Physical basis of lumen shape and stability in a simple epithelium

Abstract

A continuous sheet of epithelial cells surrounding a hollow opening, or lumen, defines the basic topology of numerous organs. De novo lumen formation is a central feature of embryonic development whose dysregulation leads to congenital and acquired diseases of the kidney and other organs. While prior work has described the hydrostatic pressure-driven expansion of lumens when they are large, the physical mechanisms that promote the formation and maintenance of small, nascent lumens are less explored. In particular, models that rely solely on pressure-driven expansion face a potential challenge in that the Laplace pressure, which resists lumen expansion, is predicted to scale inversely with lumen radius. We investigated the cellular and physical mechanisms responsible for stabilizing the initial stages of lumen growth using a 3D culture system in which epithelial cells spontaneously form hollow lumens. Our experiments revealed that neither the actomyosin nor microtubule cytoskeletons are required to stabilize lumen geometry, and that a positive intraluminal pressure is not necessary for lumen stability. Instead, our observations are in agreement with a quantitative model in which cells maintain lumen shape due to topological and geometrical factors tied to the establishment of apicobasolateral polarity. We suggest that this model may provide a general physical mechanism for the formation of luminal openings in a variety of physiological contexts. Lumen shapes for small-and intermediate-size MDCK spheroids are inconsistent with a Laplace model of lumen stabilityWe seeded MDCK cells expressing a fluorescent marker for actin filaments (Lifeact-RFP) in the recombinant extracellular matrix Matrigel. Under these culture conditions, MDCK cells spontaneously form hollow spheroids within 24 hours. To obtain high-resolution images of nascent lumens, we imaged young (18-24 hours) spheroids using lattice light sheet microscopy (LLSM). This acquisition method produced images in which the two opposing apical cortices of the lumen are clearly distinguishable and separated by ~200-300 nm (Fig. 1b,c). We infer that cellular apical surfaces are intrinsically non-adherent, as even small fluctuations in cell shape would allow apposing apical surfaces to contact and potentially adhere. This "non-stick" behavior may reflect the enrichment of negatively charged sialoglycoproteins such as