Developing Molecular Tools to Study N-Cadherin Mediated Adherens Junction Mechanobiology

Abstract

Within the body, cells constantly experience mechanical forces—from various extracellular sources and from neighboring cells via their actomyosin networks. [1]. These forces have dramatic effects on cells and tissues and affect signaling cascades, both towards maintaining physiological homeostasis, and in the pathological progression of numerous diseases [2]. Towards mediating these signals, cells have evolved numerous, specialized receptors/protein complexes capable of sensing, interpreting, and responding to these mechanical forces. The process by which cells sense and convert these mechanical signals into biochemical signaling pathways is referred to as mechanotransduction, and the study of this field as a whole is referred to as mechanobiology. Of interest to the work in this dissertation are the structures that mediate mechanotransduction at the cell-cell interface. Specifically, we are interested in adherens junction (AJs), the canonical force-sensitive structures at cell-cell interfaces. AJs are composed of clusters of classical cadherins and mediate the physical interactions between cells through the actin cytoskeleton. AJs are implicated as both critical regulators of numerous physiological processes [3] as well as the pathological progression of many diseases including atherosclerosis, fibrosis, and cancer [4-8].In the study of AJ mechanobiology, the primary focus has been on tissues that form monolayers [9], such as the epithelia and endothelia. These AJs are mediated by the classical cadherins, E-cadherin and VE-cadherin, respectively [10, 11], and, early studies in these systems have laid the groundwork for our understanding of AJ and cadherin mechanobiology. However, more recent studies have highlighted the role of AJ mechanobiology in tissues that do not form monolayers, such as those formed by vascular smooth muscle cells (VSMCs), neurons, cardiomyocytes, etc. [6]. These AJs are mediated by another classical cadherin, N-cadherin. Although these cell types do not form the prototypic cell sheets or engage in collective behaviors that were originally ascribed to be controlled by E-/VE-cadherin-mediated mechanotransmission, we are now learning that the N-cadherin mediated AJs in these tissues play equally important roles in a number of physiological and pathological processes. However, current progress in studying the in vitro mechanobiology of N-cadherin in these systems is hindered by a lack of appropriate molecular tools. Therefore, the first major goal of this dissertation was to develop the necessary tools to study N-cadherin mechanobiology in a variety of settings. To achieve this first goal, we leveraged Förster resonance energy transfer (FRET)-based molecular tension sensor (MTS) technology [12]. These MTSs have emerged in the last 15 years as valuable tools that fill the length scale gap between the mesoscopic (whole cell) and ex vivo microscopic (individual protein) studies that have dominated the last several decades of mechanobiology research [13, 14]. This class of MTSs have enabled the investigation of pico-newton (pN)-scale mechanics within living cells, thus greatly expanding the available tools to study mechanosensitive proteins in vitro [15]. We therefore sought to leverage this technology to develop a novel sensor for N-cadherin.The first results chapter of this dissertation, Chapter 3, details the engineering and validation of this novel molecular tool, the N-cadherin tension sensor: NcadTS. Successful implementation of such a molecular tool requires a rigorous series of assays and controls to ensure that the newly engineered sensor behaves like the endogenous protein of interest, and the measurements reported are both reliable and repeatable. Thus, in addition to engineering the NcadTS, we also developed several control sensors to thoroughly validate the biological and technical function of this new molecular tool. We demonstrate, in the Chapter 3, the rigorous validation of the NcadTS and the ability for this new tool to faithfully report pN-scale forces within AJs. Following validation of this tool, in Chapter 4, we sought to gain insights into the mechanobiology of N-cadherin mediated AJs. To accomplish this, we decided to study the AJs of VSMCs. Within VSMCs, N-cadherin has been linked to the control of myogenic vasomotor tone, as well as to intimal thickening during atherosclerosis [16-18]. Additionally, towards maintaining cell–cell connectivity to mediate vessel tone and structural integrity, VSMCs form several distinct morphological types of AJs [16]. Thus, the second major goal of this work was to study the mechanobiology of diverse AJ substructures, as well as to gain a more holistic understanding of VSMC AJs.Using the NcadTS in combination with the development of new analysis paradigms to study diverse AJ morphologies, we reveal new insights into the regulation of mechanical force at a variety of AJ structures. Additionally, we study the role of several perturbations to N-cadherin mechanotransmission including alteration of extracellular stiffness, and to the adhesive binding state mediating interactions of cadherins between cells. These results, in combination with work examining the force-induced turnover of N-cadherin, provide new insights into N-cadherin mechanotransmission and potential differences from other classical cadherins. The findings of this work expand our understanding of VSMC AJ mechanobiology, and raise interesting questions about how cadherins might differentially mediate mechanical force to meet the mechanical needs of the tissues they reside in.In summary, this dissertation advances our molecular understanding of N-cadherin–mediated mechanotransmission and provides a robust toolkit for probing how cell–cell forces regulate physiological and pathological processes in a wide range of cells and tissues. Future work using this tool will surely advance our understanding of physiological signaling in systems like cardiac and neuronal tissue, as well as pathological signaling cascades like fibrosis and cancer.