The β3‐integrin endothelial adhesome regulates microtubule‐dependent cell migration

Abstract

Integrin β3 is seen as a key anti‐angiogenic target for cancer treatment due to its expression on neovasculature, but the role it plays in the process is complex; whether it is pro‐ or anti‐angiogenic depends on the context in which it is expressed. To understand precisely β3's role in regulating integrin adhesion complexes in endothelial cells, we characterised, by mass spectrometry, the β3‐dependent adhesome. We show that depletion of β3‐integrin in this cell type leads to changes in microtubule behaviour that control cell migration. β3‐integrin regulates microtubule stability in endothelial cells through Rcc2/Anxa2‐driven control of active Rac1 localisation. Our findings reveal that angiogenic processes, both in vitro and in vivo, are more sensitive to microtubule targeting agents when β3‐integrin levels are reduced.

NRP2 as an Emerging Angiogenic Player; Promoting Endothelial Cell Adhesion and Migration by Regulating Recycling of α5 Integrin

“…Importantly, the disrupted ITGA5 phenotype we see following NRP2 depletion in our immortalized ECs is also present in primary ECs. Along with our previous studies demonstrating they express a canonical adhesome (Atkinson et al, 2018), this rebuts the concept that immortalized ECs are an inappropriate model for studying angiogenic processes.…”

“…To investigate this, we isolated mouse lung microvascular endothelial cells (mLMECs) from both wild-type (WT) and ITGB3-heterozygous (β3HET) mice and immortalized them with polyoma-middle-T antigen (PyMT) by retroviral transduction. As in previous studies, we decided to use β3HET cells for these analyses, rather than β3-integrin knockout (β3NULL) cells, because we have shown they are a good model for studying the role of αvβ3-integrin in cell migration, whilst evading changes arising from the complete loss of the integrin on both alleles (e.g., up-regulated VEGFR2 expression) (Ellison et al, 2015;Atkinson et al, 2018). DNA was extracted from multiple immortalized lines and analyzed by PCR to confirm their genetic status as either WT or β3HET cells (Supplementary Figure S1A).…”

“…Silencing of NRP2 in mLMECs significantly reduced the rate of both FA assembly and disassembly compared to control siRNA treated cells ( Figure 2C). We also measured FA number and size distribution in ECs using two different NRP2 siRNAs by immunolabelling for endogenous paxillin in cells that had adhered to FN for 90 min, a time that allows for mature FAs to form (Ridley et al, 2003;Schiller et al, 2013;Atkinson et al, 2018). Despite no significant difference in average cell area (Supplementary Figure S2C), FAs were significantly fewer in number and smaller in average size in NRP2 siRNA treated cells compared to control siRNA treated ECs ( Figure 2D, Supplementary Figure S2D), suggesting NRP2 depletion inhibits FA maturation.…”

NRP2 as an emerging angiogenic player; promoting endothelial cell adhesion and migration by regulating recycling of α5 integrin

“…Silencing of NRP2 in mLMECs significantly reduced the rate of both FA assembly and disassembly compared to control siRNA treated cells (Fig2A). We also measured FA number and size distribution in ECs by immunolabelling for endogenous paxillin in cells that had adhered to FN for 90 minutes, a time that allows mature FAs to form [31], [32], [33]. Despite no significant difference in average cell area (Suppl.…”

Definition of the fibroblast adhesome using multiplexed proximity biotinylation

“…The scale of the reported complexity of IACs is consistent with their substantial functional diversity. Although empirical analysis of IACs has been hampered by their lability and vicinity to the plasma membrane, advances in mass spectrometry coupled to the development of protocols to isolate IACs and adjacent material have greatly facilitated the identification of IACassociated proteins [7][8][9][10][11][12][13] . The large-scale examination of isolated IACs enabled assembly of adhesome datasets, and the bioinformatic integration of seven such analyses (of fibronectin substrate-induced IACs) defined a 'meta adhesome' of over 2,400 proteins 11 .…”