10779/rcsi.10800065.v1 Andrei I. Ivanov Andrei I. Ivanov Ann M. Hopkins Ann M. Hopkins G Thomas Brown G Thomas Brown Kirsten Gerner-Smidt Kirsten Gerner-Smidt Brian A. Babbin Brian A. Babbin Charles A. Parkos Charles A. Parkos Asma Nusrat Asma Nusrat Myosin II regulates the shape of three-dimensional intestinal epithelial cysts. Royal College of Surgeons in Ireland 2019 Actins Caco-2 Cells Cell Polarity Cell Shape Cofilin 1 Down-Regulation Epithelial Cells Heterocyclic Compounds with 4 or More Rings Humans Intestinal Mucosa Microfilaments Myosin Type II Nocodazole Organ Culture Techniques Polymers RNA Stability Type C Phospholipases Surgery 2019-11-22 17:28:54 Journal contribution https://repository.rcsi.com/articles/journal_contribution/Myosin_II_regulates_the_shape_of_three-dimensional_intestinal_epithelial_cysts_/10800065 <p>The development of luminal organs begins with the formation of spherical cysts composed of a single layer of epithelial cells. Using a model three-dimensional cell culture, this study examines the role of a cytoskeletal motor, myosin II, in cyst formation. Caco-2 and SK-CO15 intestinal epithelial cells were embedded into Matrigel, and myosin II was inhibited by blebbistatin or siRNA-mediated knockdown. Whereas control cells formed spherical cysts with a smooth surface, inhibition of myosin II induced the outgrowth of F-actin-rich surface protrusions. The development of these protrusions was abrogated after inhibition of F-actin polymerization or of phospholipase C (PLC) activity, as well as after overexpression of a dominant-negative ADF/cofilin. Surface protrusions were enriched in microtubules and their formation was prevented by microtubule depolymerization. Myosin II inhibition caused a loss of peripheral F-actin bundles and a submembranous extension of cortical microtubules. Our findings suggest that inhibition of myosin II eliminates the cortical F-actin barrier, allowing microtubules to reach and activate PLC at the plasma membrane. PLC-dependent stimulation of ADF/cofilin creates actin-filament barbed ends and promotes the outgrowth of F-actin-rich protrusions. We conclude that myosin II regulates the spherical shape of epithelial cysts by controlling actin polymerization at the cyst surface.</p>