Focal adhesions (FAs) are crucial for vascular morphogenesis, but how they influence endothelial cell behaviour in live functioning blood vessels was unknown. In a new paper in Development, Anne Lagendijk and colleagues find that functional FAs are required for endothelial cell rearrangements, in an actin-dependent manner. We caught up with the first author Tevin Chau and corresponding author Anne Lagendijk, a Group Leader at the Institute for Molecular Bioscience (IMB), University of Queensland, Australia, to find out more about their research.
Anne Lagendijk (L) and Tevin Chau (R)
Anne, can you give us your scientific biography and the questions your lab is trying to answer?
AL: I did my undergraduate studies (MSc degree in Biomedical Sciences) at the Radboud University in Nijmegen (The Netherlands). As an undergraduate, I developed a lasting fascination with developmental biology, working in the labs of Professor Peter Koopman (IMB, University of Queensland, Brisbane, Australia) on ovarian specification and with Professor Ronald Plasterk (Hubrecht Institute, Utrecht, the Netherlands) on microRNAs that control pancreas development.
For my PhD training, I stayed at the Hubrecht and joined the lab of Professor Jeroen Bakkers. I identified novel regulators that control hyaluronic acid homeostasis in the extracellular matrix of the developing zebrafish heart. These factors were proven to be essential for cardiac valve formation. After completing my PhD, I relocated back to the IMB in 2012 to work as a University of Queensland Postdoctoral Fellow in the lab of Professor Ben Hogan. I initiated a project studying mechanotransduction at endothelial cell-cell junctions in vivo, in collaboration with the lab of Professor Alpha Yap. I generated the first vertebrate FRET tension biosensor as part of this work. During this time, I also continued pursuing my interest in hyaluronic acid signalling and, in collaboration with the lab of Associate Professor Kelly Smith, identified that the hyaluronidase Tmem2 is required for sprouting angiogenesis in vivo.
I have been leading my own lab at the IMB since 2019. We use both zebrafish and 3D human micro-vessels to understand the cellular mechanisms that control blood vessel integrity, both during development and in diseases such as childhood brain cancer and familial vascular malformations. Our major focus is on genetic pathways that control endothelial cell (EC) adhesion, mechanotransduction and extracellular matrix composition.
Tevin, how did you come to work in Anne's lab and what drives your research today?
TC: I was completing a research project for my undergraduate degree with Professor Peter Koopman (IMB) on mouse sex determination when I noticed some really exciting work being done by our lab neighbour (Anne), so I decided to give zebrafish a go!
I no longer have research projects, but I wake up excited about work every morning knowing that I'm helping researchers get great imaging data and answer their very diverse research questions.
What was known about the role of FAs in vascular morphogenesis before this work?
TC & AL: Numerous studies had shown that FAs are crucial for the basic formation and integrity of blood vessels. These findings came mostly from embryonic or postnatal deletion of major FA components in mice, which results in haemorrhaging and, in some cases, changes in VE-cadherin patterning. However, it has remained to be shown precisely where, when and how EC behaviour is controlled by FAs in live developing tissues, as previous fixed time-point analyses are difficult to interpret in terms of cell dynamics and the timing of FA function.
Can you give us the key results of the paper in a paragraph?
TC & AL: By combining our novel talin1 mutant allele with the VE-cadherin transgene that we had previously established, we could now visualise cell shape and adherens junctions in ECs devoid of FAs with live imaging. Pressure inflicted on the ECs by blood flow is a major physical signal that impacts EC biology. Importantly, the Talin1-deficient ECs were experiencing normal blood flow pressure for the timepoints we analysed. We identified that FAs in wild-type ECs are extremely dynamic and that loss of these structures in talin1 mutants leads to a failure in EC rearrangements. In both the intersegmental vessels (ISVs) and in the dorsal aorta, ECs fail to elongate and linearise cell-cell junctions. We found that loss of F-actin polymerisation is essential for the process of EC elongation and when we treated talin1 mutants with a polymerisation agonist, this significantly restored the capacity of the ECs to elongate. We thereby showed that Talin1 and FAs are required in the ECs to drive cell shape changes, likely by connecting with polymerised actin networks and interpreting and controlling the major sites of tension. We think that these findings improve our understanding of the mechanical inputs controlling vessel formation and integrity and may help inform new approaches to normalise barrier function in disease.
Is the integrity of either the adherens junctions or tight junctions compromised by loss of Talin1?
TC & AL: We have not interrogated the effect of Talin1 loss on vessel barrier integrity at this stage, nor have we examined the organisation of tight junctions. We did, however, observe occasional blood pooling in somites within the trunk amongst our transplanted embryos. Such bleeding correlated with the presence of Talin1-deficient grafts in ISVs. This observation, together with the ISV regression events that we identified, suggest that, at least in the ISVs, loss of Talin1 might compromise the vessel barrier, contributing to ISV collapse.
Single intersegmental vessels of a wild-type sibling and in tln1uq1al−/− mutant embryo at 50 hpf, expressing VE-cadherin Tg(ve-cad:ve-cad-TS). False-coloured images indicate EC shapes.
Do you think FAs also control actomyosin contractility in ECs, or are the effects confined to actin rearrangements?
TC & AL: We anticipate that there would be spatial changes in actomyosin contractility when the ECs are devoid of FAs. As vinculin is recruited to FAs that are experiencing high tension, the vast number of Vinculinb-GFP-positive FAs in the ECs of the dorsal aorta [see movie 1 in Chao et al. (2022)] suggests that there are many regions of high tension across the EC membrane. In talin1 mutants these structures are lost and thus we assume that the level or the distribution of forces at the membrane is altered.
When doing the research, did you have any particular result or eureka moment that has stuck with you?
TC: The timelapse video showing Vinculinb-GFP dynamics in ECs is such a beautiful thing. I spent 2 years of cloning and screening to get this stable transgenic line. When I finally saw the expression live, it almost felt like my baby was born. Historically, the presence of FAs in ECs in vivo has been contested and thus to visualise them in action was exciting.
I showed Anne this timelapse video a few days after I captured it so, for a little while, I was the only person in the world who knew how vinculin moves in ECs of live vessels. She nearly lost it from excitement when I showed her!
For a little while, I was the only person in the world who knew how vinculin moves in ECs of live vessels
And what about the flipside: any moments of frustration or despair?
TC: The range of cell transplantation studies we carried out for the paper required a lot of practice and effort. There were some weeks where I transplanted cells from hundreds of embryos and ended up not finding any vascular grafts in the recipients. This would be a little demoralising when sitting in a dark confocal room late in the day, all by myself.
Tevin, what is next for you after this paper?
TC: I'm currently working for Nikon Australia as an Application Specialist. It's an exciting role. I get to help people generate pretty and insightful microscopy data all day long. Through this role, I now get exposed to so much cutting-edge research that extends far beyond my PhD discipline.
Anne, where will this story take your lab next?
AL: We are currently using the Vinculinb-GFP transgene to analyse how forces are distributed in ECs during vascular morphogenesis. By combining the transgene with genetic mutants that either distort the formation of FAs or adherens junctions, we would like to identify when crosstalk occurs and whether there are specific aspects of morphogenesis where this is crucial.
Finally, let's move outside the lab – what do you like to do in your spare time?
TC: Outside of the lab, I enjoy road cycling and playing cello. Riding in the countryside for miles and miles using my own strength and without the noise of an engine is very empowering. I like pushing myself and getting stronger as a rider. The cello is a beautiful instrument that has that the perfect balance of tenor and bass, and I enjoy growing my musicality and expressiveness as a cellist as I diligently practice.
AL: The absolute beauty of the Australian coastline will never stop amazing me, so my most favourite thing is to pack up and go to the coast for a weekend and enjoy time on the beach with my family. In Brisbane, I like to get into the pilates studio as frequent as I can. It is a great way for me to compensate for hours at the desk and to tune out and take my mind off work.
T.C. & A.L.: Division of Cell and Developmental Biology, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland 4072, Australia.
A.L.: School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia.
E-mail: [email protected]
