Heterogeneous focal adhesion cytoskeleton nanoarchitectures from microengineered interfacial curvature to oversee nuclear remodeling and mechanotransduction of mesenchymal stem cells

Abstract

Background: Interfacial heterogeneity is widely explored to reveal molecular mechanisms of force-mediated pathways due to biased tension. However, the influence of cell density,, curvature, and interfacial heterogeneity on underlying pathways of mechanotransduction is obscure.

Methods: Polydimethylsiloxane (PDMS)-based stencils were micropatterned to prepare the micropores for cell culture. The colonies of human mesenchymal stem cells (hMSCs) were formed by controlling cell seeding density to investigate the influences of cell density, curvature and heterogeneity on mechanotransduction. Immunofluorescent staining of integrin, vinculin, and talin-1 was conducted to evaluate adhesion-related expression levels. Then, immunofluorescent staining of actin, actinin, and myosin was performed to detect cytoskeleton distribution, especially at the periphery. Nuclear force-sensing mechanotransduction was explained by yes-associated protein (YAP) and laminA/C analysis.

Results: The micropatterned colony of hMSCs demonstrated the coincident characters with engineered micropores of microstencils. The cell colony obviously developed the heterogeneous morphogenesis. Heterogeneous focal adhesion guided the development of actin, actinin, and myosin together to regulate cellular contractility and movement by integrin, vinculin, and talin-1. Cytoskeletal staining showed that actin, actinin, and myosin fibers were reorganized at the periphery of microstencils. YAP nuclear translocation and laminA/C nuclear remodeling were enhanced at the periphery by the regulation of heterogeneous focal adhesion (FA) and cytoskeleton arrangement.

Conclusions: The characters of the engineered clustering colony showed similar results with prepared microstencils, and colony curvature was also well adjusted to establish heterogeneous balance at the periphery of cell colony. The mechanism of curvature, spreading, and elongation was also investigated to disclose the compliance of FA and cytoskeleton along with curvature microarrays for increased nuclear force-sensing mechanotransduction. The results may provide helpful information for understanding interfacial heterogeneity and nuclear mechanotransduction of stem cells.

Keywords: Cell colony; Cytoskeleton tension; Focal adhesion; Interfacial heterogeneity; Nuclear mechanotransduction.

Fig. 1

Characteristics of polydimethylsiloxane (PDMS) stencils and the characterization of cell colony of human mesenchymal stem cells (hMSCs). a Illustration of PDMS-based microstencils preparation and cell seeding; b geometrical features of microstencils, two-type microstencils were considered as D-800 and D-1500 for the diameters of 800 and 1500 μm; c digital pictures of PDMS-based microstencils, a millimeter ruler is shown at the bottom; d microscopic pictures of a clustering colony of hMSCs. The scale bar represents 1000 μm. Right two pictures for each density are the amplifications at both central and edge areas. The scale bar represents 100 μm

Fig. 2

Regulation of cell seeding density, curvature and heterogeneity on cell distribution. a Illustration of the compliance of human mesenchymal stem cells (hMSCs) at the periphery; b fluorescent pictures of blue 4’,6-diamidino-2-phenylindole (DAPI) nuclei, scale bar represents 1000 μm; c definition of central and peripheral areas in the engineered hMSCs; d density of hMSCs at both central and peripheral areas of hMSCs in D-800 and D-1500 colonies, data are presented as mean ±  standard deviation (SD), n = 5, n.s.: no significance, *p < 0.05, ***p < 0.001

Fig. 3

Evaluation of heterogeneous focal adhesions (FAs) by engineered cell density and curvature. a Illustration of the engineered FA proteins as a mechanosensor between extracellular matrices (ECMs) and cytoskeletons; b fluorescent pictures of integrin proteins (green), scale bar represents 1000 μm; c Western blot (WB) analysis of integrin and vinculin in the micropatterned human mesenchymal stem cells (hMSCs) at the central regions; d quantitative intergrin/β-actin in central regions; e quantitative vinculin/β-actin in central regions; f WB analysis of integrin and vinculin in the micropatterned hMSCs at the peripheral regions; g quantitative integrin/β-actin in peripheral regions; h quantitative vinculin/β-actin in peripheral regions, data are presented as mean ± standard deviation (SD), n = 3, ns: no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0005

Fig. 4

Regulation of interfacial heterogeneity on actin and actinin arrangement. a Fluorescent pictures of nuclei (blue), actin (red) and actinin (green), scale bar represents 1000 μm; b elongation (aspect ratio) of the engineered human mesenchymal stem cells (hMSCs) in the central regions; c elongation (aspect ratio) of the engineered hMSCs in peripheral regions, data are presented as mean ±  standard deviation (SD), n = 5, n.s.: no significance, *p < 0.05, **p < 0.01

Fig. 5

Heterogeneity of curvature-mediated myosin by the regulation of heterogeneous focal adhesion (FA) assembly in an engineered colony of human mesenchymal stem cells (hMSCs). a Fluorescent pictures of nuclei (blue), actin (red) and myosin (green), scale bar represents 1000 μm; b myosin heatmap pictures, created by overlaying more than 20 colonies into one picture, scale bar represents 1000 μm; c illustration of the regulation of FAs and cytoskeleton filaments on nuclear mechanotransduction; MLC: myosin light chain; MLCK: myosin light chain kinase; PPtase: phosphopantetheinyl transferase

Fig. 6

Heterogeneous focal adhesion (FA) assembly and cytoskeleton remodeling to regulate yes-associated protein (YAP) mechanotransduction and nuclear laminA/C organization in an engineered colony of hMSCs. a Fluorescent pictures of YAP staining (green), nuclei were stained blue, scale bar represents 1000 μm, bottom two panels are enlargement images of central and peripheral regions, scale bar represents 500 μm; b percentage of YAP nuclear location at the central regions; c percentage of YAP nuclear location at the peripheral regions, data are presented as mean ± standard deviation (SD), n = 5, n.s.: no significance, *p < 0.05, **p < 0.01, ***p < 0.001; d Western blot (WB) analysis of laminA/C in the engineered colony with low and high density; e quantitative laminA/C/β-actin in the central regions; f quantitative laminA/C/β-actin in peripheral regions, data are presented as mean ± SD, n = 3, n.s.: no significance, ***p < 0.001, ****p < 0.0005

Fig. 7

Mechanism of curvature, spreading area and aspect ratio on cytoskeleton distribution and nuclear activity in the engineered cells. a Fluorescent pictures of actin (green) and nuclei (blue) in the engineered cells, scale bar represents 50 μm; b nuclear area of the engineered cells, data are presented as mean ± standard deviation (SD), n = 5, n.s.: no significance, **p < 0.01, ***p < 0.001; c angular compliance of nuclei in the engineered cells; d fluorescent pictures of BrdU positive nuclei, BrdU is shown in green, nuclei in blue, scale bar represents 200 μm; e nuclear activity by calculating BrdU positive cells in the engineered cells, data are presented as mean ± SD, n = 5, n.s.: no significance, *p < 0.05, **p < 0.01, ***p < 0.001