How do microtubules mechanistically control sprouting angiogenesis: identification of proximal neighbors of microtubules plus-end by proximity labelling

Abstract

Many diseases, such as diabetic retinopathy or obesity-associated disorders are associated with abnormal vascular network. Understanding the mechanisms regulating blood vessel formation by angiogenesis is therefore attractive, also because cancer progression is intimately linked to vessel irrigation of the tumor. One of the first step of the angiogenic process is the sprouting of a leading endothelial cell that will migrate and guide the formation of the new blood vessel. During this sprouting process , microtubules are concentrated in the forming protrusion and this enrichment is important to polarize the protrusion in the leading cell. The microtubule network is thus important for controlling the direction of the new blood vessel in formation. Nevertheless, the precise function of microtubules in sprouting angiogenesis still needs to be elucidated. To bring some insights to answer this question, the objective of my master thesis is to determine the interactome of the peripheral ends of microtubules during protrusion formation. We used proximity labelling of endothelial cells in a 3D model of angiogenic sprouting and chose the miniTurbo biotin ligase as the labelling enzyme. To target the plus-ends of microtubules, the biotin ligase was fused to the +TIP protein EB3. Whereas the physiological behavior of this fusion construct was validated, the enzymatic activity of miniTurbo was not sufficient in HUVEC cells (Human umbilical vein endothelial cells). As a backup approach, an immunoprecipitation of EB3 interactors followed by mass spectrometry analysis was performed and led to the identification of 17 proteins after selective thresholding. Interestingly, some interactors are enriched after pro-angiogenic treatment, suggesting that microtubule plus-ends can respond to this signal by recruiting specific factors. Microtubules also seem to interact with signaling proteins that are not known to be implicated in angiogenesis and could thus be interesting to study to understand how microtubules can participate in these signaling pathways. Moreover, some identified proteins confirm the importance of microtubule organization during angiogenesis. Finally, crosstalk between microtubules and other cytoskeleton structures appears to be a key element for the regulation of angiogenesis and would deserve future investigations.