The Role of Class III Myosins in Actin Protrusion Length Regulation
Restricted (Penn State Only)
- Author:
- Cirilo, Joseph
- Graduate Program:
- Biomedical Sciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- January 23, 2024
- Committee Members:
- Thomas Spratt, Major Field Member
Christopher Yengo, Chair & Dissertation Advisor
Maria Bewley, Outside Unit Member
Lisa Shantz, Program Head/Chair
Nikolay Dokholyan, Outside Field Member - Keywords:
- myosin
actin
actin cytoskeleton
actin protrusion
filopodia
stereocilia
molecular motor
MYO3A
cell biology - Abstract:
- Actin based protrusions such as filopodia, microvilli, and stereocilia are important for various cellular processes such as motility and mechano-transduction. Despite their diverse functions, they contain a similar structure of a tightly packed, parallel bundled actin core and an arsenal of cytoskeletal binding and regulatory proteins. Amongst these cytoskeletal proteins are members of the myosin superfamily, actin-based molecular motors that utilize energy from ATP hydrolysis to generate movement and force. Many of these myosins localize to the tips of these actin protrusions and are implicated in protrusion length regulation, though biophysical models suggest actin polymerization forces alone control protrusion extension. The goal of this work was to understand how tip-localized myosins can contribute to protrusion extension, as well as how the motor properties of these myosins play a role in protrusion length regulation. To address these questions, we performed biochemical, biophysical, and cell biological analyses of the class III myosin, MYO3A, to determine how changes in the MYO3A motor properties altered its function within actin protrusions. Furthermore, we present a novel hypothesis as to how myosins at the tip of protrusions can control length regulation. MYO3A is one of two class III myosins that localizes to the tips of inner ear stereocilia, where it has been implicated in regulation of the stereocilia length. Interestingly, mutations in the MYO3A motor that alter its motor properties lead to a form of non-syndromic hearing loss, DFNB30. We characterized a dominant mutation in MYO3A, L697W, and looked at its impact on MYO3A kinetics and cellular function. We found that the mutation reduced ADP release and ATP hydrolysis, resulting in a slightreduction in duty-ratio (fraction of the ATPase cycle in which the myosin is strongly bound to actin) and slowed detachment from actin. Within COS7 cells, we found that L697W produced shorter filopodia that extended at a slower rate compared to WT MYO3A. Thus, we conclude that L697W is a “loss-of-function” mutations that leads to hearing loss by altering the ability of MYO3A to extend protrusions. Similarly, we expressed and characterized a different MYO3A hearing loss mutation, H442N, and analyzed its impact on MYO3A ATPase, in vitro motility, and cellular function. We found that this mutation increased MYO3A motor activity in vitro and resulted in protrusions that extended at a greater velocity compared to WT MYO3A. In addition, we generated chimeric myosin constructs that replaced the MYO3A motor and neck region with that of various other myosin motors and transfected each construct into COS-7 cells. Interestingly, we found a correlation between the in vitro gliding velocity of each myosin motor and the extension velocity of filopodia in cells, suggesting that the motor properties of myosins at protrusion tips are critical for length regulation. Lastly, this work contains unpublished data regarding a third mutation (V607F), a MYO3A binding partner (MORN4), and the other class III myosin, MYO3B. The V607F mutation enhanced MYO3A-associated filopodia extension, but not tip localization or lengths. WT MYO3A co-transfected with MORN4 resulted in filopodia that extended faster and were longer than either WT MYO3A or MORN4 alone. Lastly, we found that calcium enhances MYO3B ATPase, but not in vitro motility, suggesting it may increase MYO3B duty ratio independent of changes to ADP release. Furthermore, we found that COS7 cells transfected with MYO3BΔK and ESPN produced filopodia both with and without the presence of 2mM calcium. Similarly, a construct containing MYO3BΔK plus the actin binding THDII of MYO3A (MYO3BΔK.THDII) produced filopodia in both conditions. Overall, this work demonstrates how class III myosins contribute to length regulation of actin-based protrusions. Our findings build upon a fundamental mechanism by which tip-localized myosin motors may contribute to pushing force at protrusion tips. Mutations in myosin motors that alter this mechanism (i.e., by changing the intrinsic motor properties of the tip-localized myosins) may ultimately lead to diseases, such as hearing loss with MYO3A mutations. These findings lay a groundwork for future studies investigating how myosins at protrusion tips can control the length of the protrusions, as well as studies investigating the role of MYO3A in hearing loss.