trails surrounding the microtubule bundles and participated in microtubule transport.
In conclusion, our study of the emergent properties of the spatial organization of enzyme cascades and the mutualistic interactions within active systems of motor proteins and cytoskeletal filaments provides insight into both how these systems function within cells and how they can be repurposed outside of them.

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More About This Work

Academic Units
Biomedical Engineering
Thesis Advisors
Hess, Henry S.
Degree
Ph.D., Columbia University
Published Here
November 16, 2020