Regulatory mechanisms of MYO6 motor domain activity and tail interactions

The myosin super family represent a diverse group of actin-based molecular motors, driven by ATP hydrolysis. They are fundamental for a multitude of cellular processes, ranging from muscle contraction to intracellular transport to tethering. Originating from an ancestral protein, myosins have evolved into many classes with distinct structural domains and mechanochemical properties that contribute to their specialised functions. Myosins of class VI are exceptional, due to their unique reversed directionality towards to minus-end on actin filaments compared to other classes. It can function as a -retrograde- transporter or tether in a wide range of biological processes. Dysfunction of myosin VI (MYO6) is linked to multiple pathologies, such as hearing loss, hypertrophic cardiomyopathy, and various cancers. This thesis focuses on the spatial and temporal control of MYO6 and its network of interactions. First, I examined the MYO6 phosphoproteome under steady state and stimulated conditions using mass spectrometry. After identifying multiple sites of interest, the S267 site is characterised *in vitro* and *in vivo*. This reveals that S267 phosphorylation increases the motor velocity by 4-fold. The impact of the increased motor velocity can also be observed *in vivo*, as mimicking S267 phosphorylation increases *Salmonella enterica* uptake. In addition, the artificially engineered MYO6 plus-end directed mutant forms filopodia on the cell surface, which upon expression of the phosphomimetic significantly increases in number. Co-expression of the phosphomimetic and wildtype MYO6 causes a re-distribution of their localization to the filopodia tip and stalk, respectively. I also identified the kinase that phosphorylates S267 using a selection of online kinase prediction tools and curated databases. This is followed up by further validation of the kinase *in vitro*. My analysis of phosphorylation sites in the MYO6 motor domain reveals the conservation of S267 among vertebrates and Cephalochordates and a few other highly conserved phosphorylation sites. Lastly, recent advances in proximity labelling techniques allow me to determine dynamic MYO6 interactions *in vivo*, at steady state conditions and after the induction of autophagy. Overall, this study provides novel insights into the dynamic regulation of MYO6 function and activity.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• De Jonge, Janeska

Advisor dc:contributor.advisor

• Buss, Folma

• Myosin VI

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/bc8977aa-7b80-43ce-a82f-7b827b02fec7/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.108259

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/367820