Spatiotemporal organisation of protein nanoclusters in adhesion complexes

The main goal of this thesis was to contribute to the understanding of the nanoscale lateral organisation of key proteins in adhesion complexes. For this, we exploited single molecule localisation-based super-resolution microscopy STORM to visualise the lateral organisation of five key proteins of the adhesion complex: the integrins, a5ß1 and avß3, and three of their adaptor proteins: paxillin, talin, and vinculin. We first established that these proteins form nanoclusters of around 50nm size that are preserved across all five proteins. Interestingly, these nanoclusters have similar size and number of localisations regardless of their localisation on the membrane, i.e., in the different adhesion structures studied, namely, FA and fAs as well as outside, and were maintained for different cell seeding times, from 90 min to 24 h. These results suggest that nanoclustering constitutes a general mechanism of adhesion protein organisation, creating nanohubs of functional activity. When studying how protein organisation in nanoclusters changes as a function of adhesion time, we revealed a two- and a four-fold increase in the density of a5ß1 and avß3 clusters, respectively, for cells that spread for 24 h as compared to those that spread for 90 min. Further analysis suggests that the increase in density of integrin nanoclusters is due to selective targeting of new integrin nanoclusters to the basal membrane. Following on from this, we then focus on mapping the distribution of these nanoclusters, first by measuring the nearest neighbour distance; (NND) between clusters of the same protein, and second by considering the shortest distance between clusters of different proteins. We found a clear physical segregation of nanoclusters of the same protein around ~55 nm, which is established at early time points after cell seeding for a5ß1 and the adaptors and maintained after 24 h. Interestingly, avß3 nanoclusters exhibited a more random distribution at earlier seeding times and progressively reached similar lateral segregation at 24 h. Concomitant with this lateral segregation, we observed an enriched of all proteins at distances between 100-200 nm. Our observations are in line with the existence of a critical distance spacing between integrins needed for support adhesion and stabilisation of focal adhesions. Furthermore, we found that the relative distribution of nanoclusters of different proteins is predominantly random, with the exception of a5ß1 and paxillin, which organise with a separation of 50 nm. Such an unexpected random distribution between integrins and their adaptors might reflect the dynamic and short-live active state of integrins. Finally, we evaluated and described the mesoscale organisation of nanoclusters inside adhesions. Specifically, we computed the shortest distance between a nanocluster and the edge of the adhesion and studied how the distance to the edge depends on the NND between clusters of different proteins. Remarkably, we found a preference for a5ß1 nanoclusters to be at the edge of the adhesions and in close proximity to its adaptors in a peripheral belt region of the adhesions. Altogether, the results of this thesis demonstrate a clear lateral and hierarchical organisation of integrins and their adaptors inside focal adhesions. Based on our results (together with extensive literature in the field), we propose that one population of a5ß1 nanoclusters and their adaptors preferentially localise close to the edge of adhesion complexes regulating the process of adhesion. A second population of a5ß1 and most of the avß3 nanoclusters organise more randomly at the centre of the adhesions, with dynamic and brief engagement to their adaptors, likely playing a role in mechanotransduction. As a whole, we postulate that the lateral nano- and meso-scale organisation of adhesion proteins is strictly related to and important for the functions of adhesion, mechanosensing and mechanotransduction.El objetivo de esta tesis ha sido contribuir a la comprensión de la organización lateral a nanoescala de proteínas clave en complejos de adhesión. Para ello, usamos la microscopía de superresolución STORM, para visualizar con resolución espacial nanométrica la organización lateral de cinco proteínas del complejo de adhesión: dos integrinas, a5ß1 y avß3, y las proteínas adaptadoras: paxilin, talin y vinculin. En primer lugar, establecimos que estas proteínas forman nanoagregados de ~50 nm tamaño en las cinco proteínas. Curiosamente, su tamaño y número de localizaciones son similares, independientemente de su localización en la membrana, es decir, tanto en FA y fAs, así como fuera de las adhesiones, manteniéndose constantes durante diferentes tiempos de siembra celular. Estos resultados sugieren que la nanoagregación constituye un mecanismo general de organización de proteínas de adhesión, constituyendo nanocentros de actividad funcional. Además, revelamos un aumento en la densidad de los agregados de a5ß1 y avß3 en células extendidas por 24 h en comparación con 90 min, mientras que la densidad de agregados de las proteínas adaptadoras se mantuvo constante. Esta disparidad en densidades indica que solo una fracción de las integrinas interacciona con sus adaptadores, consistente con estados dinámicos de activación-desactivación de las integrinas. También nos enfocamos en mapear la distribución de estos nanoagregados, midiendo la distancia entre agregados más corta entre grupos de la misma proteína, y luego, considerando la distancia más corta entre grupos de diferentes proteínas. Encontramos una clara segregación física de agregados de la misma proteína alrededor de ~55 nm, que se establece temprano después de la siembra celular para a5ß1 y sus adaptadores, y se mantiene hasta 24 h. Curiosamente, los agregados de avß3 exhibieron una distribución más aleatoria en tiempos tempranos de siembra, alcanzando progresivamente una segregación lateral similar a 24 h. Acompañada a esta segregación lateral, observamos un enriquecimiento de todas las proteínas a distancias entre 100¿200 nm. Nuestras observaciones son consistentes con la existencia de un espaciado de distancia crítico entre las integrinas necesarias para apoyar la adhesión y estabilizar las adhesiones focales. Además, encontramos que la distribución relativa de nanoagregados de diferentes proteínas es aleatoria, lo cual podría reflejar el estado activo dinámico y de corta duración de las integrinas, de modo que con nuestras condiciones de imágenes, actualmente no podemos capturar la participación de aquellas integrinas activas dentro de la población total. Finalmente, evaluamos la organización de mesoescala de nanoagregados en FAs, específicamente, en los bordes y el centro. Sorprendentemente, encontramos una preferencia por nanoagregados de a5ß1 en el borde de las FAs y cerca de sus adaptadores, en una región periférica a los bordes. En conjunto, nuestros resultados demuestran una clara organización lateral y jerárquica de las integrinas y sus adaptadores dentro de las adhesiones focales. Proponemos que una población de nanoagregados de a5ß1 y sus adaptadores se localizan preferentemente cerca del borde de los complejos de adhesión para regular el proceso de adhesión y probablemente interaccionando activamente con la maquinaria de la actomiosina. Una segunda población de a5ß1 y la mayoría de los nano-gregados de avß3 se organizan de forma aleatoria en el centro de las FAs con una interacción dinámica y breve con sus adaptadores, posiblemente comprometidos con el proceso de mecanotransducción. En conjunto, y similar a su organización axial, postulamos que la organización lateral a nano- y meso-escala dentro de las FAs es importante para las funciones de adhesión, mecanosensibilidad y mecanotransducción.Postprint (published version

Shear forces induce ICAM-1 nanoclustering on endothelial cells that impact on T cell migration

The leukocyte specific β2-integrin LFA-1, and its ligand ICAM-1 expressed on endothelial cells (ECs), are involved in the arrest, adhesion and transendothelial migration of leukocytes. Although the role of mechanical forces on LFA-1 activation is wellestablished, the impact of forces on its major ligand ICAM-1, has received less attention. Using a parallel-plate flow-chamber combined with confocal and super-resolution microscopy, we show that prolonged shear-flow induces global translocation of ICAM-1 on ECs upstream of flow direction. Interestingly, shear-forces caused actin rearrangements and promoted actin-dependent ICAM-1 nanoclustering prior to LFA-1 engagement. T-cells adhered to mechanically pre-stimulated ECs or nanoclustered ICAM-1 substrates, developed a pro-migratory phenotype, migrated faster and exhibited shorter-lived interactions with ECs than when adhered to non-mechanically stimulated ECs, or to monomeric ICAM-1 substrates. Together, our results indicate that shearforces increase ICAM-1/LFA-1 bonds due to ICAM-1 nanoclustering, strengthening adhesion and allowing cells to exert higher traction forces required for faster migration. Our data also underscores the importance of mechanical forces regulating the nanoscale organization of membrane receptors and their contribution to cell adhesion regulation.Peer ReviewedPostprint (author's final draft

Shear forces induce ICAM-1 nanoclustering on endothelial cells that impact on T cell migration

The leukocyte specific β2-integrin LFA-1, and its ligand ICAM-1 expressed on endothelial cells (ECs), are involved in the arrest, adhesion and transendothelial migration of leukocytes. Although the role of mechanical forces on LFA-1 activation is wellestablished, the impact of forces on its major ligand ICAM-1, has received less attention. Using a parallel-plate flow-chamber combined with confocal and super-resolution microscopy, we show that prolonged shear-flow induces global translocation of ICAM-1 on ECs upstream of flow direction. Interestingly, shear-forces caused actin rearrangements and promoted actin-dependent ICAM-1 nanoclustering prior to LFA-1 engagement. T-cells adhered to mechanically pre-stimulated ECs or nanoclustered ICAM-1 substrates, developed a pro-migratory phenotype, migrated faster and exhibited shorter-lived interactions with ECs than when adhered to non-mechanically stimulated ECs, or to monomeric ICAM-1 substrates. Together, our results indicate that shearforces increase ICAM-1/LFA-1 bonds due to ICAM-1 nanoclustering, strengthening adhesion and allowing cells to exert higher traction forces required for faster migration. Our data also underscores the importance of mechanical forces regulating the nanoscale organization of membrane receptors and their contribution to cell adhesion regulation.Peer ReviewedPostprint (author's final draft

A multidisciplinary approach and consensus statement to establish standards of care for Angelman syndrome

Background: Angelman syndrome (AS) is a rare neurogenetic disorder present in approximately 1/12,000 individuals and characterized by developmental delay, cognitive impairment, motor dysfunction, seizures, gastrointestinal concerns, and abnormal electroencephalographic background. AS is caused by absent expression of the paternally imprinted gene UBE3A in the central nervous system. Disparities in the management of AS are a major problem in preparing for precision therapies and occur even in patients with access to experts and recognized clinics. AS patients receive care based on collective provider experience due to limited evidence-based literature. We present a consensus statement and comprehensive literature review that proposes a standard of care practices for the management of AS at a critical time when therapeutics to alter the natural history of the disease are on the horizon. Methods: We compiled the key recognized clinical features of AS based on consensus from a team of specialists managing patients with AS. Working groups were established to address each focus area with committees comprised of providers who manage >5 individuals. Committees developed management guidelines for their area of expertise. These were compiled into a final document to provide a framework for standardizing management. Evidence from the medical literature was also comprehensively reviewed. Results: Areas covered by working groups in the consensus document include genetics, developmental medicine, psychology, general health concerns, neurology (including movement disorders), sleep, psychiatry, orthopedics, ophthalmology, communication, early intervention and therapies, and caregiver health. Working groups created frameworks, including flowcharts and tables, to help with quick access for providers. Data from the literature were incorporated to ensure providers had review of experiential versus evidence-based care guidelines. Conclusion: Standards of care in the management of AS are keys to ensure optimal care at a critical time when new disease-modifying therapies are emerging. This document is a framework for providers of all familiarity levels