Characterization of CEACAM1 Spatial Distribution, Self-association and Dynamics using High-resolution Microscopy Techniques
CEACAMs are cell surface glycoproteins involved in homo- and heterophilic intercellular interactions that control cellular growth, differentiation, tumourigenesis, inflammation and infection. There is growing evidence that CEACAM1 isoforms regulate the cellular response to CEACAM1-mediated interactions between various cell types. Reinforcing this point, given its potential as a tumour suppressor and immunomodulator, CEACAM1 is being targeted for clinical development. However, there remain clear challenges owing to the observation that different CEACAM1 isoforms display cell-type specific expression patterns under different physiological conditions. Moreover, the ability of CEACAMs to form dimers and higher-order oligomers, which are thought to impact regulation of intercellular signals, add considerable complexity to our understanding of its functional role(s). Super resolution microscopy has enabled the quantitative characterization of the nanoscale distribution of several membrane proteins, revealing cluster hierarchies at the micro- and nano-scale. However, clustering and self-association are not necessarily the same piece of information and may have very different effects on the cellular response, so mapping the preferential nanoscale distribution of the various self-association states is of particular interest to many. This thesis reports on the development and application of a correlative STORM/homoFRET strategy to study CEACAM1 isoforms and mutants. We have further explored the use of single particle tracking (SPT) and mean square displacement analysis to characterize CEACAM1 dynamics as a function of oligomeric state and location. Building on these datasets, we have applied co-localization and co-occurrence approaches to measure CEACAM1 association to lipid-ordered regions, ezrin and actin. Finally, we present a set of preliminary methods and results that will serve to further the study of membrane protein clustering and self-association and their link to function. Together, this thesis aims to develop a more fulsome perspective on structure, association and dynamics of the CEACAM1 membrane protein while providing methodological approaches to tackle similar questions.
CEACAM1, Cell signaling, FRET, Microscopy, Single particle tracking, Super resolution
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