The Nuclear Mechanics & Genome Regulation Laboratory

GV Shivashankar's Laboratory pursues research in understanding the role of cell geometry on nuclear mechanics and genome regulation.

Cells, under physiological conditions, acquire a number of well-defined morphologies. Alterations in their shape, by mechanical microenvironment and/or cytokine signals, have profound impact on tissue homeostasis. While cells undergo changes in shape, for example, during circulation, crawling, extrusion or transmigration; the extent and duration to which such shape changes occur would have critical roles in regulating nuclear function including gene expression. In this context, how cell shape modulation alters nuclear mechanical architecture and how it integrates with the 3D organization of chromosomes and transcription networks are rather unexplored. Understanding the biophysical design principles underlying such processes will have important implications in establishing mechano-chemical routes to cellular reprogramming and in developing biomarkers for early disease diagnosis. Centered on this theme, our ongoing studies are beginning to provide a quantitative framework to explore the coupling between cell geometry and genome regulation. For these studies, we employ a multidisciplinary approach combining microfabrication techniques to sculpt single cell geometry, high-resolution microscopy, genomics and theoretical modelling.

Role of cell-geometry on nuclear and chromatin plasticity

Recent studies, including work from our own laboratory, have shown that changes in cell geometry leads to alterations in actin cytoskeletal architecture. This in turn modulates nuclear morphology via the physical links on the nuclear envelope and the lamin meshwork. However the role of cell shape regulated dynamic alterations in the cytoskeleton, on nuclear and chromatin plasticity is less understood. To address this, we use fibronectin coated micropatterns to define cell geometries with distinct cytoskeletal architecture and directly visualize the alterations in nuclear and chromatin dynamics using high resolution quantitative microscopy. The projects include probing the role of cell geometry on i) chromatin (heterochromatin and telomere) plasticity ii) its role in nuclear reprogramming.

Defining a nuclear mechanical code for genome regulation

Moodulation in cell geometric constraints has been shown to result in changes gene expression patterns. However, the critical role of 3D organization of the nuclear architecture and chromosome assembly in facilitating this genome regulation is unclear. To address this, we systematically alter fibroblast cell geometry and map whole genome transcriptome using microarray analysis. In addition we map chromosome positions using in situ hybridization techniques and directly visualize specific chromosome contacts under different geometric constraints using super-resolution microscopy. We are currently exploring i) transcription dependent reorganization of chromosome positions and functional gene clusters with altered cell-geometry ii) lamin A/C dependent active mechanisms underlying such chromosome reorganization.

Matrix and cytokine assisted nuclear mechanotransduction

Finally, recent studies have shown that, mechanical constraints in conjunction with soluble cytokine signals alter cellular behavior within the local tissue microenvironment. However the mechanisms underlying the interplay between these signals in regulating gene expression and thus cell behavior at the single-cell resolution are unexplored. A number of diseases, including fibrosis and cancer, originate at the single-cell level within the tissue microenvironment and therefore a quantitative understanding of the modular codes underlying these processes would be essential to develop therapeutic models. In these projects we study i) matrix and cytokine induced nuclear mechanotransduction ii) its role in chromosome contact maps and gene expression.

Nuclear microrheology and single-cell disease diagnostics

Cell-geometric constraints have profound impact on cytoskeletal organization thus influencing nuclear positioning and its microrhelogical response. For this, a number of cytoskeletal-to-nuclear linking proteins have been shown to be critical in regulating nuclear homeostatic balance. Quantitative analysis of these alterations could potentially serve as quantitative physical biomarkers of various diseases including Cancer. Based on this, we are developing miniaturized single-cell assays systems to define novel paradigms in early diagnostics. In these projects we study i) impact of cell geometry on nuclear positioning and its microrhelogy ii) implementing high-content and high-throughput nuclear biomechanical disease diagnostic device platforms.