Sedzinski Group: Mechanics of Tissue Homeostasis
"Our group is interested in understanding mechanics of epithelial tissue homeostasis and morphogenesis. Particularly, we want to determine both the mechanics and molecular regulation of epithelial cell renewal. For this, we study how forces generated by the actomyosin cytoskeleton and adhesion molecules shape and move epithelial cell progenitors within tissues. We use high-resolution microscopy to image dynamics of progenitor cells, biophysical and theoretical methods to describe the forces, and genetic to perturb the system.
Development and homeostasis of epithelial sheets depend upon the regular addition of new cells and removal of old cells. The last decade has seen an explosion of interest in the mechanisms governing the homeostatic extrusion or delamination of cells from epithelia. These studies reveal a complex interplay between molecular signals and cell mechanics, and underscore the importance of epithelial cell shape in epithelial homeostasis. Despite the intense focus on the actomyosin-generated forces and cell removal from epithelia, the converse process by which a new cell is added to an existing epithelium has been largely ignored. This gap in our knowledge is significant because homeostasis of many epithelial tissues involves the addition of new cells from basally-located progenitors. Indeed, such basal stem cells have been described in the airway, olfactory epithelium, cornea, and prostate, among others. In such multilayered tissues, progenitor cells must move apically and insert individually and seamlessly into the epithelial sheet, a process that must be exquisitely coordinated in order to maintain epithelial function.
Apical emergence – a new cell behavior
During epithelial cell renewal, a nascent cell must not only define an apical domain, but must also build a new apical surface of sufficient size to accommodate its function (e.g. directional beating in a ciliated cell, luminal secretion in a secretory cell, etc.). This process of apical surface emergence can be envisioned perhaps most simply as the converse of apical constriction, a well-defined cell behavior underlying epithelial cell extrusion and epithelial folding. While molecular and mechanical aspects of apical constriction have been extensively studied, far less is known of apical emergence.
One major unresolved question concerns force generation during apical emergence of individual cells. What are the mechanisms that expand the growing apical surface? Moreover, how do new cells generate force to displace the neighboring cells that abut them? These questions about addition of new cells to epithelia are important because the answer will provide an essential complement to the burst of recent studies elucidating mechanisms of force generation during extrusion of old cells from epithelia and most importantly will provide key insights into mechanisms of epithelial homeostasis.
Figure 1. Apical emergence is an essential step for epithelial cell renewal.
A) Schematics of epithelial cell renewal. Basally localized progenitors move (step 1 to 3) from basal to apical layer of a multilayered epithelium. Upon docking at the tricellular junction, cell progenitors integrate within an existing epithelium by expanding their apical domain (step 3 to 4). B) We use Xenopus laevis mucociliary epithelium as a model system to study epithelial cell renewal in vivo (picture courtesy of Wallingford lab). C) Schematics of apical emergence. Green – cell progenitor, grey - existing epithelium. Apical domain of the progenitor cell expands displacing the neighboring cells. D, E) Image sequence of apically emerging multiciliated cell (MCC) (visualized with LifeAct-GFP, green) within Xenopus laevis epidermis (visualized with LifeAct-RFP). Note that cell-type specific promoters allow for targeted protein expression to MCCs of surrounding cells only.
Theoretical model of apical emergence
In collaboration with Dr. Edouard Hannezo we develop a minimal theoretical model of apically emerging cell. The model says that actin-based pushing (effective 2D Pressure), pulling (Λ), and contractile (𝛾) forces control expansion of the apical domain. The respective contributions of these forces can be assessed by analyzing the shape and dynamics behavior of the apical cell perimeter.
Figure 2. Theoretical model of apical emergence.
A) Apical domain expanding forces: effective 2D Pressure (2D Pressure) and neighboring junctional pulling forces (Λ) acting against cortical tension (𝛾) and elasticity from the surrounding cells (E). B) Model predicts shape changes of the apical domain and expansion dynamics. When considering cell autonomous forces within the cell apex, apical surface perimeters are rounded when pushing forces dominate, but are polygonal when pulling forces are in excess. Moreover, apical perimeters collapse when cortical tension (𝛾) exceeds the pushing forces (2D). For intermediary pressure, third cell behavior appears, where the apical area overshoots its steady state value, then undergoes damped oscillations.
Mechanism of apical actin assembly
Mature multiciliated cells are characterized by a complex and well-defined apical actin network. This stereotypic pattern is generated by the action of actin-binding proteins, such as actin nucleators and cross-linkers. The coordinated action of these proteins could generate the pushing force that expands apical surface. We use TIRF and super-resolution microscopy to image the process of apical actin assembly.
Figure 3. Assembly of the apical actin network expands the apical domain of a progenitor cell. A) Schematics of the apical actin network assembly. B) Imaging of de novo actin network assembly in vivo.
Tu, F., Sedzinski, J., Ma, Y., Marcotte, E.M., and Wallingford, J.B. (2018). Protein localization screening in vivo reveals novel regulators of multiciliated cell development and function. Journal of Cell Science 131, doi:10.1242/jcs.206565.
Sedzinski, J., Hannezo, E., Tu, F., Biro, M., and Wallingford, J.B. (2017). RhoA regulates actin network dynamics during apical surface emergence in multiciliated epithelial cells. Journal of Cell Science 130, 420-428, doi:10.1242/jcs.194704. Correction in Journal of Cell Science 130, 1017. doi: 10.1242/jcs.202234.
Sedzinski, J., Hannezo, E., Tu, F., Biro, M., and Wallingford, J.B. (2016). Emergence of an Apical Epithelial Cell Surface In Vivo. Developmental Cell 36, 24-35, doi:10.1016/j.devcel.2015.12.013.
Sedzinski, J., and Wallingford, J. (2015). Planar Pol(o)arity. Developmental Cell 33, 494-495, doi:10.1016/j.devcel.2015.05.022.
Turk, E., Wills, A.A., Kwon, T., Sedzinski, J., Wallingford, J.B., and Stearns, T. (2015). Zeta-Tubulin Is a Member of a Conserved Tubulin Module and Is a Component of the Centriolar Basal Foot in Multiciliated Cells. Current Biology 25, 2177-2183, doi:10.1016/j.cub.2015.06.063.
Sedzinski, J., Biro, M., Oswald, A., Tinevez, J.Y., Salbreux, G., and Paluch, E. (2011). Polar actomyosin contractility destabilizes the position of the cytokinetic furrow. Nature 476, 462-466, doi:10.1038/nature10286.
Epithelial cell renewal in vivo
Click on the movie to open on YouTube
Apical emergence of a multiciliated cell
(MCC) within Xenopus laevis epidermis
(green – MCC progenitor, magenta – goblet cells)
Automatic segmentation of apically emerging cell. We use image-processing software developed by Dr. Maté Biro to quantify geometrical and intensity parameters during apical emergence.
Apical emergence of a multiciliated cell (MCC) within Xenopus laevis epidermis upon expression of a dominant negative (DN) RhoA