Deputy Director, Mechanobiology Institute and IFOM-NUS Chair Professor
The nucleus is often thought of as a rigid, spherical structure that is solely responsible for storing the cell’s genetic material; DNA. Such a notion stems from early observations of cells fixed to microscopy slides. However, advances in the imaging of living cells have revealed that the nucleus is in fact a highly dynamic structure, and one that can adopt different shapes and sizes. What geometry it adopts will depend on the shape of the cell, and the environment in which the cell is growing. This characteristic is called nuclear plasticity or deformability.
Importantly, the shape and size that a nucleus adopts has a profound impact on the life of the cell, well beyond how it appears under a microscope. It will, in fact, affect how the DNA is stored, how it is unpacked, and which genes are decoded and when. A question that has remained unanswered however, is how forces from outside the cell regulate the dynamics of the nucleus, and hence, the DNA housed within. Clues that answer these questions have however begun to emerge through the work of Makhija et al, from the lab of GV Shivashankar, Deputy Director at the Mechanobiology Institute (MBI), National University of Singapore.
These findings were recently published in the Proceedings of the National Academy of Sciences. [Makhija E, Jokhun DS, Shivashankar GV, Nuclear deformability and telomere dynamics are regulated by cell geometric constraints. Proc Natl Acad Sci U S A. 2016 Jan 5; 113(1):E32-40. doi: 10.1073/pnas.1513189113].
Nuclear Mechanics & Genome Regulation Laboratory
Shivashankar’s lab 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.
The four major research directions in our laboratory include:
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
Modulation 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
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.
Aradhana Bharati, Kamal Sharma, Karthik Damodharan, Yejun Wang
Shiyuan Wing, Neetu Govindan, Marco Monticelli
Aninda Mitra, Bibhas Roy
Aneesh Sathe, Mallika Nagarajan
Cell geometry orients and repositions chromosomes to regulate genomic programs, Yejun Wang, Mallika Nagarajan, Caroline Uhler and G.V.Shivashankar (2016 – under review)
Nuclear positioning and its translation dynamics is regulated by cell geometry K. Radhakrishna, Saradha V. Pathy and G.V.Shivashankar (2016 – under review)
Cell-geometric-constraints differentially regulate TNFα-mediated gene expression programs, Aninda Mitra, Saradha.V. Pathy, Prasuna Rao and G.V.Shivashankar (2016 – under review)
Geometric confinement of cells induce nuclear reprogramming, Bibhas Roy, Prasuna Rao and G.V.Shivashankar (2016 – under review)
Geometric control and modeling of genome reprogramming, Caroline Uhler and G.V. Shivashankar. BioArchitecture. 2016 Jul 19:1-9.
Nuclear plasticity and telomere dynamics is modulated by extra-cellular matrix constraints, Ekta Makhija, Jokhun Kamal and G.V.Shivashankar, Proceedings of the National Academy of Sciences-USA (2016) 113(1):E32-40.
Matrix Mechanics Controls FHL2 Movement to the Nucleus to Activate p21/CDKN1A Expression, Naotaka Nakazawa, Aneesh R. Sathe, G. V. Shivashankar and Mike Sheetz Proceedings of the National Academy of Sciences-USA (2016 – in press)
Nuclear transport of paxillin depends on focal adhesion dynamics and FAT Domains, Aneesh Sathe, G.V.Shivashankar and Mike Sheetz, Journal of Cell Science (2016 – in press)
Chromosome intermingling – the physical basis of chromosome organization in differentiated cells, Shovamayee Maharana, K.Venkatesan Iyer, Nikhil Jain, Mallika Nagarajan, Yejun Wang and G.V.Shivashankar Nucleic Acids Research, (2016) doi: 10.1093/nar/gkw131
Regulation of nuclear morphology by actomyosin components and cell geometry, Ramdas NM, Qingsen Li, Shivashankar GV Proc IEEE Eng Med Biol Soc. 2015:342-5.
Novel localization of formin mDia2: importin β-mediated delivery to and retention at the cytoplasmic side of the nuclear envelope, Shao X, Kawauchi K, Shivashankar GV, Bershadsky AD. Biol Open. 2015 4(11):1569-75.
Actin cytoskeleton differentially alters the dynamics of lamin A/C, HP1α and H2B core histone proteins to remodel chromatin condensation state in living cells, Kee Chua Toh, Nisha Ramdas and G.V.Shivashankar Integrative Biology, 2015 (10):1309-17.
Mechanobiology Institute, Ministry of Education Tier-3 Co-Investigator Grant & IFOM-MBI Joint Research Laboratory, Singapore.
Prof.G.V.Shivashankar is currently the Deputy Director of Mechanobiology Institute, National University of Singapore. Shivashankar’s laboratory is focused on understanding the role of cell geometry on nuclear mechanics and genome regulation in living cells using a multi-disciplinary approach. He carried out his PhD research at the Rockefeller University (1994-1999) and Postdoctoral research at NEC Research Institute, Princeton USA (1999-2000). He started his laboratory at the National Center for Biological Sciences, TIFR- Bangalore, India (2000-2009) before relocating to a tenured faculty position at the National University of Singapore in 2009. His scientific awards include; the Birla Science Prize (2006), The Swarnajayanthi Fellowship (2007) and was elected to the Indian Academy of Sciences (2010). He Edited the Methods in Cell Biology series book on “Nuclear Mechanics and Genome Regulation” (2010), Elsevier Press. More recently he also Heads the Joint Research Laboratory with FIRC Institute of Molecular Oncology (IFOM), Milan, Italy and was appointed as an IFOM-NUS Chair Professor in 2014.