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The spacing of hair in mammals and feathers in birds is one of the most apparent morphological features of the skin. This pattern arises when uniform fields of progenitor cells diversify their molecular fate while adopting higher-order structure. Using the nascent skin of the developing chicken embryo as a model system, we find that morphological and molecular symmetries are simultaneously broken by an emergent process of cellular self-organization. The key initiators of heterogeneity are dermal progenitors, which spontaneously aggregate through contractility-driven cellular pulling. Concurrently, this dermal cell aggregation triggers the mechanosensitive activation of β-catenin in adjacent epidermal cells, initiating the follicle gene expression program. Taken together, this mechanism provides a means of integrating mechanical and molecular perspectives of organ formation.

Background The pathways that control protein transport across the blood–brain barrier (BBB) remain poorly characterized. Despite great advances in recapitulating the human BBB in vitro, current models are not suitable for systematic analysis of the molecular mechanisms of antibody transport. The gaps in our mechanistic understanding of antibody transcytosis hinder new therapeutic delivery strategy development. Methods We applied a novel bioengineering approach to generate human BBB organoids by the self-assembly of astrocytes, pericytes and brain endothelial cells with unprecedented throughput and reproducibility using micro patterned hydrogels. We designed a semi-automated and scalable imaging assay to measure receptor-mediated transcytosis of antibodies. Finally, we developed a workflow to use CRISPR/Cas9 gene editing in BBB organoid arrays to knock out regulators of endocytosis specifically in brain endothelial cells in order to dissect the molecular mechanisms of receptor-mediated transcytosis. Results BBB organoid arrays allowed the simultaneous growth of more than 3000 homogenous organoids per individual experiment in a highly reproducible manner. BBB organoid arrays showed low permeability to macromolecules and prevented transport of human non-targeting antibodies. In contrast, a monovalent antibody targeting the human transferrin receptor underwent dose- and time-dependent transcytosis in organoids. Using CRISPR/Cas9 gene editing in BBB organoid arrays, we showed that clathrin, but not caveolin, is required for transferrin receptor-dependent transcytosis. Conclusions Human BBB organoid arrays are a robust high-throughput platform that can be used to discover new mechanisms of receptor-mediated antibody transcytosis. The implementation of this platform during early stages of drug discovery can accelerate the development of new brain delivery technologies.

Actomyosin stress fibers (SFs) play key roles in driving polarized motility and generating traction forces, yet little is known about how tension borne by an individual SF is governed by SF geometry and its connectivity to other cytoskeletal elements. We now address this question by combining single-cell micropatterning with subcellular laser ablation to probe the mechanics of single, geometrically defined SFs. The retraction length of geometrically isolated SFs after cutting depends strongly on SF length, demonstrating that longer SFs dissipate more energy upon incision. Furthermore, when cell geometry and adhesive spacing are fixed, cell-to-cell heterogeneities in SF dissipated elastic energy can be predicted from varying degrees of physical integration with the surrounding network. We apply genetic, pharmacological, and computational approaches to demonstrate a causal and quantitative relationship between SF connectivity and mechanics for patterned cells and show that similar relationships hold for nonpatterned cells allowed to form cell–cell contacts in monolayer culture. Remarkably, dissipation of a single SF within a monolayer induces cytoskeletal rearrangements in cells long distances away. Finally, stimulation of cell migration leads to characteristic changes in network connectivity that promote SF bundling at the cell rear. Our findings demonstrate that SFs influence and are influenced by the networks in which they reside. Such higher order network interactions contribute in unexpected ways to cell mechanics and motility.

One of the most exciting recent developments in cell biology is the recognition that the geometry, topography, and elasticity of the extracellular matrix (ECM) can provide signals to cells that can affect cell physiology via a process called tensional homeostasis–the ability of a cell to balance tractional forces against the ECM. Actomyosin stress fibers (SFs) directly connect the cell to its ECM via focal adhesions and are responsible for force generation and transmission. Despite the importance of these structures, we lack a clear understanding of how the geometry of SFs, particularly their length, regulates force generation. Furthermore, current techniques used to study SF mechanical properties consider SFs as isolated bodies that lack connections with the remaining cytoskeletal elements. As a result, we lack the understanding of how tension is distributed across an individual SF that is part of a two-dimensional (2D) network.In this dissertation, we sought to understand SF viscoelasticity by considering an SF as part of a network by combining subcellular laser ablation (SLA) and single cell micropatterning. First, we show that longer SFs dissipate increased elastic energy after SLA. Second, we show that in addition to single SF geometry, the architecture of the connecting SF network also regulates the elastic energy dissipated by a single SF after SLA. We developed a computational model that takes in consideration the geometry of the surrounding SFs and can recapitulate SLA retraction kinetics. We then sought to understand the parameters that contribute to the observed differences in internal SF architectures and show that the initial cell binding position and spreading history on the micropattern may predict the final SF configuration. Finally, we expanded the theme of connectivity to study connections between cytoskeletal elements such as SFs and intermediate filaments. We show that vimentin interacts with central SFs and regulates SLA retraction kinetics. The work presented in this thesis provides a new framework of thinking about SFs as part of a 2D network both during cell spreading and during “steady state”.Moreover, myosin in SFs can be phosphorylated via two parallel pathways: Rho Associated Kinase (ROCK) and Myosin Light Chain Kinase (MLCK). While each pathway regulates the formation of SFs in different cellular compartments, it is unclear how each kinase contributes to the viscoelastic properties of SFs found throughout the cell. The relationship between these pathways and SF formation have been explored through pharmacological inhibition making it hard to determine whether SF properties can be tuned according to the amount of myosin phosphorylation. To study these questions, we combine SLA with synthetic biology tools and show that gradient expression of each kinase results to gradient changes in viscoelastic properties of SFs within specific cellular compartments. Specifically, MLCK induces mono-phosphorylation of myosin which primarily localizes in peripheral SFs, altering their viscoelasticity whereas ROCK induces myosin di-phosphorylation which primarily localizes and controls the viscoelastic properties of central SFs.Overall, this thesis represents a systematic attempt to combine single cell micropatterning and SLAtools to relate single SF geometry and architecture of the connecting SF network to single SF mechanics. Our findings enhance our understanding of how cells distribute tension across a network of SFs and other cytoskeletal structures, addressing a fundamental open question in cell biology. We also provide further insight into the mechanistic pathways of SF tension generation by ascribing the viscoelasticity of SFs found in different cellular compartments to the activity of specific kinases.

Recent advancements in engineering Complex in vitro models (CIVMs) such as Blood-brain barrier (BBB) organoids offer promising platforms for preclinical drug testing. However, their application in drug development, and especially for the regulatory purposes of toxicity assessment, requires robust and reproducible techniques. Here, we developed an adapted set of orthogonal image-based tissue methods including hematoxylin and eosin staining (HE), immunohistochemistry (IHC), multiplex immunofluorescence (mIF), and Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) to validate CIVMs for drug toxicity assessments. We developed an artificial intelligence (AI) algorithm to increase the throughput and the reliability of histomorphologic evaluations of apoptosis for in vitro toxicity studies. Our data highlight the potential to integrate advanced morphology-based readouts such as histological techniques and digital pathology algorithms for use on CIVMs, as part of a standard preclinical drug development assessment. The graphical abstract was partially created with biorender.com. Advanced Complex in vitro models (CIVMs) like Blood-brain barrier (BBB) organoids show promise for preclinical drug testing. However, robust and reproducible techniques are crucial for the acceptance of CIVMs in drug development processes, especially for toxicity assessments which are highly regulated by health authorities. We developed orthogonal image-based readouts on histological sections to enable the use of BBB organoids for future compound toxicity assessment. A newly established artificial intelligence (AI) algorithm provides automated and label-free detection of apoptotic cells in drug screening using BBB organoids and provides an alternative killing assay on single cell resolution (40×) to current standards.