Epithelial Cell Chirality Revealed by Three-Dimensional Spontaneous Rotation

Abstract

Our understanding of the left-right (LR) asymmetry of embryonic development, in particular the contribution of intrinsic handedness of the cell or cell chirality, is limited due to the confounding systematic and environmental factors during morphogenesis and a ack of physiologically relevant in vitro 3D platforms. Here we report an efficient two-layered biomaterial platform for determining the chirality of individual cells, cell aggregates, and self-organized hollow epithelial spheroids. This bioengineered niche provides a uniform defined axis allowing for cells to rotate spontaneously with a directional bias toward either clockwise or counterclockwise directions. Mechanistic studies reveal an actin-dependent, cell-intrinsic property of 3D chirality that can be mediated by actin cross-linking via α-actinin-1. Our findings suggest that the gradient of extracellular matrix is an important biophysicochemical cue influencing cell polarity and chirality. Engineered biomaterial systems can serve as an effective platform for studying developmental asymmetry and screening for environmental factors causing birth defects.

Keywords: biomaterial; cell chirality; cell polarity; left–right asymmetry; tissue morphogenesis.

Fig. 1.

Spontaneous rotation of self-organized microspheroids is chiral. (A) Schematic depicting the 3D cell chirality assay for epithelial microspheroids. Individual epithelial cells were embedded between a 100% Matrigel base layer and a 2% Matrigel top layer and then allowed to proliferate and form microspheroids containing a lumen after 5 d of culture. The spontaneous chiral rotation of a multicellular hollow spheroid about the z-axis is defined by the hydrogel gradient. (B) Confocal imaging of MDCK microsphere surface at day 5 shows a honeycomb-like assembly of cells with ZO-1 localized at the nodes. (C) A confocal cross-section through the middle of the microspheroid reveals a hollow lumen. (D and E) Cell alignment along the middle region of the microtissue coils around the structure toward the poles at the top and the bottom of the spheroid in 3D reconstructions of confocal imaging. (F) The microspheroids undergo spontaneous in-plane (x-y) rotation, with a bias toward the CCW direction. (Scale bars: 10 µm.) **P < 0.01.

Fig. 2.

The 3D rotational direction of hollow microspheroids switched from CCW to CW under Lat A treatment. (A) The fraction of microspheroids undergoing CW or CCW rotation. (B) The fraction of microspheroids undergoing rotation, complex rotation, or no rotation. (C) The corresponding table showing the number of spheroids in different modes of motion. The boldface red numbers in C represent a statistically significant bias toward the specific direction. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.

The 3D rotation of individual cells is CW-biased under Lat A treatment. (A) The fraction of individual cells undergoing CW or CCW rotation. (B) The fraction of cells undergoing rotation, complex rotation, or no rotation. (C) The corresponding table showing the number of cells in different modes of motion. The boldface red numbers in C represent a statistically significant bias toward the specific direction. *P < 0.05; **P < 0.01; ***P < 0.001. (D) Overlay of live fluorescence and phase time-lapse imaging of the cells without Lat A (blue, nuclei; red, Golgi apparatuses), with nuclear movement tracked with green lines. (E) Corresponding phase-contrast images with the cells tracked as shown in green. (Scale bars: 20 μm.)

Fig. 4.

Directional bias of rotational behavior by individual cells can be regulated by α-actinin-1. (A and D) MDCK cells transiently transfected with pEGFP-N1 α-actinin-1 (D) exhibited a predominantly CW rotation, in contrast to those transfected with the control plasmid, pEGFP-N1 (A). (B and E) Quantification of the total EGFP fluorescence intensity per cell reveals that the CW-rotating MDCK cells exhibited greater average expression of α-actinin-1 compared with the cells identified with CCW rotational motion (E), while there was no significant difference between CW- and CCW-rotating cells transfected with the control plasmid (B). (C and F) Histograms displaying the distribution of CCW and CW cells transfected with pEGFP-N1 (C) or pEGFP-N1 α-actinin-1 plasmids (F) with the increase in fluorescence intensity. Each bar represents an individual CW (orange) or CCW (blue) cell, sorted in ascending order of EGFP fluorescence expression. Cells expressing higher levels of α-actinin-1 expression rotated mostly CW, while there were nearly equal instances of CW and CCW rotation in the cells with lower α-actinin-1 expression. MDCK cells transfected with the control plasmid exhibited a slight bias toward CCW, which did not change with changes in fluorescence intensity. ***P < 0.001.