A High-Resolution Subcellular Map of Proteins in Cells with Motile Cilia

Motile cilia are complex structures regulated by thousands
of genes,
essential for various physiological functions like respiration and
reproduction. Their dysfunction can result in severe conditions like
primary ciliary dyskinesia (PCD), highlighting the need for a deeper
molecular understanding of their specific ciliary compartments. Interestingly,
ciliated cells harbor multiple proteins with limited evidence on biological
function, as defined by Functional Evidence (FE) scores, a grading
system developed by the Human Proteome Project (HPP). Building upon
the stringent antibody validation pipeline of the Human Protein Atlas
(HPA) project, we developed a high-throughput workflow that combines
a novel multiplex immunohistochemistry protocol with image analysis
to investigate protein expression and subcellular localization in
motile ciliated cells across five human tissues: nasopharynx, bronchus,
fallopian tube, endometrium, and cervix. We spatially mapped >180
proteins, out of which 73% have FE scores 2–5, suggesting that
further evidence is needed to establish these proteins’ biological
function. Notably, expression patterns varied between tissues, suggesting
that motile cilia proteins are not universally expressed across the
different epithelia. Our pipeline constitutes a promising resource
for comprehensive mapping of the motile cilia proteome, and a first
step toward identifying cilia proteins for functional studies to understand
the molecular mechanisms underlying ciliopathies.