We propose to identify the effects of altered gravity on nuclear morphology and cytoskeletal organization in human breast epithelial cells flown in parabolic flight. Cells flown will be cultured in a specially designed hardware to control for temperature and humidity, and the cells will be fixed on flight at different timepoints. High resolution imaging of cell shape and nuclear shape will be carried out using a laser scanning confocal microscope. Image analysis software will be used for three- dimensional shape reconstruction and quantification of shape parameters. We will characterize the mechanical forces acting on the nucleus using a Fluorescence Resonance Energy Transfer (FRET) probe biosensor to investigate the effect of altered gravity on the nuclear tension. Finally, we will identify the genes that are differentially expressed in LINC disrupted cells to understand the mechanotransduction pathway in sensing altered gravity.

The LINC complex (Linker of Nucleoskeleton and Cytoskeleton) connects the nucleus and the surrounding cytoskeleton to mechanically couple the nuclear lamina with the cytoskeleton. Recent advances in our understanding of how nuclear envelope and nucleoskeleton associated elements contribute to both health and disease make it clear that cells require a functional mechanical coupling mediated by the LINC complex to respond normally to external mechanical forces. This research will answer the following question: Does altered gravity induce changes in force transmission through the LINC complex and LINC-associated gene expression by analyzing fibroblasts subjected to microgravity of parabolic flight?

Post-flight analysis

i. Microscopy

Completed imaging all the samples transfected with control and FRET tension sensors with laser scanning confocal microscope. Completed imaging samples stained for nucleus and actin cytoskeleton from the first parabolic flight. The samples were collected at 1st, 7th, and 15th parabolas, inflight controls and ground controls.

We are currently performing quantitative analysis on the acquired images.

ii. RNA isolation and RNAseq analyses

RNA was successfully isolated and RNA sequencing performed at the Interdisciplinary Center for Biotechnology Research (ICBR) University of Florida. Results are currently being analyzed to extract patterns of gene expression.

We propose to identify the effects of altered gravity on nuclear morphology and cytoskeletal organization in fibroblasts flown in parabolic flight. Cells flown will be cultured in a specially designed hardware to control for temperature and humidity, and the cells will be fixed on flight at different timepoints. High resolution imaging of cell shape and nuclear shape will be carried out using a laser scanning confocal microscope. Image analysis software will be used for three- dimensional shape reconstruction and quantification of shape parameters. We will characterize the mechanical forces acting on the nucleus using a Fluorescence Resonance Energy Transfer (FRET) probe biosensor to investigate the effect of altered gravity on the nuclear tension. Finally, we will identify the genes that are differentially expressed in LINC disrupted cells to understand the mechanotransduction pathway in sensing altered gravity.