Abstract
Epithelial cells form tissues with many functions, including secretion and environmental separation and protection. Glandular epithelial tissues comprise cysts and tubules that are formed from a polarized, single-epithelial cell layer surrounding a central, fluid-filled lumen. The pathways regulating key processes in epithelial tissue morphogenesis such as mitotic spindle formation are incompletely understood, but are important to investigate, as their dysregulation is a signature of epithelial tumors. Here, we describe a signaling axis that manifests in a defect in mitotic spindle orientation during epithelial growth and cystogenesis. We found that activation of the small GTPase ADP-ribosylation factor 6 (ARF6) results in the sustained internalization of cell-surface components such as the cMet receptor and the cell-adhesion molecule E-cadherin. The spindle orientation defect arising from elevated levels of ARF6-GTP required an increase in cMet endocytosis, but was independent of E-cadherin internalization or elevated extracellular signal-regulated kinase (ERK) activity resulting from internalized receptor signaling on endosomes. Misorientation of the mitotic spindle resulted in the development of epithelial cysts with structural abnormalities, the most conspicuous of which was the presence of multiple intercellular lumens. Abnormal mitotic spindle orientation was necessary but insufficient to disrupt glandular development, as blocking the strong prosurvival signal resulting from ERK hyperactivation yielded structurally normal cysts despite continued manifestation of spindle orientation defects. Our findings highlight a previously unknown link between ARF6 activation, cMet receptor internalization, and mitotic spindle orientation during epithelial glandular morphogenesis.
Highlights
Epithelial cells form tissues with many functions, including secretion and environmental separation and protection
In light of previous research demonstrating that depletion of Cdc42–GTP results in the formation of multilumen cysts stemming from abnormal mitotic spindle orientation [7, 13], we examined the amount of active Cdc42 in MDCKARF6–GTP cells
MDCKARF6–GTP cells treated with PD98059 had an average spindle angle of 14.9°, on par with untreated cells showing an average angle of 18.3° (Fig. 7E). Taken together these results demonstrate the importance of tightly controlled endocytosis and intracellular signaling to epithelial glandular morphogenesis, as blocking the long-lived extracellular signal-regulated kinase (ERK) signal emanating from signaling endosomes is sufficient to rescue glandular architecture even in the presence of abnormal mitotic spindle orientation
Summary
Epithelial cells form tissues with many functions, including secretion and environmental separation and protection. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Madin-Darby canine kidney (MDCK) cells can be grown in 3D organotypic culture to form highly polarized cysts [2, 3] Using this system we have previously reported on the development of aberrant glandular structures when epithelial cells are grown with sustained activation of ARF6. Constitutive and sustained ARF6 activation leads to the disassembly of cell– cell contacts followed by internalization of both E-cadherin and growth factor receptors that accumulate in signaling endosomes These signaling endosomes can serve as platforms for hyperactive ERK signaling, which results in the formation of epithelial cysts with multiple lumens that contain surviving, matrix-deprived cells [4]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
