Cytoskeletal Mechanism in Cell Chirality Reversal

細胞骨架調控細胞手性逆轉的機制研究

Student thesis: Doctoral Thesis

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Award date3 Aug 2023

Abstract

Research on cell behavior in relation to biochemical molecules has been carried out to understand the cell response in adhesion, polarity, motility, and spreading. Chirality or left-right (LR) asymmetry of cells is one of the recent research topics on biological properties that affect cell differentiation, tissue morphology, and organ positioning. The intrinsic property of LR asymmetry arises from molecule handiness, such as righthand bias in actin polymerization, and can be diversified across different cell types. However, the biochemistry environment and molecular factors controlling the diverse chirality are not thoroughly investigated. Here, this thesis explores the tissue, cellular, and cytoskeleton levels of LR asymmetry and chirality by providing a specific adhering surface with common myoblast and fibroblast cell lines as the biological model.

In the first project, C2C12 myoblast subculturing conditions demonstrate the organization of cell alignment is significantly influenced by the actin cytoskeleton, which plays a critical role in tissue morphology. The cell alignment dependence on substrate stiffness is observed after a low-density subculture. Cells previously conditioned with different subculturing densities behave differently in aspect ratio and intercellular alignment, which are controlled by the actin cytoskeleton. Different actin structure of single cells induced by subculturing densities shows that actin organization controls the chirality expression and the alignment of cells on micropatterned stripe. Furthermore, replating of the cell under subculturing conditions supplemented with actin-inhibiting drugs shows the lingering effects on cell alignment and actin cytoskeleton arrangement. This work benefits the reconstruction of tissue morphology by highlighting the importance of subculturing conditions in the actin structure of single cells, contributing to the self-organization of cell alignment.

The second project reveals that the cell projection area and actin structure determine the expression of single-cell chirality during cell spreading. Understanding single-cell chirality is crucial as single-cell chirality propagates and affects tissue alignment. In this study, the chirality of the nucleus rotation reverses from clockwise (CW) to anticlockwise (ACW) during spreading. Controlling the cell projection area with an isotropic circular interface mimics the chirality reversal, showing the cell spreading area directly influences the expression of cell chirality. This phenomenon is observed consistently across different cell types. The actin structure, radial fiber, shows the same ACW chiral direction with nucleus rotation when spreading on the large pattern. Meanwhile, almost no observable radial fiber or chiral structure is found on the small pattern. Recovery of the actin structure on the small pattern by drug treatment reverses the chirality from CW to ACW, suggesting the role of radial fibers on ACW chirality. This study indicates the importance of cell projection area and actin structure for nucleus rotation chirality.

The third project demonstrates different classes of actin, the radial fiber and the transverse arc, contribute to ACW and CW chirality in nucleus rotation, respectively. Radial fiber and transverse arc form a chiral pattern on an isotropic circular island. With live cell imaging, the reduction of radial fiber elongation rate attenuates ACW chirality, indicating the radial fiber attributes to ACW chirality. On the other hand, the CW chirality is inhibited with the disruption of the contractile component in the transverse arc, by suppressing myosin activity with molecule inhibition or gene silencing of myosin-related proteins, mDia2 and Tpm4. Additionally, the transverse arc locates immediately underneath the plasma membrane. Because force can be generated against a rigid surface, the actomyosin contraction of the transverse arc appears to be responsible for CW chirality. Overall, manipulating the cell projection area or altering one class of actin shows the critical role of radial fiber and transverse arc in the expression of cell chirality.

Together, the work suggested a suitable culturing condition to preserve the chiral behavior, and a new mechanism of actomyosin structure controls cell chirality, providing more insight into LR asymmetry at molecular and cellular levels. This work can benefit the chirality research in future tissue engineering and regeneration medicine.