THE NOVEL DESMOSOMAL PROTEIN POF1B IS ESSENTIAL FOR CELL-CELL ADHESION STRENGTH

POF1B is a candidate gene for premature ovarian failure (POF); it is mainly expressed in polarized epithelial tissues, but its function in these tissues and the relationship with the disorder are unknown. In polarized epithelial MDCK cells the human stably expressed POF1B showed a tight junction localization that was lost by the POF1B R329Q variant associated with POF. Although the apico-basal polarity markers and ultrastructure of the tight junctions were maintained in cells expressing the mutant, tight junction assembly, as well as the organization of the monolayer appeared altered. Moreover, cells expressing the POF1B R329Q variant showed defects in ciliogenesis and cystogenesis as a result of misorientation of primary cilia and mitotic division. All of these defects were explained by interference of the mutant with the content and organization of F-actin at the junctions. Subsequently, by means of morphological and biochemical criteria we documented that POF1B is actually a desmosome- associated protein. Both in Caco-2 human intestinal cells and in stratified HaCaT keratinocyte cell lines, indeed, endogenous POF1B colocalized with desmoplakin and plakophilin 2 at the desmosomal plaque and in cytoplasmic particles aligned along intermediate filaments. POF1B co-fractionated with desmosomes and intermediate filament components and showed properties characteristic of desmosomes (i.e. detergent insolubility and calcium independence). Furthermore, POF1B was required for desmosome assembly and function, as the stable downregulation of the protein in HaCaT cells caused a decrease in desmosome number and size, and these desmosomes had very weak electron dense plaques. Among the cell-cell adhesion structures, desmosomes are the most essential for mechanical coupling. The reduced capability of POF1B-silenced keratinocytes to respond to mechanical stress revealed the protein's crucial role in these junctions. Moreover, altered desmosomes in POF1B- downregulated keratinocytes were associated with altered cell proliferation and differentiation. The localization of POF1B in simple and stratified epithelia, as well as its relocation to desmosomes in human skin tumors, further indicated the protein's role in desmosome function, and suggested its involvement in human diseases associated with impairment of these junctions.