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A Micropatterning Strategy to Study Nuclear Mechanotransduction in Cells.

ABSTRACT: Micropatterning techniques have been widely used in biology, particularly in studies involving cell adhesion and proliferation on different substrates. Cell micropatterning approaches are also increasingly employed as in vitro tools to investigate intracellular mechanotransduction processes. In this report, we examined how modulating cellular shapes on two-dimensional rectangular fibronectin micropatterns of different widths influences nuclear mechanotransduction mediated by emerin, a nuclear envelope protein implicated in Emery-Dreifuss muscular dystrophy (EDMD). Fibronectin microcontact printing was tested onto glass coverslips functionalized with three different silane reagents (hexamethyldisilazane (HMDS), (3-Aminopropyl)triethoxysilane (APTES) and (3-Glycidyloxypropyl)trimethoxysilane (GPTMS)) using a vapor-phase deposition method. We observed that HMDS provides the most reliable printing surface for cell micropatterning, notably because it forms a hydrophobic organosilane monolayer that favors the retainment of surface antifouling agents on the coverslips. We showed that, under specific mechanical cues, emerin-null human skin fibroblasts display a significantly more deformed nucleus than skin fibroblasts expressing wild type emerin, indicating that emerin plays a crucial role in nuclear adaptability to mechanical stresses. We further showed that proper nuclear responses to forces involve a significant relocation of emerin from the inner nuclear envelope towards the outer nuclear envelope and the endoplasmic reticulum membrane network. Cell micropatterning by fibronectin microcontact printing directly on HMDS-treated glass represents a simple approach to apply steady-state biophysical cues to cells and study their specific mechanobiology responses in vitro.

SUBMITTER: Bautista M

PROVIDER: S-EPMC6952994 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Artificial small-diameter vascular grafts remain an unmet need in modern medicine, due to the thrombosis and neointimal hyperplasia that plague currently available synthetic devices. Tissue engineering techniques, including in vitro endothelialization, could offer a solution to this problem. A potential minimally invasive source of patient autologous endothelium is endothelial colony-forming cells (ECFCs), endothelial-like outgrowth products of circulating progenitors. While ECFCs respond to shear stress similar to mature endothelial cells (ECs), their response to luminal topographic micropatterning (TMP), a biomaterial modification with the potential to flow-independently, enhance the attachment, migration, gene expression, and function of mature ECs, remains unstudied. In this study, case-matched carotid endothelial cells (CaECs) and blood-derived ECFCs are statically cultured on polyurethane substrates with micropatterned pitches (pitch = peak to peak distance) ranging from 3-to 14 μm. On all pattern pitches tested, both CaECs and ECFCs showed significant and robust alignment to the angle of the micropatterns. Using a novel cell-by-cell image analysis technique, it was found that actin fibers similarly and significantly aligned to the angle of micropatterned features on all pitches tested. Microtubules analyzed through the same novel approach showed significant alignment on most pitches examined, with a greater variation in fiber angle overall. Interestingly, only CaECs showed significant cellular elongation, and notably to a lower degree than previously seen either in vivo due to flow or in vitro due to spatial growth restriction micropatterning, but consistent with earlier studies of TMP. Neither cell type displayed any significant micropattern-driven changes in the expression of KLF-2 or the downstream adhesion molecules it regulates. These results demonstrate that TMP flow-independently affects ECFC morphology, but that alignment alone is insufficient to drive protective changes in EC and ECFC function.

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A Micropatterning Strategy to Study Nuclear Mechanotransduction in Cells.

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