Biology subjects

Ghagre, A.

Publications and source records attributed to Ghagre, A..

3 recordsLinked to original sources

bioRxiv2020

Pattern-based Contractility Screening (PaCS), a reference-free traction force microscopy methodology, reveals contractile differences in breast cancer cells

The sensing and generation of cellular forces are essential aspects of life. Traction Force Microscopy (TFM) has emerged as a standard broadly applicable methodology to measure cell contractility and its role in cell behavior. While TFM platforms have enabled diverse discoveries, their implementation remains limited in part due to various constraints, such as time-consuming substrate fabrication techniques, the need to detach cells to measure null force images, followed by complex imaging and analysis, and the unavailability of cells for post-processing. Here we introduce a reference-free technique to measure cell contractile work in real-time, with basic substrate fabrication methodologies, simple imaging, and analysis with the availability of the cells for post-processing. In this technique, we confine the cells on fluorescent adhesive protein micropatterns of a known area on compliant silicone substrates and use the cell deformed pattern area to calculate cell contractile work. We validated this approach by comparing this Pattern-based Contractility Screening (PaCS) to conventional bead-displacement TFM and show quantitative agreement between the methodologies. Using this platform, we measure the contractile work of highly metastatic MDA-MB-231 breast cancer cells is significantly higher than non-invasive MCF-7 cells. PaCS enables the broader implementation of contractile work measurements in diverse quantitative biology and biomedical applications.

biophysics↗

bioRxiv2020

Lamin A redistribution mediated by nuclear deformation determines dynamic localization of YAP

YAP is a key mechanotransduction protein with essential roles in diverse physiological processes. Dysregulation in YAP activity is associated with multiple diseases such as atherosclerosis, fibrosis, and cancer progression. Here we examine the physical stimuli that regulate dynamic YAP translocation to the nucleus. Through a combination of biophysical studies, we demonstrate that YAP localization is insensitive to cell substrate stiffness, but strongly determined by cellular contractile work, which in turn deforms the nucleus. We show that nuclear deformation from LINC-mediated cytoskeletal contractility or extracellular osmotic forces triggers YAP nuclear localization. By modulating the expression of lamin A and nuclear stiffness, we illustrate that nuclear rigidity modulates deformation-mediated YAP nuclear localization. Finally, we show that nuclear deformation causes relocalization of lamin A from the nuclear membrane to the nucleoplasm, and this is essential in allowing YAP to enter the nucleus. These results reveal key physical nuclear deformation mechanics that drive YAP nuclear import.

biophysics↗

bioRxiv2019

Spatial distribution of lamin A determines nuclear stiffness and stress-mediated deformation

The nucleus is the largest organelle and information center of the cell; while diverse cellular components have been identified as mechanotransduction elements, the deformation of the nucleus itself is emerging as a critical mechanosensory mechanism, suggesting that the nuclear stiffness is essential in determining responses to intracellular and extracellular stresses. The nuclear membrane protein, lamin A, is known to be a dominant component in nuclear stiffening; however, the quantitative relationship between lamin A expression and nuclear deformation is still unclear. Here we measure the nuclear moduli as a function of lamin A expression and applied stress, revealing a linear dependence of bulk modulus on lamin A expression. We also find that the bulk modulus is anisotropic, with the vertical axis of the nucleus being more compliant than the minor and major axis. To examine how lamin A influences nuclear mechanics at the sub-micron scale we correlated the spatial distribution of lamin A with 3D nuclear envelope deformation, revealing that local areas of the nuclear envelope with higher expression levels of lamin A have correspondingly lower local deformations, and that increased lamin A expression levels result in a narrower distribution of smaller deformations. These findings describe the complex dispersion of nuclear deformations as a function of lamin A expression and distribution and implicate a role in mechanotransduction.

bioengineering↗