Investigating the Trafficking Status of Caveolin-3 Missense Variants

Abstract

Caveolae are small invaginations of the cell membrane which help regulate many cellular
functions, such as signal transduction and membrane trafficking. Essential to the formation
of caveolae are caveolins, oligomeric membrane proteins. Caveolin-3 (Cav-3) (encoded by
the CAV3 gene) is the predominant isoform in myocytes. Missense variants in CAV3 are
associated with a variety of disease phenotypes including long QT syndrome, hypertrophic
cardiomyopathy, hyperCKemia, rippling muscle disease, and limb-girdle muscular dystrophy.
The pathogenic mechanisms of many CAV3 variants are largely unknown, and there is a lack
of experimental data that quantitatively assesses the impact of variants on the trafficking of
Cav-3 to the cell membrane.
Therefore, this project uses a quantitative imaging-based assay to determine if specific
missense variants of Cav-3 affect its trafficking to the cell surface. To investigate the cellular
localisation of Cav-3, transfected Human Embryonic Kidney-293 cells were immunostained
and imaged using confocal microscopy. Co-localisation analysis quantitatively characterised
the variants’ trafficking status. Additionally, AlphaMissense (a machine-learning model) was
used as an experimental tool to explore CAV3 gene pathogenicity predictions and how these
may correlate to trafficking status.
Co-localisation analysis revealed that Cav-3 variants S61R, L79R, L87P, and P105L had a
significantly different cellular localisation profile compared to wild-type, whereas D8N, K30R,
E34K, V37L, A46T, and A85T did not differ significantly from wild-type. Furthermore, a key
co-localisation parameter (Pearson’s Correlation Coefficient (plasma membrane vs Cav-3))
demonstrated a significant negative correlation with AlphaMissense scores. These findings
provide valuable insight into the impact of variants on Cav-3 trafficking and highlight the
potential of integrating computational tools with quantitative imaging to better understand
Cav-3 disease mechanisms.

Date of Award	20 Jan 2026
Original language	English
Awarding Institution	University of Bristol
Supervisor	Stephen C Harmer (Supervisor) & Andrew F James (Supervisor)