title: Analysis of Myosin II Minifilament Orientation at Epithelial Zonula Adherens.
abstract: Non-muscle myosin II (NMII) form bipolar filaments, which bind F-actin to exert cellular contractility during physiological processes (Vicente-Manzanareset al., 2009). Using a combinatorial approach to fluorescently label both N- and C-termini of the NMII heavy chain, recent works have demonstrated the ability to visualize NMII bipolar filaments at various subcellular localizations (Ebrahimet al., 2013; Beachet al., 2014). At the zonula adherens (ZA) of epithelia, NMII minifilaments bind the circumferential actin bundles in a pseudo-sarcomeric manner (Ebrahimet al., 2013), a conformation required to maintain junctional tension and tissue integrity (Ratheeshet al., 2012). By expressing green fluorescent protein (GFP)-NMIIA heavy chain and immunolabel it using a NMIIA C-terminus specific antibody, we were able to visualize the NMII minifilaments bound to F-actin bundles in Caco-2 cells (Michaelet al., 2016), as previously reported (Ebrahimet al., 2013; Beachet al., 2014). In addition, we designed an FIJI/MATLAB analysis module to quantify the size, distance and alignment of these minifilaments with respect to junctional F-actin at the ZA. Measurements of the dispersion of minifilaments angles were proven to be a useful parameter that closely correlated to the extent of contractility at junctions (Michaelet al., 2016).
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, Guy's Campus, London, UK.
authors.forename: Magdalene
authors.initials: M
authors.lastname: Michael
authors.affiliation: Divisions of Cell Biology and Molecular Medicine, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane, Queensland, Australia.
authors.forename: Xuan
authors.initials: X
authors.lastname: Liang
authors.affiliation: Divisions of Cell Biology and Molecular Medicine, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane, Queensland, Australia.
authors.forename: Guillermo A
authors.initials: GA
authors.lastname: Gomez
keywords: Actin organization; Adherens junctions; Myosin II minifilaments; Structured illumination microscopy