Nanomechanics of Soft and Living Matters

Abstract

This section contains abstracts of projects done for completion of my MS thesis. Project 1: Molecular interaction of SARS-CoV-2 mutants binding to the human ACE2 receptor using AFM The main project of this thesis is dedicated to examining the intricate interaction dynamics of SARS-CoV-2 virus mutants, specifically the Wuhan strain and the highly transmissible Omicron variant, with their cellular entry point, the human Angiotensin-Converting Enzyme 2 (ACE2) receptor. Through the application of Atomic Force Microscopy (AFM), this study aims to meticulously compare the binding strengths of these variants with the ACE2 receptor, offering insights into their respective kinetic profiles. By analyzing force-distance curves obtained through AFM measurements and employing the Bell-Evans model, the project endeavors to unravel the specific molecular interactions playing the binding process between the viral spike protein and the ACE2 receptor. This comprehensive investigation not only deepens our understanding of viral-host interactions of mutants but also holds promise for informing the development of targeted vaccine strategies aimed at changed kinetic off rates of viral variants with small mutations. We have shown experimentally the decrease in kinetic off rate for omicron variant. Project 2: Viscoelastic measurements of L Cells and their Clathrin Light Chain Knockout Variants using AFM Nanoindentation The actin cytoskeleton plays an important role in various cellular and biological processes such as cell polarity, cell migration, and intracellular and extracellular trafficking, thereby maintaining cell shape and structure of a cell. The role of actin in endocytosis is well established, particularly in the context of clathrin mediated endocytosis (CME). CME is the major endocytic pathway involved in the maintenance of mouse embryonic stem cell (mESC) pluripotency. The central component of CME is the clathrin triskelion, which is composed of three molecules of the clathrin heavy chain (CHC), each of which is associated with the smaller clathrin light chain (CLC). In mammals, there are two clathrin light chains, CLCa and CLCb whose individual physiological roles are poorly understand. CLCs have been shown to help recruit and organize actin to sites of endocytosis in differentiated cells, especially under conditions of high membrane tension. The organization of actin cytoskeleton in distinct in different cell type. Using the CRISPR-Cas9 genome editing system, we have generated CLC knock out L - cell lines. We show that in the absence of CLCa, CLCb or both the light chains show reduction in viscoelasticity of cells. Project 3:Quantitative evaluation of collagen organisation and its regulation by mouse fibroblasts in 3D hydrogels using AFM Cells surrounded by the extracellular matrix (ECM) can sense mechanical cues from their micro-environment that are converted to a biochemical signal and executed as a biological response such as alteration of protein expression (Intracellular) or remodelling of the extra-cellular matrix (Extracellular). 3D collagen hydrogels with changing collagen concentration and embedded wild-type mouse fibroblasts (WT MEFs) provide a controlled system to evaluate the cell-matrix interaction. A small change in collagen concentration affects collagen organization in 3D hydrogels and differentially affects collagen organization in gels embedded with cells. Caveolin-1 lined caveolae are plasma membrane-invaginated nano-domains that are mechanosensitive and play a vital role in cellular mechano-protection. Caveolin-1 deficient fibroblasts (Cav-1 Null MEFs) differentially remodel their immediate microenvironment compared to WT MEFs. The effect of the differential collagen organization by WT MEFs and Cav-1 Null MEFs in stiffness has been investigated by using AFM using soft cantilevers and large silicon bead glued on it, 3D hydrogels with variable collagen concentration at different time points has been studied.