Collections > Electronic Theses and Dissertations > Functions of Myosin-X in Polarized Epithelial Cells
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Myosin-X (Myo10) is an unconventional myosin, known functions of which have been largely deduced through studies in non-polarized cells. Myo10 is well-known for localizing to the tips of filopodia and has important roles in filopodial formation. Myo10 is expressed at high levels in epithelial tissues such as kidney, yet relatively little is known about the functions of Myo10 in polarized epithelia. Here, we determine that Myo10 localizes to the basolateral domain of polarized epithelial cells and has important functions in junction assembly and epithelial morphogenesis. Polarized epithelial cells have apical and basolateral domains, which are separated by the apical cell junction. Normal functions of polarized cell junctions, such as barrier formation and maintenance of cell polarity, are critical. Dysfunction is commonly observed in inflammatory diseases and infection where the epithelial barrier is breached, and in ischemic states and invasive cancers, where malignant cells undergo epithelial to mesenchymal transition. Thus, it is important to identify molecules that mediate these processes in polarized epithelia. In kidney, we found that Myo10 is expressed at the basolateral domain. We then used MDCK cells as a cell culture model to generate stably expressing GFP-Myo10 and Myo10 knockdown lines. Consistent with our results in vivo, GFP-Myo10 localized to the lateral membrane during junction assembly. Importantly knockdown led to a delay in junction formation and increased paracellular permeability. Furthermore, Myo10 knockdown resulted in abnormal multiple lumen formation during cystogenesis. In fully polarized cells, GFP-Myo10 showed striking localization to the tips of basolateral filopodia. Intriguingly, GFP-Myo10 HMM (heavy mero-myosin, a construct lacking the tail) was found at apical microvilli. Yet, localization experiments using GFPMyo10 and anti-Myo10 antibodies showed full-length Myo10 is not detected in microvilli. Moreover, knockdown studies indicated that Myo10 does not appear to function in the formation of microvilli. Nonetheless, the apical localization of GFP-Myo10 HMM led us to investigate apico-basal targeting of Myo10. The tail of Myo10 localized to the basolateral membrane, and deletion of the pleckstrin homology (PH) domains resulted in localization to the apical microvilli. Additionally, disruption of phosphatidylinositol binding partially redistributed Myo10 to apical microvilli. Our results suggest Myo10 targeting to basolateral filopodia is regulated by PH domains of the tail. Recent studies have revealed that basolateral filopodia are critical for normal tissue patterning, yet relatively few proteins are known to localize to and function in basolateral filopodia. Given the importance of Myo10 in filopodia and the functions we have discovered here in polarized epithelia, it is likely that Myo10 mediates critical processes in basolateral filopodia, a subject that beckons further investigation.