Elsevier

Journal of Biomechanics

Volume 48, Issue 10, 16 July 2015, Pages 1796-1803
Journal of Biomechanics

Mechanical trapping of the nucleus on micropillared surfaces inhibits the proliferation of vascular smooth muscle cells but not cervical cancer HeLa cells

https://doi.org/10.1016/j.jbiomech.2015.05.004Get rights and content

Abstract

The interaction between cells and the extracellular matrix on a topographically patterned surface can result in changes in cell shape and many cellular functions. In the present study, we demonstrated the mechanical deformation and trapping of the intracellular nucleus using polydimethylsiloxane (PDMS)-based microfabricated substrates with an array of micropillars. We investigated the differential effects of nuclear deformation on the proliferation of healthy vascular smooth muscle cells (SMCs) and cervical cancer HeLa cells. Both types of cell spread normally in the space between micropillars and completely invaded the extracellular microstructures, including parts of their cytoplasm and their nuclei. We found that the proliferation of SMCs but not HeLa cells was dramatically inhibited by cultivation on the micropillar substrates, even though remarkable deformation of nuclei was observed in both types of cells. Mechanical testing with an atomic force microscope and a detailed image analysis with confocal microscopy revealed that SMC nuclei had a thicker nuclear lamina and greater expression of lamin A/C than those of HeLa cells, which consequently increased the elastic modulus of the SMC nuclei and their nuclear mechanical resistance against extracellular microstructures. These results indicate that the inhibition of cell proliferation resulted from deformation of the mature lamin structures, which might be exposed to higher internal stress during nuclear deformation. This nuclear stress-induced inhibition of cell proliferation occurred rarely in cancer cells with deformable nuclei.

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)

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