Probing single-molecule dynamics of molecular motors and gene editing proteins

Single molecule fluorescence imaging is a powerful tool for studying protein dynamics. I have leveraged the power of single molecule imaging in studying two proteins systems: Molecular Motors and Gene Editing Proteins. Kinesin walks on cellular roads called microtubules and transports cellular cargos. Kinesin is one of the many types of molecular motors present in living cells. Generally, multiple kinesin motors are present on a single cargo during transport. Therefore, it is important to fundamentally understand how multiple motors work with each other during transport of the cargo to its destination. For understanding this, we design an in vitro assay, that we call force gliding assay. With this assay, dynamics of multiple kinesins (from 2-8) and its effect on cargo velocity can be simultaneously tracked when these kinesins transport a common cargo. Furthermore, we show that kinesins dynamically interact to overcome roadblocks. Consequently, multiple kinesins acting as a team may play a significant role in facilitating smooth cargo motion in a dense environment. (Chapter 2) Next, we wanted to quantify the forces exerted by individual motors during collective transport. Existing force sensing techniques cannot quantify the forces exerted by individual molecular motors during multi-motor transport. We combined fluorescence and denatured ssDNA-based design to develop Real-Time Force Sensing of Individual Motors (RT-FSIM) assay that can measure forces exerted by individual kinesin motors in real-time during collective cargo transport. Kinesins primarily exerted less than 1 pN forces for transporting a common microtubule. Our assay also enabled force sensing of multiple kinesins in the presence of roadblocks. Our assay is high-throughput, versatile and is applicable to other molecular motors such as dynein and myosin. (Chapter 3) Discovery of gene editing proteins has revolutionized the biotechnology field. Now we have capability to edit any gene in the mammalian genome that has numerous applications in therapeutics, agriculture and disease prevention. We study two such gene editing systems: clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and transcription activator-like effector nuclease (TALEN). Genome editing critically relies on selective recognition of target sites with minimal off-target effects. However, despite recent progress, the underlying search mechanism of genome editing proteins is not fully understood in the context of cellular chromatin environments. Here, we use single-molecule imaging in live cells to directly study the behavior of Cas9 and TALEN. It is known that Cas9 typically outperforms TALEN in open genomic regions (euchromatin) of the mammalian nucleus, but Cas9 is inhibited by nucleosomes and fails to efficiently edit DNA-targets in closed chromatin (heterochromatin). Our results show that Cas9 is less efficient in heterochromatin compared to TALEN because Cas9 becomes encumbered by local searches on non-specific sites in these regions. Using sequencing-based editing analysis, we find up to fivefold increase in editing efficiency for TALEN compared to Cas9 in heterochromatin regions. Overall, our results show that nuclease-deficient versions of Cas9 and TALEN use a combination of 3-D and local searches to identify target sites, and the macroscopic nature of local search determines editing outcomes of the genome-editing proteins. Taken together, our results suggest that TALEN is a more efficient gene-editing tool than Cas9 for applications in heterochromatin. (Chapter 4)LimitedAuthor requested closed access (OA after 2yrs) in Vireo ETD syste