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Endothelial cell invasiveness is controlled by myosin IIA-dependent inhibition of Arp2/3 activity

Ana M. Figueiredo, Pedro Barbacena, Ana Russo, Silvia Vaccaro, Daniela Ramalho, Andreia Pena, Aida P. Lima, Rita R. Ferreira, Fatima El-Marjou, Yulia Carvalho, Francisca F. Vasconcelos, Ana-Maria Lennon-Duménil, Danijela M. Vignevic, Claudio A. Franco

Preprint posted on September 08, 2020 https://www.biorxiv.org/content/10.1101/2020.09.08.287466v1

Exploring mechanisms of endothelial cell invasive behaviour.

Selected by Mariana De Niz

Background

   The formation of new blood vessels from pre-existing ones – a term called sprouting angiogenesis – is a fundamental process for homeostasis, and for various pathologies. Sprouting angiogenesis depends on the ability of a specialized endothelial cell (called tip cell) to invade and migrate into tissues. During migration, endothelial tip cells adopt polarized membrane protrusions which are believed to be key for invasion and guidance. Altogether, a balance of different filamentous actin networks and their regulators shapes the type of cellular protrusions formed (i.e. lamellipodia or filopodia), and hence the mode of cell migration. Despite the key role of tip cell invasive properties, it is still not fully understood how actomyosin and its regulators affect cell shape and protrusive behaviour, and how different membrane protrusions influence endothelial cell migration in vivo. In their work, Figueiredo, Barbacena et al (1) investigated specialized membrane protrusions on endothelial tip cells and their physiological relevance for endothelial cell invasion and angiogenesis in vivo, Moreover, the authors proposed an integrative model of how endothelial tip cells invade tissues.

Figure 1. Working model for the fine-tuning between filopodia and LLPs in endothelial tip cells. From (Ref1.)

Key findings and developments

Actin dynamics and membrane protrusions. Lamellipodia and filopodia are two types of membrane protrusions driven by actin dynamics. On one hand, formin-dependent linear actin arrays promote filopodia formation. On the other hand, actin-related protein 2/3 (Arp2/3) complex-dependent dendritic actin arrays promote lamellipodia formation. Non-muscle myosin-II (MII)-dependent contractility has been associated with the inhibition of protrusions and the promotion of membrane blebbing, while the isoform MIIA has been shown to promote filopodia stability.

The VEGF signaling pathway. CDC42 and RAC1 (small GTPases) are downstream of VEGF, and are master regulators of endothelial tip cell membrane protrusions. Moreover, VEGF also activates serum response factor (SRF), a transcription factor highly expressed by endothelial tip cells. Together with another player called myocardin-related transcription factor (MRTF), SRF regulates endothelial invasive behaviour.

Role of MII in endothelial tip cells. The authors began by analysing the function of MII in endothelial cells, focusing first on identifying the location and expression levels of isoforms MIIA and MIIB. Both, MIIA and MIIB were found to be highly expressed on endothelial tip cells, and to be enriched at the base of filopodia protrusions. Spatially, the high correlation of MIIA and MIIB with phospho-myosin light chain (pMLC) and filamentous actin suggest that endothelial tip cells have high levels of actomyosin contractility at their leading edges. The authors went on to generate single and double knock-outs of MIIA and/or MIIB, and found significant phenotypic differences to WT cells. Single deletion of MIIA led to significant and cell-specific changes in tip cell morphology including enlarged membrane protrusions and a severe decrease in filopodia number. Single deletion of MIIB led to a small decrease in the number of filopodia in endothelial tip cells. In neither case was filamentous actin compromised. Conversely, the double mutants lacking MIIA and MIIB show an extremely severe phenotype, whereby endothelial cells lacked filopodia, and had long and thin membrane protrusions attributed to a complete disruption of actomyosin contractility.

The authors went on to explore the dynamics of membrane protrusions of endothelial tip cells in vivo in mouse retinas, and found important differences between WT and MIIA-deficient cells. On one hand, WT cells displayed high rates of filopodia protrusions, while a small proportion of cells displayed also short-lived membrane ruffling both at the filopodium’s base and along the filopodium’s lateral membrane. MIIA-deficient cells were also able to protrude filopodia, but with highly altered dynamics including extensive filopodia membrane ruffling. The authors confirmed an association between membrane ruffling and actin polymerization at the base of the filopodia, and conclude altogether, that a key function of MIIA in tip cells is to negatively regulate the formation of long protrusions.

Exploring long lamellipodia projections (LLP). The authors named naturally occurring projections in WT cells LLPs, and found that a) they correlate with regions where endothelial tip cells contact with non-vascular extracellular matrix (ECM) and b) are associated with increased levels of active integrin beta 1 (ITGB1), a marker for matrix-bound focal adhesions (crucial for endothelial cell migration and invasion). This localization was altered in MIIA-deficient tip cells. The authors conclude that LLPs are tip cell-specific protrusions derived from filopodia, and that they may play important roles in migration into non-vascular ECM. The next step was to investigate the mechanism driving LLP formation, and the authors began by exploring the role of Arp2/3-dependent dendritic actin networks. Endothelial cells lacking a functional Arp2/3 were unable to invade avascular areas, and had impaired LLP formation. Moreover, they inversely mirrored the phenotype observed in MIIA-deficient cells in terms of filopodia and LLP number and morphology, without MIIA distribution being altered.  This led the authors to conclude that the Arp2/3 complex is key for LLP formation and for endothelial tip cell invasiveness.

Balance between filopodia and LLPs in endothelial tip cells. The authors then investigated how the balance between filopodia and LLPs is established. The first hypothesis tested was that MIIA could balance the ability of tip cells to form filopodia over LLPs via regulation of the Arp2/3 complex. The authors used pharmacological, genetic and optogenetic manipulations to explore LLPs, lamellipodia and filopodia formation. Their findings support the hypothesis that MII-activity promotes filopodia formation by limiting the activation of Arp2/3. This was further tested by generating a double KO for MIIA and Arp2/3, which led to 4 key conclusions: a) that Arp2/3 activity is necessary for endothelial tip cell invasiveness and formation of pro-invasive LLPs; b) that LLPs derive from filopodia; c) that filopodia are not required for tip cell migration; d) that MIIA enables filopodia formation by restricting Arp2/3 activation in endothelial tip cells in vivo.

Mechanism of MIIA-dependent control of Arp2/3 activity. Arp2/3 is activated by complexes downstream of Rac1 and Cdc42 (previously introduced above). A trigger for Rac1/Cdc42 activation and migration is signaling from immature focal adhesions at the leading edge of cells. The authors observed that Arp2/3-dependent LLPs correlated with local invasion into extravascular matrices, suggesting a feedback loop derived from integrin signaling from focal adhesions. This was supported by the fact that ITGB1-KO had striking similarities to Arp2/3-deficient cells in terms of filopodia morphology, lack of LLPs, and loss of invasiveness potential. The authors explored the hypothesis that focal adhesions promote the crosstalk between MIIA and Arp2/3 at the leading edge of tip cells. MIIA-deficient tip cells had a significant reduction of mature focal adhesions, and signals for integrins were not enriched at the base of the filopodia, but were instead diffuse over the LLPs. This altogether suggested that inhibition of MIIA leads to significant differences in integrin location and activation state. Altogether the mechanism suggested is that immature adhesions activate Arp2/3, and MIIA inhibits the signaling axis by promoting maturation of focal adhesions. Further work investigated the interplay between Rac1, Arp2/3 and integrin by pharmacological and genetic manipulation. This led to the conclusion that MIIA balances LLPs and filopodia formation by promoting focal adhesion maturation, thereby limiting Arp2/3 activation through inhibition of Rac1 activation downstream of integrin-βPIX in nascent adhesions.

What I like about this preprint

   I like vascular biology and I like that the authors a) address an important gap in understanding the role of cellular protrusions (i.e. lamellipodia or filopodia) on cell migration, and b) propose a model which explains the mechanisms by which such protrusions are regulated and their link to cell invasiveness and migration. I enjoyed reading the manuscript, and I think it integrates a wide plethora of tools to address the questions and hypotheses raised.

References

  1. Figueiredo, Barbacena, et al, Endothelial cell invasiveness is controlled by myosin IIA-dependent inhibition of Arp2/3 activity, bioRxiv, 2020.

 

Posted on: 14th December 2020

doi: https://doi.org/10.1242/prelights.26392

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Author's response

Pedro Barbacena, Ana Figueiredo and Claudio Franco shared

Open questions 

1.Firstly, this is very interesting work! You begin by discussing the role of sprouting angiogenesis in health and disease, and mention several types of pathology whereby sprouting angiogenesis is important such as cancer, diabetic retinopathy, and cardiovascular diseases. How does the new model you propose here, with its various players, fit with the different pathologies – that is, is sprouting angiogenesis in health and disease regulated by the same mechanisms? Or should we envisage that sprouting angiogenesis could be altered in many different ways in disease?

In a disease context, sprouting angiogenesis could be dysregulated in different ways. The underlying mechanisms pathological angiogenesis can be distinguished in two classes, intrinsic or extrinsic (Fonseca et al. 2020/10.1530/VB-20-0007). Nevertheless, we strongly believe that the role of LLP and filopodia for invasion and migration, is conserved. Therefore, we expect that in a pathological angiogenesis, both MIIA and Arp2/3 will regulate the formation of LLPs and filopodia, and thus influence the invasion and migration of endothelial cells in a conserved way.

2.Following from the question above: is it known whether endothelial tip cell behaviour (and the regulations you study in your work) is conserved across all types of vasculature, and across all organs?

The vascular biology community has showed that the main mechanisms regulating angiogenesis are conserved between species, organs and diseases. However, there are some examples that do not follow this rule, such as in the bone vasculature (Ramasamy, S. et al. 2016/10.1038/ncomms13601; Watson EC, Adams RH 2018/10.1101/cshperspect.a031559; Sivaraj KK, Adams RH 2016/10.1242/dev.136861). Given that our observations are based on core regulators of the actin cytoskeleton, we expect that the mechanisms regulating tip cells invasiveness that we have discovered will be conserved in other organs and types of vasculature.

3.Perhaps a naïve question, but you explore here the mechanisms regulating how endothelial tip cells invade tissues. How does your model fit with other possible regulators of cell migration, such as chemokines and cytokines, both in health and disease?

In this report, we have explored the mechanism of tip cell invasion through an actin perspective. Arp2/3 and MII are both controlled by external signaling, including chemokines and cytokines, mainly by activation of many members of the Rho GTPase family. Thus, external signaling will play a fundamental role in regulating the tip cell invasion in health and disease.

4.You began your work by comparing localizations of MIIA and MIIB, but did not explore the role of MIIB further in various points. Is there a level of compensation upon the loss of each other?

As we have described in the manuscript, MIIA and MIIB have redundant functions in endothelial cells. MIIB-iECKO mice do not show strong phenotypes, but showed a small decrease in number of filopodia. In contrast, MIIA-iECKO has a much stronger phenotype, with a severe decrease in the number of filopodia and a concomitant increase in LLPs. Double KO mice evidenced an even stronger phenotype, when compared with the MIIA alone, with almost no filopodia. Yet, retinas from double KO mice also showed a dramatic reduction of vessel width and marked changes in endothelial cell shape, a phenotype not seen in MIIA-iECKO or MIIB-iECKO alone. These results could possibly reflect complete disruption of the actomyosin contractility in the system. Thus, it is clear that MIIA and MIIB have redundant functions in endothelial cells. However, the function of MIIA in the formation and regulation of filopodia and LLPs is much more pronounced than MIIB, thus we decided to further investigate MIIA alone.

5.Are there naturally occurring diseases derived from mutations of any of the players you investigated in your work? What is the outcome if so?

It is known that several mutations in the MYH9 gene can lead to premature release of platelets from the bone marrow, macrothrombocytopenia, and cytoplasmic inclusion bodies within leukocytes. Syndromes like May-Hegglin anomaly, Epstein syndrome, Fechtner syndrome, and Sebastian platelet syndrome, define different clinical manifestations of MYH9 gene mutations (Althaus K 2010/ 10.1159/000320335). Moreover, different studies associated MYH9 with cancer cell migration, invasion, and metastasis. For instance, high levels of MYH9 are correlated with high malignancy and metastatic potential, in different type of cancers (Betapudi V 2006/ 10.1158/0008-5472.CAN-05-4236; Katono K 2015/10.1371/journal.pone.0121460).

In line with this, the inappropriate function of the Arp2/3 complex and its regulators can lead to disease. One example is WAS, a rare recessive X-linked genetic disorder that involves defects in blood-cell function and leads to susceptibility to infection and internal haemorrhages (Thrasher AJ 2002/10.1038/nri884). Arp2/3 complex dysfunction is also associated with cancer metastasis, which relies on the capability of cancer cells to migrate away from primary tumors and invade healthy tissues. The expression of Arp2/3, N-WASP and WAVE2 is upregulated in some tumor tissues and invasive cells, what as associated with an increased motility and invasiveness (Semba S 2006/10.1158/1078-0432.CCR-05-2566; Wang W 2005/10.1016/j.tcb.2005.01.003).

However, mutations in genes involved in the Arp2/3 or MII complexes have not been associated with endothelial dysfunction to our knowledge.

6.What are the limitations of the model you propose, namely, what is left still unexplained in the understanding of the mechanisms regulating sprouting angiogenesis, and what are main discrepancies (if any) with other proposed models?

Classically, filopodia are considered the first point of cell contact within the external environmental factors, being responsible for the cellular direction and guidance. In a study published on Development in 2013, Phng LK et al claimed that filopodia are in fact not essential for endothelial cell guidance or migration in zebrafish embryos, bringing controversy to the field. In our study, we have observed that endothelial-specific deletion of myosin IIA (MIIA-iECKO) results in the inhibition of filopodia formation and stability. However, this translated into a modest delay in the capacity of endothelial cells to migrate and invade. Our results confirmed the original observation by Phng and colleagues using a different approach and a different animal model.

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