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Analysis of junctional and F-actin dynamics during blood vessel morphogenesis

Wiesner, Cora. Analysis of junctional and F-actin dynamics during blood vessel morphogenesis. 2020, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_13631

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Abstract

Organ morphogenesis relies on dynamic cell behaviors, which are highly coordinated to ensure a functional cellular organ architecture. During vascular morphogenesis, the process of angiogenesis is driven by cell migration, cell shape changes and cell rearrangements. Here, a dynamic balance between inter-endothelial cell adhesion and plasticity allows angiogenic sprouting while maintaining the endothelial seal.
Previous analyses on blood vessel formation and anastomosis in zebrafish have shown that junctional remodeling is central to many aspects of morphogenetic endothelial cell-cell interactions. In particular, the adhesion molecule VE-cadherin (Cdh5) is essential for coordinated cell shape changes during multicellular tube formation, as loss of VE-cadherin was shown to inhibit cell rearrangements (Sauteur et al., 2014). This study also proposed an active, force generating function for VE-cadherin in this process. This hypothesis is supported by our study showing that cell elongation is mediated by junction-based lamellipodia (JBL), which are thought to provide a tractile force for junction elongation (Paatero et al., 2018).
In my thesis, my goal was to further analyze the molecular mechanisms which underly JBL function. In particular I focused on molecular players that influence F-actin dynamics or contractility (Arp2/3, Rac1 and ROCK) in order to identify critical players in the process of the cell elongation movement. Furthermore, I elucidated, how JBL might generate motile forces and how these forces are transmitted onto endothelial cell junctions (e.g. VE-cadherin)
In the course of my experiments I identified the actomyosin contractility as an important basis for junctional ring elongation. Inhibition of ROCK did interfere with the correct localization of junctional protein ZO1 as it is abrogated formation of double junctions- a hallmark during JBL oscillations and an indispensable step to since it leads to the formation of a new attachment site. Furthermore, I found that the establishment of differential VE-cadherin tension is also ROCK-dependent, which might provide the basis for junctional remodeling. Junctional rearrangements were not only impaired after inhibition of ROCK. Also, interference with the F-actin dynamics significantly altered junctional ring elongation. Arp2/3 (and concomitant formation of branched F-actin networks) is necessary for maintaining junctional stability and responsible for the correct localization of F-actin during the process, whereas Rac1 mostly seem to play a role in the induction phase of the JBL. Last but not least I generated two new transgenic fish lines (fli:iRFP-UCHD and kdrl:mCherry-PA-Rac1), which will open up a whole lot of new possibilities for future experiments. Making use of the photoactivatable Rac1 will give manifold new insights into processes during vascular morphogenesis, which underly Rac1 activity (sprouting, anastomosis, etc.).
In summary JBL function and subsequent endothelial cell rearrangements rely on a tight interplay between generation and maintenance of a dynamic F-actin cytoskeleton and regulation of junctional proteins. The F-actin cytoskeleton furthermore provide a basis for local force generation, which is reflected in differential VE-cadherin tension and thus exert mechanical forces, which in turn are a major driver of the process of junctional ring elongation. Last but not least my experiments suggest, that JBL formation and local protrusion formation might be a general mechanism of endothelial cells to induce cell movements and cell shape changes (e.g. in the dorsa aorta).
Advisors:Affolter, Markus and Gerhardt, Holger
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Growth & Development > Cell Biology (Affolter)
UniBasel Contributors:Affolter, Markus
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:13631
Thesis status:Complete
Number of Pages:1 Online-Ressource (IX, 232 Seiten)
Language:English
Identification Number:
Last Modified:05 Aug 2020 04:30
Deposited On:04 Aug 2020 12:44

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