Epithelium comprises the majority of metazoan structures and perform important physiological functions such as protection barrier, secretion, and selective absorption. They are highly polarized and the plasma membranes are separated into apical, lateral, and basal sides. Tight junctions form a continuous belt at the sub-apical location at the borders of two cells as a fence function to maintain the polarity of membrane proteins and seal the paracellular space between cells. The tight junctions are linked to actin cytoskeleton through an adaptor proteins. We found that the tight junctions form tortuous structure as Madin Darby Canine Kidney (MDCK) cells grow into higher confluency on a 2D transwell or as a cyst in 3D matrigel. When we perturbed actin-myosin contractility of MDCK cells by small molecules Y27632, blebbistatin, and ML-7, the tight junctions become less tortuous, and cell shape changes in terms of height and the apical area. We developed 3D image analysis to quantify the tortuosity of tight junctions and proposed that the morphological change of tight junctions can be indication of apical constriction force and cell-cell tension. By constructing a simple theoretical model by surface evolver to explain the morphology of tight junction affected by the interplay between apical, and lateral tension. Using the endogenous cellular structure for quantify intercellular force is non-perturbative and the gained knowledge can be used to test current theoretical models which explains the epithelial cell shapes based on basal, lateral, and apical tensions.
3D Time-lapse of the actins, and tight junctions of a cyst treated with Y-27632, I took it using confocal spinning disk. Notice how the tight junctions become less tortuous.
So I made a macro to segment, and skeletonize tight junction signals in Imaris then wrote a program to quantify curvature in Matlab.
I also segmented actin signals in the cyst with Avizo to quantify cellular shape. I found there’s a correlation between tight junction tortuousity, cell apical, lateral, and basal area, and its height.
I also ablated actins using 2-photon microscope to show that apical area expands, while the tortuosity of tight junctions becomes less.
Then here comes the magical part, our simulation in surface evolver is similar with our experimental observation! Tight junctions become less tortuous with less apical surface tension.
In summary, we provided evidence that actin-myosin in the apical part produces surface tension (A) in contrast to a long known “purse-string” model (B) where line tension dominates.
BTW, I also have observed similar changes in cell shape, and tight junction tortuousity in 2D culture which is amazing! I have tons of supporting experiments, and we can talk about it. I did this work during my MSc in Physics at Soft matter and Biophysics laboratory in Institute of Physics, Academia Sinica under the supervision of Dr. Keng-hui Lin, and support of a talented undergraduate student Ching-Chung Hsueh. I presented this work in Biophysical Journal conference, and won the student poster award.
(January 2016 – June 2017)