Mechanical trapping of the nucleus on micropillared surfaces inhibits the proliferation of vascular smooth muscle cells but not cervical cancer HeLa cells
Introduction
Cells sense the external environment and translate this information into biochemical signals that induce various cell responses. The transport of macromolecules between the nucleus and cytoplasm is believed to have a central role in these intracellular signal transduction processes (Terry et al., 2007); nuclear transport of proteins and RNA through the nuclear pore complex is mediated by the biochemical activation of transport receptors, and the machinery that mediates nucleocytoplasmic exchange directly affects gene expression in cells (Pemberton and Paschal, 2005).
Recent studies have suggested that biophysical and biomechanical cues are also important factors for directing cell events, such as cell proliferation (Ingber, 1990), differentiation (Engler et al., 2006), apoptosis (Chen et al., 1997), and gene expression (Smith et al., 2000). The nucleus is the largest and stiffest organelle in eukaryotic cells (Dahl et al., 2008) and is exposed to the mechanical forces transmitted through the cytoskeleton from outside the cell (Lombardi et al., 2011) and the intracellular tension generated by the actin–myosin contractile cytoskeleton via the LINC complex (Nagayama et al., 2011b; Anno et al., 2012). The nucleus itself has been proposed to act as a cellular mechanosensor, and the changes in nuclear shape or volume induced by the area controlling cell adhesion possibly affect the regulation of cell proliferation (Roca-Cusachs et al., 2008, Versaevel et al., 2012). Mechanical properties of the nucleus and its deformability have been also suggested to be involved in cell functions; the nuclei in undifferentiated stem cells deform more readily than those in differentiated cells (Pajerowski et al., 2007), and metastatic cancer cells achieve tissue invasion through the dense extracellular matrix owing to nuclear deformability (Mak et al., 2013). However, the investigation of nuclear mechanosensing is still very recent and numerous questions regarding the physiological roles of the nuclear deformation phenomenon remain unanswered.
Here we investigated the effects of nuclear deformation on cellular events, such as cell proliferation, using microfabricated cell culture substrates with an array of micropillars. The nuclei in the cells showed dramatic deformation with features that matched the surface topography of the micropillar substrates. Thus, we used vascular smooth muscle cells (SMCs) and HeLa cells to determine the differential effects of the micropillar-induced nuclear deformation on normal healthy cells and metastatic cancer cells, respectively. We measured the proliferation of cells on the micropillar substrates and quantified their nuclear morphology, nuclear mechanical properties, and deformability, and we discussed the effects of nuclear trapping with the micropillar substrates on cell proliferation processes.
Section snippets
Preparation of the specimen cells
Porcine aortic SMCs were used as the healthy cells. They were obtained by an explant method described previously (Nagayama et al., 2006), and cultured in a standard culture medium, i.e., Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, JRH Bioscience, Lenexa, KS, USA), penicillin (100 unit/ml) and streptomycin (100 µg/ml, Sigma, St. Louis, MO, USA) at 37 °C in 5% CO2 and 95% air. The cells were passaged repeatedly at a 1:4
Results and discussion
Typical fluorescent images of SMCs and HeLa cells cultured on the PDMS pillars and flat substrates are shown in Fig. 2. Both types of cells spread completely between the fibronectin-coated pillars at day 1, leading to strong deformations of their nuclei (Fig. 2C and G). On the pillar substrates, the nuclei in SMCs were entirely inserted into the grooves between the pillars and they appeared to be “trapped” mechanically on the array of pillars, although they remained in an elongated shape (Fig. 2
Conflict of interest statement
The authors declare that they have no conflict of interest with regard to this manuscript.
Acknowledgments
This work was supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (Nos. 24680051, 25111711, and 26560207 to K.N., and No. 22127008 to T.M.). The authors would like to thank Mrs. Emi Nagayama for her technical help for image analysis.
References (35)
- et al.
Role of nesprin-1 in nuclear deformation in endothelial cells under static and uniaxial stretching conditions
Biochem. Biophys. Res. Commun.
(2012) - et al.
In the middle of it all: mutual mechanical regulation between the nucleus and the cytoskeleton
J. Biomech.
(2010) - et al.
A thin-layer model for viscoelastic, stress-relaxation testing of cells using atomic force microscopy: do cell properties reflect metastatic potential?
Biophys. J.
(2007) - et al.
Flow-induced hardening of endothelial nucleus as an intracellular stress-bearing organelle
J. Biomech.
(2005) - et al.
Matrix elasticity directs stem cell lineage specification
Cell
(2006) - et al.
Inhibitory activity of a heterochromatin-associated serpin (MENT) against papain-like cysteine proteinases affects chromatin structure and blocks cell proliferation
J. Biol. Chem.
(2002) - et al.
Lamins A and C but not lamin B1 regulate nuclear mechanics
J. Biol. Chem.
(2006) - et al.
The interaction between nesprins and sun proteins at the nuclear envelope is critical for force transmission between the nucleus and cytoskeleton
J. Biol. Chem.
(2011) - et al.
Barrier-to-autointegration factor – a BAFfling little protein
Trends Cell Biol.
(2007) - et al.
Heterogeneous response of traction force at focal adhesions of vascular smooth muscle cells subjected to macroscopic stretch on a micropillar substrate
J. Biomech.
(2011)
Stress fibers stabilize the position of intranuclear DNA through mechanical connection with the nucleus in vascular smooth muscle cells
FEBS Lett.
Hydrostatic pressure influences morphology and expression of VE-cadherin of vascular endothelial cells
J. Biomech.
Micropatterning of single endothelial cell shape reveals a tight coupling between nuclear volume in G1 and proliferation
Biophys. J.
Influence of lamin A on the mechanical properties of amphibian oocyte nuclei measured by atomic force microscopy
Biophys. J.
BAF: roles in chromatin, nuclear structure and retrovirus integration
Trends Cell. Biol.
Facultative heterochromatin: is there a distinctive 15 molecular signature?
Mol. Cell
Geometric control of cell life and death
Science
Cited by (29)
Changes in the intra- and extra-mechanical environment of the nucleus in Saos-2 osteoblastic cells during bone differentiation process: Nuclear shrinkage and stiffening in cell differentiation
2023, Journal of the Mechanical Behavior of Biomedical MaterialsCitation Excerpt :Moreover, modulation of the F-actin cytoskeleton has been found to influence complex pathways in some processes of cellular differentiation, including osteogenesis (Vinall et al., 2002; Zhang et al., 2006; Lim et al., 2000). However, limited information is available regarding the effect on the mechanical environment of the intracellular nucleus, such as the mechanical properties of the nucleus and intracellular forces exerted on the nucleus, which have recently been found to be involved to a large degree in various cellular functions (Woychek and Jones, 2019; Renkawitz et al., 2019; Versaevel et al., 2012; Nagayama et al., 2015). Accordingly, in this study, we investigated the changes in the mechanical environment of the nucleus during osteogenic differentiation in human osteoblast-like cells (Saos-2), in which differentiation was induced by cultivation in the osteogenic differentiation medium.
Micropillar-based phenotypic screening platform uncovers involvement of HDAC2 in nuclear deformability
2022, BiomaterialsCitation Excerpt :Nevertheless, the striking results with both molecules targeting HDAC2 confirmed the specific involvement of HDAC2 in the control of nuclear shape on micropillars, with the induction of a strong nuclear rounding effect at low micromolar concentrationsfor WT161 (EC50 2 μM) (Fig. 6F, n = 6) and FK228 outperforming TSA in terms of potency with an EC50 of 41 nM (Fig. 6G, n = 6). Micropillared surfaces are a well-established technique to promote self-induced deformations to the nucleus as the result of cellular adaptation to pseudo-3D (2.5D) environments [14–27,31–36]. Relative to low-throughput assays testing individual nuclear physical parameters such as atomic force microscopy, micropipette suction and optical/magnetic tweezers, 2.5D substrates are suitable for high-throughput analytical approaches, especially when paired with imaging [37].
Macroscopic and microscopic analysis of the mechanical properties and adhesion force of cells using a single cell tensile test and atomic force microscopy: Remarkable differences in cell types
2020, Journal of the Mechanical Behavior of Biomedical MaterialsCitation Excerpt :These cellular stiffness and adhesion strength might be reflected cell type difference which is deeply involved in cellular physiological functions. Based on the fundamental research for cell-type differences in mechanical characteristics, we previously found that the nucleus in cervical cancer HeLa cells was significantly softer than that in VSMCs (Nagayama et al., 2015). Such mechanical differences in intracellular organelles may influence the whole cell mechanical properties and their physiological functions.
Cell engineering: Biophysical regulation of the nucleus
2020, BiomaterialsSquare prism micropillars on poly(methyl methacrylate) surfaces modulate the morphology and differentiation of human dental pulp mesenchymal stem cells
2019, Colloids and Surfaces B: BiointerfacesCitation Excerpt :Indeed, a negative correlation was found between the specific growth rates and the average nuclear deformation values on the substrates during Days 3–7 (Fig. 5B). Nagayama et al. also reported inhibition of the proliferation of vascular smooth muscle cells on micropillar decorated surfaces, which they explained by the condensation of the chromatin in the deformed nuclei [33]. Specific growth rates between days 7 and 14 on untreated P4G4 and P8G8 surfaces leveled with those on the unpatterned and P16G16 surfaces (Supplementary Table 3), probably because of the gradual disappearance of nuclear deformations on P4G4 and P8G8 as the cells crawled onto the pillar tops at later time points.