Mechanotransduction of substrate stiffness in endothelial cell collective migration

Endothelial damage during life-saving percutaneous angioplasty contributes to re-stenosis rates of nearly 20% within 5 years. Re-endothelialization, the collective endothelial cell migration over exposed extracellular matrix (ECM) and stent struts, can restore a continuous, functional endothelium. During atherosclerotic disease, vascular ECM becomes stiffer. ECM stiffness affects epithelial cell collective migration in other pathogenic contexts. However, substrate stiffness effects on endothelial cell collective migration have yet to be explored. We developed quantitative computational image processing algorithms for assessing collective migration. We then used these image analysis techniques to measure the effect of substrate stiffness on critical aspects of porcine aortic endothelial cell (PAEC) two-dimensional collective migration: (1) migration distance, (2) directedness, and (3) togetherness. PAEC were seeded on collagen-coated polyacrylamide hydrogels (4-50 kPa) in a 5 mm cloning ring and then allowed to migrate outwards. We found that migration distance increased with substrate stiffness and that there was a concomitant increase in PAEC alignment. We found that decreased togetherness on stiffer substrates led to enhanced proliferation at the migratory interface. We used the specific Rho kinase (ROCK) inhibitor Y27632 to show that ROCK-mediated contractility limited endothelial cell collective migration on soft substrates. We observed that PAEC secrete and remodel fibronectin on collagen-coated substrates. Interestingly, α5 integrin, but not fibronectin, was important for directed collective migration on stiff substrates. These findings provide insight into how substrate stiffness affects endothelial cell collective migration. This work will inform how the mechanical properties of tissue and tissue engineered construct could be designed to promote a functional endothelium.Ph.D., Biomedical Engineering -- Drexel University, 201

Centrosomal microtubule nucleation regulates radial migration of projection neurons independently of polarization in the developing brain

Cortical projection neurons polarize and form an axon while migrating radially. Even though these dynamic processes are closely interwoven, they are regulated separately-the neurons terminate their migration when reaching their destination, the cortical plate, but continue to grow their axons. Here, we show that in rodents, the centrosome distinguishes these processes. Newly developed molecular tools modulating centrosomal microtubule nucleation combined with in vivo imaging uncovered that dysregulation of centro-somal microtubule nucleation abrogated radial migration without affecting axon formation. Tightly regu-lated centrosomal microtubule nucleation was required for periodic formation of the cytoplasmic dilation at the leading process, which is essential for radial migration. The microtubule nucleating factor g-tubulin decreased at neuronal centrosomes during the migratory phase. As distinct microtubule networks drive neuronal polarization and radial migration, this provides insight into how neuronal migratory defects occur without largely affecting axonal tracts in human developmental cortical dysgeneses, caused by mutations in g-tubulin.ISSN:0896-6273ISSN:1097-419

FGF signaling and cell state transitions during organogenesis

Organogenesis is a complex choreography of morphogenetic processes, patterns and dynamic shape changes as well as the specification of cell fates. Although several molecular actors and context-specific mechanisms have already been identified, our general understanding of the fundamental principles that govern the formation of organs is far from comprehensive. The application of the concept of ‘rebuild it to understand it’ from synthetic biology represents a promising alternative to the classical approach of ‘break it to understand it’ in order to distill biological understanding from complex developmental processes. According to this ‘rebuilding’ concept, in this study we sought to develop an experimental approach to induce the formation of organs from progenitor cells ‘on demand’ and to investigate the minimum requirements for such a process. The zebrafish lateral line chain cells are a powerful in vivo model for our study because they are a group of naïve multipotent progenitor cells that display mesenchyme-like features. In order to bring these cells to form organs, we used the well-known role of the FGF signaling pathway as a driver of organogenesis in the lateral line and developed an inducible and constitutively active form of the fibroblast growth factor receptor 1a (chemoFGFR). The cell-autonomous induction of this chemoFGFR in chain cells effectively triggered the formation of fully mature organs and thus enabled spatial and temporal control of the organogenesis process. Next, we asked what it takes to form an organ de novo. We used a combination of real-time microscopy, single cell tracking, polarity quantification, and mosaic analysis to study the cell behaviors that result from chemoFGFR induction. The picture that emerges from these analyses is that de novo organs form through a genetically encoded self-assembly process that is based on the pattern of chemoFGFR induction. In this scenario, cells expressing chemoFGFR aggregate into clusters and epithelialize as they sort out of non-expressing cells. We found that this sorting process occurs through cell rearrangement and slithering, which involves an extensive remodeling of the cell-cell contacts. Chain cells that do not express chemoFGFR can envelop these chemoFGFR expressing cell clusters and form a rim at the cluster periphery. This multi-stage process leads to the establishment of the inside-outside pattern of de novo organs, which is used as a blueprint for cell differentiation. In summary, in this study we provide insights into the mechanisms involved in the self-assembly of organs from a naïve population of progenitor cells

The role of calcium signaling analysis in myeloid leukocyte activation and interactions in inflammation

Inflammation is the pathophysiologic basis of many diseases that are main causes of mortality. Caused by environmental or microbial factors, inflammation leads to the destruction of tissue around the primary focus and expands to other tissues when not resolved in a timely manner. The body’s mechanism for inflammation resolution is based on an interplay of immune cells that first drive a pro-inflammatory response necessary for recruiting neutrophils and macrophages that phagocytose debris and dendritic cells that relay information to lymphocytes that build up the body’s adaptive immunity. Neutrophils are key to this process as they are the first cell type to recognize damage and pathogen associated molecular patterns (DAMPs and PAMPs). Eventually, they undergo necrosis amplifying the chemokine and cytokine gradient needed for added neutrophil swarming and other cells’ recruitment. In addition to the release of chemokine cues, neutrophil necrosis results in the release of reactive oxygen species that is an additional mediator of tissue damage. Although the literature hints at a cellular interplay as mechanism to prevent excessive neutrophil-induced tissue damage, there is to date no tool to investigate intravital leukocyte activation patterns in vivo in this setting. For this purpose, we turned to calcium (Ca++) signaling since it is known to be involved in most aspects of cellular function. By using myeloid leukocyte specific Ca++ reporter strains together with in vivo two-photon and spinning disk confocal microscopy in a sterile inflammation mouse model, we developed an image analysis algorithm of frequency spectra that offers the following insights: macrophages react to sterile inflammation by Ca++ transients in a distinct spatiotemporal pattern and neutrophils vary their intracellular dynamics during the migration cascade in a Gαi-protein-coupled receptor dependent manner. Furthermore, during resolution of inflammation we observed tissue macrophages (TMs) physically contacting injury-bound neutrophils with their dendrites and instructing them to withdraw. Ca++ signal analysis displays that both cell types undergo cellular activation adjustments during interaction. Finally, we uncovered that the HMGB1-TLR4 axis is responsible for TM dendrite formation and that LFA-1 mediates neutrophil interaction with these dendrites. Therefore, we have uncovered a mechanism how tissue inflammation is limited through macrophage-neutrophil interactions

Three-Dimensional Biomimetic Patterning to Guide Cellular Migration and Organization

This thesis develops a novel photopatterning strategy for biomimetic scaffolds that enables spatial and biochemical control of engineered cellular architectures, such as the microvasculature. Intricate tools that allow for the three dimensional (3D) manipulation of biomaterial microenvironments will be critical for organizing cellular behavior, directing tissue formation, and ultimately, developing functional therapeutics to treat patients with critical organ failure. Poly(ethylene glycol) (PEG) based hydrogels, which without modification naturally resist protein adsorption and cellular adhesion, were utilized in combination with a two-photon laser patterning approach to covalently immobilize specific biomolecules in custom-designed, three-dimensional (3D) micropatterns. This technique, known as two-photon laser scanning lithography (TP-LSL), was shown in this thesis to possess the capability to micropattern multiple different biomolecules at modular concentrations into a single hydrogel microenvironment over a broad range of size scales with high 3D resolution. 3D cellular adhesion and migration were then explored in detail using time-lapse confocal microscopy to follow cells as they migrated along micropatterned tracks of various 3D size and composition. Further, in a valuable modification of TP-LSL, images from the endogenous microenvironment were converted into instructions to precisely direct the laser patterning of biomolecules within PEG-based hydrogels. 3D images of endogenous microvasculature from various tissues were directly converted into 3D biomolecule patterns within the hydrogel scaffold with precise pattern fidelity. While tissue engineers have previously demonstrated the formation of vessels through the encapsulation of endothelial cells and pericyte precursor cells within PEG-based hydrogels, the vessel structure had been random, uncoordinated, and therefore, ultimately non-functional. This thesis has utilized image guided TP-LSL to pattern biomolecules into a 3D structure that directs the organization of vessels to mimic that of the endogenous tissue vasculature. TP-LSL now stands as a valuable tool to control the microstructure of engineered cellular architectures, thereby providing a critical step in the development of cellularized scaffolds into functional tissues. Ultimately, this thesis develops new technologies that advance the field of regenerative medicine towards the goal of engineering viable organs to therapeutically treat the 18 patients who die every day waiting on the organ transplant list

Roles of microtubules and microtubule regulators in collective invasive migration of drosophila border cells

Ph.DDOCTOR OF PHILOSOPH

Miro1-dependent Mitochondrial Dynamics in Parvalbumin Interneurons

Parvalbumin interneurons are fast-spiking inhibitory cells that have been implicated in the generation of rhythmic network activity in the γ-frequency band (30-80Hz) by coordinating principal cell activity. In order to sustain their high firing rates, parvalbumin interneurons have a high mitochondrial content reflecting their large energy utilization. Therefore, parvalbumin interneurons are more susceptible to incidents of mitochondrial impairment as mitochondria provide the predominant source of energy. Miro1 is a Ca²⁺-sensing adaptor protein that links mitochondria to the trafficking apparatus, for their microtubule-dependent transport along axons and dendrites, in order to meet the metabolic needs of the cell. Here, we explore the role of Miro1 in parvalbumin interneurons and how changes in the mitochondrial distribution could alter network activity. To investigate mitochondrial dynamics we generated a mouse line where mitochondria are specifically labelled in parvalbumin interneurons. The Cre- recombinase is expressed exclusively in parvalbumin interneurons and excises a termination signal upstream of a fluorescent reporter allowing for the visualisation of mitochondria only in these cells. We further crossed this line with the Miro1(f/f) mouse, generating a transgenic mouse where Miro1 was conditionally knocked-out exclusively in parvalbumin interneurons. Specifically we investigated the conditional removal of Miro1 in mitochondrial dynamics in parvalbumin interneurons. Using live-imaging of ex-vivo organotypic brain cultures, we demonstrated a reduction in mitochondrial trafficking in parvalbumin interneurons in the hippocampus under basal conditions. This lead to accumulation of mitochondria in the soma and their depletion from synaptic terminals. Loss of Miro1 resulted in alterations in axonal but not dendritic branching in parvalbumin interneurons. This was accompanied by altered synaptic transmission and increased frequency of γ-oscillations in hippocampal brain slices. In this study, we show for the first time that Miro1 and Miro1-dependent mitochondrial positioning are essential for correct parvalbumin interneuron function and network activity

Studies on the function of PRG2/PLPPR3 in neuron morphogenesis

Neuron development follows a multifaceted sequence of cell migration, polarisation, neurite elongation, branching, tiling, and pruning. The implementation of this sequence differs between neuronal cell types and even in individual neurons between sub-compartments such as dendrites and axons. Membrane proteins are at a prime position in neurons to couple extrinsic morphogenetic signals with their intrinsic responses to orchestrate this defined morphological progression. The Phospholipid phosphatase-related / Plasticity-related gene (PLPPR/PRG)-family comprises five neuron-enriched and developmentally regulated membrane proteins with functions in cellular morphogenesis. At the start of this project, no publication had characterised the function of PLPPR3/PRG2 during neuron development. The presented work describes PLPPR3 as an axon-enriched protein localising to the plasma membrane and internal membrane compartments of neurons. Mutagenesis studies in cell lines establish the plasma membrane localisation of PLPPR3 as a regulator of its function to increase filopodia density (Chapter 2). Furthermore, the generation of a Plppr3-/- mouse line using CRISPR/Cas9 genome editing techniques (Chapter 3) enabled characterising endogenous phenotypes of PLPPR3 in neurons. In primary neuronal cultures, PLPPR3 was found to specifically control branch formation in a pathway with the phosphatase PTEN, without altering the overall growth capacity of neurons (Chapter 4). Loss of PLPPR3 specifically reduced branches forming from filopodia without affecting the stability of branches. This precise characterisation of PLPPR3 function unravelled the existence of parallel, independent programs for branching morphogenesis that are utilised and implemented differentially in developing axons and dendrites (Chapter 5). Furthermore, this thesis establishes multiple tools to study PLPPR3, the membrane lipid phosphatidylinositol-trisphosphate, and neuron morphogenesis by providing molecular tools, protocols, and semi-automated and automated image analysis pipelines (Appendix Chapter 7) and discusses experiments to test, refine and extend models of PLPPR3 function (Chapter 6). In summary, this thesis generated and utilised several tools and a Plppr3-/- mouse model to characterise PLPPR3 as a specific regulator of neuron branching morphogenesis. This precise characterisation refined and expanded the understanding of axon-specific branching morphogenesis.Nervenzellen entwickeln ihre komplexe Morphologie durch das Zusammenwirken diverser molekularer Entwicklungs-Programme der Zellkörper-Migration, der Polarisierung und der Morphogenese durch Wachstum, Verzweigung, Stabilisierung und Koordinierung ihrer Neuriten. Dabei unterscheidet sich die exakte Implementierung zwischen Nervenzell-Typen und selbst innerhalb einzelner Zellen zwischen Axonen und Dendriten. Diese unterschiedliche Morphogenese wird dabei speziell durch Membranproteine stark beeinflusst, die durch ihre Präsenz an der Plasmamembran Zell-extrinsische Signale mit den Zell-intrinsischen Morphogeneseprogrammen verbinden und beeinflussen. Die Familie der Phospholipid phosphatase-related / Plasticity-related gene (PLPPR/PRG) Proteine umfasst fünf Nervenzell-spezifische Membranproteine mit Effekten auf die Morphologie von Zellen. Zu Beginn dieses Projektes hatte noch keine Studie die Funktion des Familienmitglieds PLPPR3/PRG2 in Nervenzellen untersucht. Diese Dissertation beschreibt die Lokalisation von PLPPR3 an der Plasmamembran und in Zell-internen Membranstrukturen von Nervenzellen. Experimente in Zellkultur zeigen eine erhöhte Filopodien-Dichte nach Überexpression von PLPPR3, Mutagenese-Studien deuten eine strikte Kontrolle der Plasmamembran-Lokalisation an (Kapitel 2). Die Generierung einer Plppr3 Knockout Mauslinie mittels CRISPR/Cas9 Genom-Modifizierung (Kapitel 3) erlaubte eine Charakterisierung der endogenen Funktion von PLPPR3 in Nervenzellen. In Primärzellkultur von Nervenzellen des murinen Hippocampus zeigte sich, dass PLPPR3 im Zusammenspiel mit der Phosphatase PTEN spezifisch die Verzweigung von Nervenzellen kontrolliert, ohne deren Wachstumspotential global zu verändern (Kapitel 4). Dadurch kann PLPPR3 als ein Schalter zwischen Verzweigung und Verlängerung eines Nervenzell-Fortsatzes agieren. Der Verlust von PLPPR3 verursachte reduzierte spezifisch die Anzahl an Verzweigungen, die aus Filopodien entstanden, ohne dabei die Stabilität dieser Verzweigungen zu beeinflussen. Die präzise Charakterisierung dieser Funktion von PLPPR3 deckte auf, dass Verzweigungen von Nervenzell-Fortsätzen durch voneinander unabhängige Entwicklungsprogramme ausgebildet und stabilisiert werden können (Kapitel 5). Diese Programme werden von Axonen und Dendriten in unterschiedlicher Weise eingesetzt. Zusätzlich etabliert diese Arbeit sowohl diverse molekulare Werkzeuge und Visualisierungs-Protokolle zur Analyse von PLPPR3 und dem Membranlipid Phosphatidylinositol-Trisphosphat, als auch automatisierte Quantifizierungssoftware zur Studie der Nervenzellmorphologie (Appendix-Kapitel 7). Abschließend entwickelt und verfeinert die Dissertation mögliche Modelle zur PLPPR3-Funktion und zeigt experimentelle Strategien auf, um diese Modelle besser charakterisieren zu können (Kapitel 6). Zusammenfassend wurden in dieser Promotionsarbeit diverse Experimental- und Analyse-Strategien und eine Plppr3-/- Mauslinie entwickelt und genutzt, um PLPPR3 als einen spezifischen Regulator der Nervenzell-Morphogenese zu etablieren. Diese präzise Charakterisierung des PLPPR3 Phänotyps erlaubte zusätzlich eine Verfeinerung und Erweiterung der Erkenntnisse zur Axon-spezifischen Entwicklung von Verzweigungen

Eph-ephrin signalling in cell sorting and directional migration

An important problem in developmental biology is to understand how precise patterns of cell types are maintained during development. Eph receptor tyrosine kinases and ephrins have key roles in stabilising these patterns of cell organisation and segregation during development and can restrict the movement of cells by promoting cell repulsion. Previous work by Alexei Poliakov in the Wilkinson lab has shown that Eph-ephrin signalling leads to directional persistence of migration, and modelling suggests that this can contribute to cell segregation. In order to test experimentally the contribution of directional persistence in cell segregation, I have used and developed in vitro assays to dissect the roles of EphB2-ephrinB1 signalling in cell segregation, boundary sharpening and directional persistence. In these assays, stable HEK293 cell lines expressing EphB2 or ephrinB1 are mixed in cell culture and this leads to segregation of the two cell populations. Plating these cells either side of a removable barrier and allowing migration of cells towards each other leads to the formation of a sharp boundary on interaction. Analysis of cell behaviour shows EphB2 cells to move more persistently after interaction with ephrinB1 cells. To analyse how EphB2-ephrinB1 interactions lead to directional persistence of migration, my studies have focussed on the role of components potentially involved in directional persistence that act downstream of EphB2-ephrinB1 signalling, including the planar cell polarity (PCP) pathway (Dishevelled and Daam1) and core polarity components such as the PAR proteins (PAR-3 and PAR-6B). The PCP and PAR components were all found to have roles in cell segregation, as siRNA-mediated knockdown of each of these components disrupted EphB2-ephrinB1 mediated cell segregation and boundary sharpening. However, cell behaviour studies showed that only Dishevelled and PAR-6B have roles in EphB2-ephrinB1 mediated directional persistence, whilst Daam1 knockdown has no effect on the migratory response of cells. PAR-3 knockdown affects the basal ability of cells to migrate, potentially due to its role in establishing front-rear polarity. Taken together, these findings can be explained by a model in which Dishevelled and PAR-6B have a role in EphB2-ephrinB1 mediated directional persistence required for cell segregation and boundary sharpening. I propose that Daam1 may function in the contact inhibition of locomotion between cells also required for segregation