BEGIN:VCALENDAR VERSION:2.0 PRODID:Icfo X-PUBLISHED-TTL:P1W BEGIN:VEVENT UID:69ee030eef041 DTSTART:20220228T090000Z SEQUENCE:0 TRANSP:OPAQUE LOCATION:ICFO Auditorium and Online (Teams) SUMMARY:ICFO | SARAH KEARY CLASS:PUBLIC DESCRIPTION:The main goal of this thesis was to contribute to the understan ding of the nanoscale lateral organisation of key proteins in adhesion com plexes. For this\, we exploited single molecule localisation-based super-r esolution microscopy STORM to visualise the lateral organisation of five k ey proteins of the adhesion complex: the integrins\, &alpha\;5&beta\;1 and &alpha\;v&beta\;3\, and three of their adaptor proteins: paxillin\, talin \, and vinculin.\nWe first established that these proteins form nanocluste rs of around 50nm size that are preserved across all five proteins. Intere stingly\, these nanoclusters have similar size and number of localisations regardless of their localisation on the membrane\, i.e.\, in the differen t adhesion structures studied\, namely\, FA and fAs as well as outside\, a nd 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 activi ty. When studying how protein organisation in nanoclusters changes as a fu nction of adhesion time\, we revealed a two- and a four-fold increase in t he density of &alpha\;5&beta\;1 and &alpha\;v&beta\;3 clusters\, respectiv ely\, 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 t o the basal membrane.\nFollowing on from this\, we then focus on mapping t he distribution of these nanoclusters\, first by measuring the nearest nei ghbour distance\; (NND) between clusters of the same protein\, and second by considering the shortest distance between clusters of different protein s. We found a clear physical segregation of nanoclusters of the same prote in around ~55 nm\, which is established at early time points after cell se eding for &alpha\;5&beta\;1 and the adaptors and maintained after 24 h. In terestingly\, &alpha\;v&beta\;3 nanoclusters exhibited a more random distr ibution at earlier seeding times and progressively reached similar lateral segregation at 24 h. Concomitant with this lateral segregation\, we obser ved an enriched of all proteins at distances between 100-200 nm. Our obser vations are in line with the existence of a critical distance spacing betw een integrins needed for support adhesion and stabilisation of focal adhes ions. Furthermore\, we found that the relative distribution of nanocluster s of different proteins is predominantly random\, with the exception of &a lpha\;5&beta\;1 and paxillin\, which organise with a separation of 50 nm. Such an unexpected random distribution between integrins and their adaptor s might reflect the dynamic and short-live active state of integrins.\nFin ally\, we evaluated and described the mesoscale organisation of nanocluste rs inside adhesions. Specifically\, we computed the shortest distance betw een a nanocluster and the edge of the adhesion and studied how the distanc e to the edge depends on the NND between clusters of different proteins. R emarkably\, we found a preference for &alpha\;5&beta\;1 nanoclusters to be at the edge of the adhesions and in close proximity to its adaptors in a peripheral belt region of the adhesions.\nAltogether\, the results of this thesis demonstrate a clear lateral and hierarchical organisation of integ rins and their adaptors inside focal adhesions. Based on our results (toge ther with extensive literature in the field)\, we propose that one populat ion of &alpha\;5&beta\;1 nanoclusters and their adaptors preferentially lo calise close to the edge of adhesion complexes regulating the process of a dhesion. A second population of &alpha\;5&beta\;1 and most of the &alpha\; v&beta\;3 nanoclusters organise more randomly at the centre of the adhesio ns\, 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 mecha notransduction.\n \;\nThesis Director: Prof Dr. Marí\;a Garc&iac ute\;a-Parajo\nThesis Co-Director: Dr. Felix Campelo\n \; DTSTAMP:20260426T122030Z END:VEVENT END:VCALENDAR