Microrobotic tools for mechanical characterization and stimulation: from single cells to organisms

Abstract

Physical forces regulate the behavior of cells and tissues and are essential in cell organization and morphogenesis. Especially in plants, where pressurized cells are surrounded by a rigid cell wall that constricts cell expansion, growth is a highly coordinated process of stress and strain. With the advent of new biomechanical tools, forces at the cellular and multicellular level can finally be directly measured. This spatial force resolution is crucial to study how cells’ perception of mechanical cues leads to the cascade of biochemical and electrical signals that ultimately regulates the behavior, functions, and mechanical properties of cells. This thesis investigates biomechanical processes with an interdisciplinary approach that combines engineering sciences, biology, and physics. Newly engineered microrobotic tools for mechanical characterization and stimulation of cells and tissues, together with analytical and numerical models, allow us to assess the intricate interplay between extracellular forces, intracellular pressure, and cell wall elasticity. Biochemical and genetic techniques enable us to monitor the physiological processes controlling the cellular response to mechanical cues. In the first chapter, we explore the theoretical background of mechanobiology, the study of mechanics in biology at the cellular level, encompassing the highly integrated genetic, biochemical, and biomechanical processes involved in growth and morphogenesis. In chapter two, we introduce the cellular force microscope (CFM), an existing microrobotic platform to study the mechanobiology of cells. The chapter presents this system and the improvements made to it over the course of this project. We integrated the CFM with an inverted microscope for fluorescence imaging, which is essential to correlate cytomechanics with biochemical signaling. A newly modular CFM-structure can be rearranged to fit the requirements of different biological samples, and the operational mode (e.g., microindentation, relaxation test) can be programmed as needed. The third chapter documents mechanical characterizations at the microscale using different adaptations of the CFM, each tailored to a specific task. We present a configuration for non-perpendicular indentation using a dualforce read-out, and a multiple-CFM configuration to separately measure turgor pressure and cell wall elasticity in growing pollen tubes (PTs). We measure the mechanical properties of plants at the single- and multi-cellular level, using PTs and roots, respectively. Additionally, we demonstrate the applicability of the system for use in animal tissues with nematode embryos. In the fourth chapter, we investigate the effect of external mechanical cues on plants at the cell level using PTs, and at the organ level using Venus flytraps. We show that PTs literally feel their way through their environment to avoid obstacles as they deliver male gametes to the ovule. We measure their force sensitivity to understand this remarkable behavior. In Venus flytraps, we show for the first time a direct correlation between the magnitude of the mechanical stimulus in vivo and the electrical response that leads to trap closure. In contrast to the generally accepted idea that two hair deflections are needed to initiate trap closure, we show that under certain conditions, a single deflection is sufficient. Major conclusions and research contributions provided by this work are highlighted in the fifth chapter.