Dissecting cellular organization using innovative microscopy

Understanding the structural organization of cells is essential to elucidate their biological function in health and disease. In the first part of this thesis, we develop Expansion Microscopy (ExM) approaches to visualize cellular organization. While light microscopy is a powerful tool to study cell biology, its power to resolve subcellular structures is limited. In ExM, the effective resolution of light microscopy is increased by embedding biological specimen in a swellable hydrogel followed by isotropic physical expansion of cells and tissues. In chapter 2, we introduce Tenfold Robust Expansion (TREx) microscopy, a novel method that enables single-step tenfold expansion of both cells and tissues and offers the possibility to combine selective labeling of proteins with ultrastructural context. For biological reproducibility, quantitative accuracy, and diagnostic and pathology applications of ExM, robust characterization of the exact expansion factor and of possible local deformations are essential. In chapter 3, we introduce GelMap, the first quality control mechanism for ExM that can be readily adopted in existing workflows. GelMap introduces a fluorescent grid into the pre-expanded hydrogel that scales with the expansion factor and deforms with anisotropy. We show this intrinsic calibration can be used to accurately report the local expansion factor and can be used to computationally correct deformations that occur during expansion. In chapter 4, we apply TREx to visualize SARS-CoV-2 infection in human airway cells, underscoring how ExM can aid biological discovery. While electron microscopy is traditionally used to visualize intracellular viral replication cycle ultrastructure, here we use TREx to observe the formation of swollen multivesicular organelles in SARS-CoV-2 infected cells. In addition, we report other structural rearrangements such as clustering of cilia and the appearance of membrane protrusions in infected cells. In the second part of this thesis, we use a combination of innovative light microscopy techniques to dissect the organization of the microtubule (MT) cytoskeleton in T cells. Upon activation, T cells form a specialized structure called immunological synapse (IS). IS formation is driven by large-scale reorganization of both the actin and microtubule cytoskeleton, including polarization of the centrosome towards the IS. In chapter 5, we use a combination of live cell and fixed cell imaging, including ExM, to show that motor protein kinesin-4 KIF21B is a major regulator of MT dynamics in T cells, as KIF21B is needed to restrict MT growth for efficient remodeling of the MT network upon activation. In chapter 6, we explore the mechanism of force generation for centrosome polarization by the motor protein dynein. We validate strategies to synchronize T cell activation for large populations of cells and use these approaches to demonstrate that dynein is recruited to the membrane of the IS, and its localization is influenced by actin. We expect that these tools can be used in future studies to elucidate the role of dynein during centrosome translocation. Together, the approaches presented in this thesis will aid future research into how form and function in biology are connected.