Characterizing the mechanisms of adhesion initiation and strengthening of integrins to the extracellular matrix proteins

Abstract

Eukaryotic cells are decorated with a plethora of transmembrane proteins, which aid interactions with the environment rich in diverse components. In a homeostatic cell, these interactions are specific and tightly regulated to facilitate various cellular processes including cell adhesion, migration and proliferation. Cell adhesion is pivotal in the development and in the systematic functioning of the organisms. Decades of impeccable research in adhesion biology achieved many milestones and simultaneously opened-up a Pandora’s box asking more complex questions in the field, necessitating future researches. Our research has shed some light on some of the pertinent questions in the field of cell adhesion. One of the widely used approaches in studying cellular adhesion is single cell adhesion study. Single cell adhesion studies are performed on a variety of cellular phenotypes: round cells to study early onset of adhesion initiation and flattened cells to study adhesion maturation. Therefore, single cell adhesion studies provide diverse insights into the mechanism of the cell adhesion. The first chapter of this thesis contributes to the technical advances in the field of atomic force microscope (AFM) based single cell adhesion studies. The study enumerates the advantages and the limitations of AFM based single cell adhesion studies performed on a round cell and a flattened cell. The second part of the chapter introduces a new methodology devised to study single cell adhesion and traction, using an AFM. Single cell adhesion studies, such as single cell force spectroscopy (SCFS) and traction force microscopy, provide information on adhesion and traction forces during cell adhesion formation. Broadly, living adherent cells can mechanically sense, generate and regulate adhesion and traction. A logical combination of existing tools and methodologies was devised, which provided the direct correlation between adhesion and traction as a living cell comes in contact with the extracellular matrix and/or other cells and initiate cell adhesion and traction. Next chapters dig deep into the field of cell adhesion and focus on the role of integrins in regulating cell adhesion. Integrins are one of the key cell adhesion molecules, which provide anchorage and mediate signals across the plasma membrane. They allow cells to interact with the other cells and with the extracellular environment. Integrins recognise different extracellular matrix proteins like fibronectin, collagen, laminin etc., with high affinity and specificity. The binding of integrins to their specific ligands initiates signal transduction pathways that facilitate cell adhesion. Integrins mediate bi-directional signalling, inside–out and outside-in signalling. Not only a specific integrin can bind multiple ligands but also a specific ligand can be bound by multiple integrins. This complexity in the integrin-mediated adhesion influences the specificity of an integrin to the extracellular matrix protein and also the functioning of other integrin types binding to the same extracellular matrix protein, broadly classified as an integrin crosstalk. How different types of integrins, binding to the same (or different) extracellular matrix proteins, interact among themselves and regulate functioning of another integrin type is still not well understood. With this study, we examine the role of two fibronectin-binding integrins, α5β1 and αV-class integrins in the cell adhesion and their subsequent crosstalk at the early onset of adhesion and during adhesion maturation (chapter 3). In the first part of this chapter, we investigate how α5β1 and αV-class integrins bind the abundant extracellular matrix protein fibronectin and regulate fibroblast adhesion in the early phases of attachment (<120 s). By combining SCFS with various optical microscopy approaches, we quantify the early adhesion formation of engineered mouse fibroblasts expressing α5β1 and/or αV-class integrins. We observe a differential integrin-dependent adhesion wherein fibronectin-binding integrin classes establish distinct adhesion profiles when a fibroblast initiates adhesion to fibronectin. By measuring the binding probability of each fibronectin-binding integrin, we unravel an exclusive relationship between α5β1 and αV-class integrins wherein they both initially compete to bind the RGD-domain of fibronectin and eventually αV-class integrins win this competition. Later, upon activation of αVclass integrins, fibroblasts considerably strengthen adhesion to fibronectin. Our study reveals that after engaging to fibronectin, αV-class integrins send cues to α5β1 integrins to establish new adhesion sites and to strengthen fibroblast adhesion. Surprisingly, this regulation appears to signal across the cell and thus α5β1 integrins establish additional adhesion sites to fibronectin, away from those formed by αV-class integrins: crosstalk. Since integrin adhesion involves cascade of signalling events comprising of various integrin associated proteins, we were intrigued by the role of signalling pathways in our observed crosstalk. We determine that fibronectin-bound αV-class integrins crosstalk with α5β1 integrins via RhoA/ROCK/myosin-II and Arp2/3-mediated signalling pathways. The observed crosstalk induces the clustering of α5β1 integrins and eventually maturates adhesion. Our data suggests that this dual role of the two integrin classes, i.e. initial competition to bind fibronectin and their subsequent crosstalk, enables α5β1 and αV-class integrins to co-operate and govern the assembly focal adhesions in the mammalian cells. In the second part of the chapter 3, we elucidate the importance of the synergy site in fibronectin and the subsequent crosstalk between αV-class and α5β1 integrins in adhesion maturation. Adjacent to the RGD site is the so-called synergy site that additionally interacts with the α5-subunit of α5β1 integrins and not with the αV-class integrins. In this work, genetic studies on mice were complemented with the AFM based SCFS studies. The SCFS was employed to determine the binding probability of the α5β1 and αV-class integrins for the fibronectin, which was isolated from mice with or without the synergy site-mutation. The results revealed that theinitial binding of α5β1 to fibronectin (on-rate) occurs in a synergy site-independent manner, whereas the formation of strengthened bonds with fibronectin (off-rate) was synergy site-dependent. Further, chapter 4 gives insights into the much-debated topic in the field of integrin activation wherein the synergistical roles of the two-key players, talin and kindlin, are still unclear. Talin and kindlin contribute to integrin activation and signalling, however it is unclear to what extent and how. AFM based SCFS was employed to determine the fibronectin-adhesion exhibited by the fibroblasts in the absence of talin and kindlin. Therefore, we used engineered talin null and kindlin null fibroblasts and compared their fibronectin-adhesion to the control parental fibroblasts. Our results revealed the importance of kindlin during adhesion initiation and contributed the mechanistic insights into the synergy of talin and kindlin in adhesion initiation. The last part of this thesis discusses the contribution of this work to the field of integrin-mediated cell adhesion and paves the path to hypothesize the possible future projects. Briefly, all the studies described in this thesis comprise the interaction of αV-class and α5β1 integrins with their ligand in the mouse kidney fibroblasts that appears to be a basic cellular mechanism to assemble focal adhesions to the extracellular matrix. We believe that these studies contribute to the research in the cellular processes including growth, differentiation and therapeutics.