INVESTIGATING THE ROLE OF DISORDER IN MYOSIN CARGO RECOGNITION AND BINDING

Myosin 10 (Myo10) cargo binding is essential for the proper functioning of multiple cellular signaling processes, including cellular migration and neuronal development. Despite prior investigations into the molecular mechanisms of Myo10 cargo binding, a complete picture of the underlying protein dynamics is still missing. This thesis clarifies the molecular underpinnings of Myo10 cargo-binding dynamics, with particular emphasis on how protein structure and intrinsic disorder mediate specific protein-protein interactions. The thesis begins with a project that establishes a quantitative understanding of Myo10 localization by counting Myo10 protein numbers in fixed and live cells. This work provides insight into the crowded molecular environment within thin cellular protrusions (e.g., filopodia) and shows how local Myo10 concentrations could dictate interactions with its cargo, the plasma membrane, and actin. The central project in this thesis focuses on two cargo receptors, DCC and β1 integrin, to understand the dynamics of cargo-bound Myo10. Using a multi-pronged approach combining hydrogen-deuterium exchange mass spectrometry (HDX-MS), cross-linking mass spectrometry (XL-MS), live-cell imaging, and super-resolution microscopy, we show that Myo10 decodes intrinsically disordered region (IDR) elements through two complementary mechanisms: disorder-to-order transitions and disordered binding. These findings illustrate a tunable, multivalent binding strategy that enables Myo10 to selectively coordinate diverse signaling cargos, demonstrating how regulated disorder within IDRs is one mechanism underlying protein binding and cellular signaling. In addition to these two primary bodies of work, the thesis presents two methodologies that were developed and essential for carrying out this research. One method describes how to quantify intracellular distributions of HaloTag-labeled proteins using SDS-PAGE and epifluorescence microscopy. The other method details how to measure the HDX of low-affinity protein complexes using a one-chain protein construct, by which two proteins are expressed from the same plasmid and physically connected by a flexible linker. Overall, these studies advance our understanding of both the physical constraints of packing Myo10, its cargo, and other filopodia-associated proteins into narrow membrane compartments, and the molecular strategies Myo10 employs to facilitate weak yet functionally critical interactions with DCC and β1 integrin. More broadly, this work reveals how protein concentration and their intracellular localization shape distinct IDR binding modalities that support dynamic and selective cargo coordination for cellular signaling.