Abstract
The epidermis functions as a critical barrier protecting the body from environmentalinsults, a function dependent upon proper execution of keratinocyte terminal differentiation. This
process is tightly regulated by calcium (Ca²⁺)-dependent molecular events that enable dramatic
alterations in keratinocyte structure and behavior. Despite decades of research, the molecular
mechanisms that translate Ca²⁺ signals into the polarized secretion of lamellar bodies (LBs),
specialized organelles essential for barrier formation, remain incompletely understood. This
thesis identifies FLOWER (FWE), a small transmembrane protein with no prior known function
in skin, as a novel regulator of Ca²⁺-dependent LB trafficking during epidermal differentiation
and demonstrates its relevance to both homeostatic barrier formation and disease.
Using computational modeling and experimental validation, the canonical human FWE
isoform, hFWE4, was revealed to assume a four-transmembrane domain structure positioning N-
and C-terminal tails cytosolically. This topology enabled interaction with vesicular trafficking
machinery and facilitated rapid, AP-2-dependent endocytosis from the plasma membrane. In
contrast, the non-canonical isoform hFWE3 was retained in the endoplasmic reticulum with no
apparent trafficking function.
Investigations into FWE expression in human epidermis revealed robust upregulation in
terminally differentiating keratinocytes of the upper stratum spinosum and stratum granulosum,
where FWE localized to LBs. Genetic ablation of FWE in epidermal organoids impaired barrier
function and dysregulated expression of genes involved in cornification and LB biology.
Proteomic profiling of hFWE4-positive vesicles identified extensive trafficking machinery
including Rab GTPases, SNARE complexes, and diverse LB cargo. hFWE4 potentiation of
intracellular Ca²⁺ release facilitated surface trafficking of LB cargo. FWE-positive LBs exhibited
striking mislocalization in Darier and Grover disease, providing support for the clinical relevance
of FWE-dependent vesicular trafficking.
Investigation of FWE in cutaneous squamous cell carcinoma (cSCC) revealed expression
in highly differentiated tumor regions. FWE knockout altered keratinization patterns and
impaired terminal differentiation in xenografts, while ectopic hFWE4 expression induced cell
cycle arrest and differentiation. FWE expression reliably distinguished well-differentiated from
poorly-differentiated tumors, suggesting utility as a prognostic biomarker.
Collectively, this work established FWE as a multifunctional regulator of Ca²⁺-dependent
LB trafficking essential for epidermal barrier formation and positioned it as a promising
therapeutic target and diagnostic marker in cutaneous disease.