Modulation of nuclear membrane repair machinery by nano-needle arrays

Abstract

Abnormal nuclear structure and temporary nuclear membrane rupture have been identified by recent studies as common characteristics in cancer cells. However, cancer cell lines have been found to repeatedly proliferate after nuclear membrane rupture, implicating a mechanism of over-activated nuclear membrane repair outside of the normal cell cycle. Therefore, the modulation of nuclear membrane repair machinery has emerged as a new target for cancer therapeutics. The identification of therapeutic targets depends on the visualization, characterization and mechanistic understanding of repair machinery in health and disease, with single cell resolution. Current methods for investigating nuclear repair machinery suffer from limitations in spatial resolution, throughput, control and reproducibility of activated repair mechanisms. In this work, we propose a new platform consisting of arrays of nano-scale needles with varying geometries to induce sharp curvature and potentially rupture of the nuclear membrane. We used immunocytochemistry to visualize a nuclear DNA repair protein (KU80) that mislocalized to the cytoplasm as a marker of nuclear membrane rupture, and Lamin A/C to observe nuclear membrane shape on the nanoneedles. In comparison to a flat portion of the nanopillar array, the nuclear content of seeded cells are displaced spatially where nanostructures were present. Additionally, we observed frequent ruptures of the nuclear membrane of cells on nanoneedles. Our results imply that high curvature results in higher frequencies of nuclear membrane rupture, potentially activating repair machinery at the tips of nanoneedles. Thus, designing optimal geometries of nanoneedles can provide a platform with improved spatial resolution, throughput, control and reproducibility for inducing membrane rupture, and activating specific repair machinery. Ultimately, our platform can enable more efficient and controlled investigations into the nuclear membrane rupture and repair process, leading to new discoveries and research avenues for modulating this process in cancer.