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Vijaykrishna Raghunathan, Clayton T. McKee, Elizabeth J. Tocce, Paul Nealey, Paul Russell, Christopher J. Murphy; Human Corneal Epithelial Nuclei Align To Nantopography. Invest. Ophthalmol. Vis. Sci. 2011;52(14):298.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal epithelium adheres to the stroma via the basement membrane (BM) that presents biophysical cues in the form of topography and compliance. Extracellular biophysical cues can modulate the morphology of cells and their nuclei through dynamic cytoskeletal changes, and initiate signaling cascades that in turn affect adhesion, migration, and gene expression. Nuclear morphology is an important factor influencing nuclear function, such as chromatin remodeling, nucleocytoplasmic transport and RNA trafficking. We investigated the effects of nano and sub-micron-scale topographic features on human corneal epithelial cell (HCEC) nuclear and cellular morphology and alignment.
HCECs were harvested from corneas and cultured in EpiLifeTM basal medium, for 12 h on flat NOA81 polyurethane substrates that were either planar or topographically patterned with alternating parallel ridges and grooves of various pitch. The pitch sizes were 400 nm, 800 nm, 1200 nm, 1600 nm, 2000 nm and 4000 nm. After 12 h in culture, cells were fixed with 4% paraformaldehyde, labeled with TRITC-phalloidin (f-actin) and DAPI (nucleus), and imaged by epifluorescence microscopy. To determine the alignment of single cells and their corresponding nuclei to the topography, an algorithm was developed to match the cytoskeleton to their nuclei, and measure their orientation and ratio of major to minor axis.
The orientation was observed to be dependent on the pitch size. HCECs preferentially align perpendicular to the topography with a maximum at 800 nm pitch; and with increasing pitch size, they become aligned parallel to the pitch with a maximum observed at 4000 nm. Nuclear orientation largely followed the same trend as the cell overall with the exception of those on 400 nm pitch. On this smallest biomimetic size scale some nuclei oriented perpendicular to the topography with their cytoskeleton aligned parallel. Both, cytoskeleton and nuclei were elongated on topography compared with those on flat surfaces. However, the ratio of major to minor axis of nuclei aligned parallel to the topography were significantly longer than those aligned perpendicular.
Our results demonstrate that the topographic scale mimicking those found in the native BM regulate cytoskeleton and nuclear alignment differentially. These finding suggest the topographic features of the corneal BM may have a potent effect on cytoplasmic and nuclear interaction in HCECs and may influence gene expression.
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