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Actin Cytoskeleton Self-Organization in Single Epithelial Cells And © 2019. Published by The Company of Biologists Ltd | Journal of Cell Science (2019) 132, jcs220780. doi:10.1242/jcs.220780 RESEARCH ARTICLE Actin cytoskeleton self-organization in single epithelial cells and fibroblasts under isotropic confinement Salma Jalal1, Shidong Shi1, Vidhyalakshmi Acharya1, Ruby Yun-Ju Huang2,3,4, Virgile Viasnoff1,5,6, Alexander D. Bershadsky1,7,* and Yee Han Tee1,* ABSTRACT integrin-mediated adhesions. In contrast, fibroblasts are embedded Actin cytoskeleton self-organization in two cell types, fibroblasts and in and attached to the extracellular matrix of connective tissue and epitheliocytes, was studied in cells confined to isotropic adhesive do not readily adhere to neighboring cells. The difference between islands. In fibroblasts plated onto islands of optimal size, an initially morphology and migratory characteristics of epithelial cells and circular actin pattern evolves into a radial pattern of actin bundles that fibroblasts are accompanied by differences in their actin undergo asymmetric chiral swirling before finally producing parallel cytoskeleton organization. The most prominent element of the linear stress fibers. Epitheliocytes, however, did not exhibit succession actin cytoskeleton in epithelial cells is the adhesion belt associated – through all the actin patterns described above. Upon confinement, the with the cell cell junctions and located at the apical zone of the cells actin cytoskeleton in non-keratinocyte epitheliocytes was arrested at the (Braga, 2016; Volberg et al., 1986; Zhang et al., 2005). In typical circular stage, while in keratinocytes it progressed as far as the radial fibroblasts, the actin cytoskeleton is organized into parallel linear – pattern but still could not break symmetry. Epithelial–mesenchymal actomyosin bundles (stress fibers) associated with cell matrix transition pushed actin cytoskeleton development from circular towards adhesions (focal adhesions) (Burridge, 2017; Geiger, 1979; Heath – radial patterns but remained insufficient to cause chirality. Knockout of and Dunn, 1978). Consistently, upon epithelial mesenchymal cytokeratins also did not promote actin chirality development in transition (EMT) the circumferential bundles in epithelial cells are keratinocytes. Left–right asymmetric cytoskeleton swirling could, succeeded by a system of parallel stress fibers (Boyer et al., 1989; however, be induced in keratinocytes by treatment with small doses Haynes et al., 2011). Actin dynamics and several actin regulatory of the G-actin sequestering drug, latrunculin A in a transcription- proteins have been shown to be involved in EMT (Gardberg et al., independent manner. Both the nucleus and the cytokeratin network 2013; Li et al., 2010; Mori et al., 2009; Shankar and Nabi, 2015). followed the induced chiral swirling. Development of chirality in It is well documented that outside-in signaling from adhesion keratinocytes was controlled by DIAPH1 (mDia1) and VASP, proteins receptors directs the formation of the actin cytoskeleton within involved in regulation of actin polymerization. cells (Braga, 2016; Geiger et al., 2009). Therefore, it is possible that differences in actin cytoskeleton self-organization between epithelial This article has an associated First Person interview with the first author cells and fibroblasts are determined by the differences in available – – – of the paper. extracellular adhesions (cell cell and cell matrix versus cell matrix only). On the other hand, it is also possible that the actin cytoskeleton KEY WORDS: Actin cytoskeleton, Self-organization, Epithelial cell, itself has intrinsically different morphogenetic potentials in these two Fibroblast, Epithelial–mesenchymal transition cell types. To reveal the difference in the morphogenetic potential of the actin cytoskeleton independently of differences in adhesion INTRODUCTION conditions, we study the self-organization of the actin cytoskeleton in Differences in the morphogenetic behavior between fibroblasts and a standardized microenvironment provided by microfabricated epithelial cells have been well documented. In vivo, epithelial cells adhesive patterns (Théry, 2010; Théry et al., 2006). Under such possess apico-basal polarity and are organized into sheets of conditions, cells are deprived of adhesions to neighboring cells and cells that are connected to each other via E-cadherin-mediated only contact extracellular matrix proteins (specifically fibronectin or cell–cell junctions and attached to the basement membrane via collagen). To dissect the processes of actin cytoskeleton self- organization from potential interplay with cell shape changes during migration, we confined cells to isotropic circular adhesive islands so 1 Mechanobiology Institute, National University of Singapore, Singapore 117411. that they acquire discoid morphology and do not change shape. 2Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599. 3Department of Obstetrics & Gynaecology, National University We have previously used this system of isotropic confinement Hospital, Singapore 119228. 4Department of Anatomy, Yong Loo Lin School of to investigate the process of actin cytoskeleton self-organization Medicine, National University of Singapore, Singapore 117599. 5Centre National Pour la Recherche Scientifique, Singapore 117411. 6Department of Biological that leads to formation of parallel linear stress fibers in isolated Sciences, National University of Singapore, Singapore 117558. 7Department of fibroblasts (Tee et al., 2015). This setting revealed unique patterns Molecular Cell Biology, Weizmann Institute of Science, Rehovot 7610001, Israel. of actin development with initially symmetric circular patterns *Authors for correspondence ([email protected]; consisting of circumferential bundles that progressed into radial [email protected]) patterns composed of bundles of actin anchored at focal adhesions that grow radially inwards from the cell periphery. These patterns Y.H.T., 0000-0001-9779-0915 were followed by a symmetry-breaking process that produced a This is an Open Access article distributed under the terms of the Creative Commons Attribution novel chiral pattern in which all radial bundles of actin tilted in one License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. direction. This tilting converts the centripetal actin flow into a swirling motion that persists until arrays of parallel stress fibers are Received 25 May 2018; Accepted 24 January 2019 formed (the linear pattern). Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs220780. doi:10.1242/jcs.220780 We systematically compared actin cytoskeleton self-organization was less than a quarter, while the majority of cells were able to between epithelial cells and fibroblasts, as well as between different develop the chiral pattern (Fig. 1B,C). types of epithelial cells. We found that the morphogenetic repertoire Transition from the circular to radial patterns of actin involves of actin cytoskeleton patterns in epithelial cells was limited to the growth of RFs anchored at focal adhesions. To further probe the circular actin pattern in non-keratinocyte epitheliocytes and how spread area affects actin cytoskeleton self-organization, we extended to the radial actin pattern in keratinocytes, but did not examined its effects on focal adhesions as visualized by paxillin include the chiral actin pattern present in fibroblasts. immunostaining (Fig. 1D,E). Total focal adhesion area in fibroblasts The presence of the cytokeratin network in epithelial cells, its confined to adhesive islands increased with projected cell area. depletion during EMT and absence in fibroblasts, makes its There was a strong statistically significant increase of total focal interplay with the actin cytoskeleton a possible candidate to adhesion area in cells on patterns with areas of 500, 750, 1000 and explain the differences in the actin cytoskeleton development 1500 µm2 (Fig. 1D). Of note, the increase in total focal adhesion between the two cell types. However, we demonstrated that the area was somewhat greater than the increase in projected cell area. In pattern of actin cytoskeleton self-organization in keratinocytes does particular, there is a mild statistically significant increase between not require cytokeratin filaments. Growth factor-induced EMT the total focal adhesion area normalized to the projected cell area pushed development of the actin cytoskeleton in some epithelial between patterns with areas of 500 and 1000 µm2 (Fig. S1D). The cells from circular towards radial actin patterns but was insufficient increase in total focal adhesion area approached a plateau at the to induce chiral actin pattern development. island size of 1500 µm2, so that the total focal adhesion area in cells We found, however, that chiral actin pattern development can be on islands of larger sizes (2000 and 2500 µm2) remained constant induced in keratinocytes by treatment with low doses of the G-actin (Fig. 1D), while normalized total focal adhesion area decreased sequestering drug latrunculin A. Both nuclei and the cytokeratin (Fig. S1D). network followed the chiral actin swirling without impeding it. The induction of chiral actin patterns was independent of transcriptional Non-keratinocyte epithelial cells confined to circular changes but involved at least two major regulators
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