Clara L. Essmann

Ephrin-B2 regulates VEGFR2 function in developmental and tumour angiogenesis

The formation and guidance of specialized endothelial tip cells is essential for both developmental and pathological angiogenesis. Notch-1 signalling regulates the generation of tip cells, which respond to gradients of vascular endothelial growth factor (VEGF-A). The molecular cues and signalling pathways that control the guidance of tip cells are poorly understood. Bidirectional signalling by Eph receptors and ephrin ligands represents one of the most important guidance cues involved in axon path finding. Here we show that ephrin-B2 reverse signalling involving PDZ interactions regulates endothelial tip cell guidance to control angiogenic sprouting and branching in physiological and pathological angiogenesis. In vivo, ephrin-B2 PDZ-signalling-deficient mice (ephrin-B2ΔV) exhibit a reduced number of tip cells with fewer filopodial extensions at the vascular front in the mouse retina. In pathological settings, impaired PDZ signalling decreases tumour vascularization and growth. Mechanistically, we show that ephrin-B2 controls VEGF receptor (VEGFR)-2 internalization and signalling. Importantly, internalization of VEGFR2 is necessary for activation and downstream signalling of the receptor and is required for VEGF-induced tip cell filopodial extension. Together, our results suggest that ephrin-B2 at the tip cell filopodia regulates the proper spatial activation of VEGFR2 endocytosis and signalling to direct filopodial extension. Blocking ephrin-B2 reverse signalling may be an attractive alternative or combinatorial anti-angiogenic therapy strategy to disrupt VEGFR2 function in tumour angiogenesis.

Grb4 and GIT1 transduce ephrinB reverse signals modulating spine morphogenesis and synapse formation.

Dendritic spines are small protrusions emerging from dendrites that receive excitatory input. The process of spine morphogenesis occurs both in the developing brain and during synaptic plasticity. Molecules regulating the cytoskeleton are involved in spine formation and maintenance. Here we show that reverse signaling by the transmembrane ligands for Eph receptors, ephrinBs, is required for correct spine morphogenesis. The molecular mechanism underlying this function of ephrinBs involves the SH2 and SH3 domain–containing adaptor protein Grb4 and the G protein–coupled receptor kinase–interacting protein (GIT) 1. Grb4 binds by its SH2 domain to Tyr392 in the synaptic localization domain of GIT1. Phosphorylation of Tyr392 and the recruitment of GIT1 to synapses are regulated by ephrinB activation. Disruption of this pathway in cultured rat hippocampal neurons impairs spine morphogenesis and synapse formation. We thus show an important role for ephrinB reverse signaling in spine formation and have mapped the downstream pathway involved in this process.

Serine phosphorylation of ephrinB2 regulates trafficking of synaptic AMPA receptors

Plasticity in the brain is essential for maintaining memory and learning and is associated with the dynamic membrane trafficking of AMPA receptors. EphrinB proteins, ligands for EphB receptor tyrosine kinases, are transmembrane molecules with signaling capabilities that are required for spine morphogenesis, synapse formation and synaptic plasticity. Here, we describe a molecular mechanism for ephrinB2 function in controlling synaptic transmission. EphrinB2 signaling is critical for the stabilization of AMPA receptors at the cellular membrane. Mouse hippocampal neurons from conditional ephrinB2 knockouts showed enhanced constitutive internalization of AMPA receptors and reduced synaptic transmission. Mechanistically, glutamate receptor interacting proteins bridge ephrinB ligands and AMPA receptors. Moreover, this function involved a regulatory aspect of ephrinB reverse signaling that involves the phosphorylation of a single serine residue in their cytoplasmic tails. In summary, our findings uncover a model of cooperative AMPA receptor and ephrinB reverse signaling at the synapse.

Loss of PHD3 allows tumours to overcome hypoxic growth inhibition and sustain proliferation through EGFR

Solid tumours are exposed to microenvironmental factors such as hypoxia that normally inhibit cell growth. However, tumour cells are capable of counteracting these signals through mechanisms that are largely unknown. Here we show that the prolyl hydroxylase PHD3 restrains tumour growth in response to microenvironmental cues through the control of EGFR. PHD3 silencing in human gliomas or genetic deletion in a murine high-grade astrocytoma model markedly promotes tumour growth and the ability of tumours to continue growing under unfavourable conditions. The growth-suppressive function of PHD3 is independent of the established PHD3 targets HIF and NF-κB and its hydroxylase activity. Instead, loss of PHD3 results in hyperphosphorylation of epidermal growth factor receptor (EGFR). Importantly, epigenetic/genetic silencing of PHD3 preferentially occurs in gliomas without EGFR amplification. Our findings reveal that PHD3 inactivation provides an alternative route of EGFR activation through which tumour cells sustain proliferative signalling even under conditions of limited oxygen availability.

In-vivo high resolution AFM topographic imaging of Caenorhabditis elegans reveals previously unreported surface structures of cuticle mutants

Atomic force microscopy (AFM) is a powerful method for topographic imaging of surfaces with nanometer resolution. AFM offers significant advantages over scanning electron microscopy (SEM) including the acquisition of quantitative 3D-images and biomechanical information. More importantly, for in-vivo biological imaging, AFM does not require sample dehydration/labeling. We show for the first time high-resolution topographical images of the cuticle of the model organism C. elegans under physiological conditions using AFM. C. elegans is used extensively for drug screening and to study pathogen adherence in innate immunity; both applications highly depend on the integrity of the nematodes cuticle. Mutations affecting both drug adsorption and pathogen clearance have been proposed to relate to changes in the cuticle structure, but never visually examined in high resolution. In this study we use AFM to visualize the topography of wild-type adult C. elegans as well as several cuticle collagen mutants and describe previously unseen anatomical differences.

Natural Infection of C. elegans by an Oomycete Reveals a New Pathogen-Specific Immune Response

In its natural habitat, the nematode Caenorhabditis elegans encounters a plethora of other organisms, including many that are pathogenic [1, 2]. The study of interactions between C.xa0elegans and various pathogens has contributed to characterizing key mechanisms of innate immunity [2-4]. However, how C.xa0elegans recognizes different pathogens to mount pathogen-specific immune responses remains still largely unknown [3, 5-8]. Expanding the range of known C.xa0elegans-infecting pathogens and characterizing novel pathogen-specific immune responses are key steps toward answering this question. We report here that the oomycete Myzocytiopsis humicola is a natural pathogen of C.xa0elegans, and we describe its infection strategy. We identify axa0new host immune response to pathogen exposure that involves induction of members of a previously uncharacterized gene family encoding chitinase-like (CHIL) proteins. We demonstrate that this response is highly specific against M.xa0humicola and antagonizes the infection. We propose that CHIL proteins may diminish the ability of the oomycete to infect by hindering pathogen attachment to the host cuticle. This work expands our knowledge of natural eukaryotic pathogens of C. elegans and introduces a new pathosystem to address how animal hosts recognize and respond to oomycete infections.

Activation of RHO-1 in cholinergic motor neurons competes with dopamine signalling to control locomotion

The small GTPase RhoA plays a crucial role in the regulation of neuronal signalling to generate behaviour. In the developing nervous system RhoA is known to regulate the actin cytoskeleton, however the effectors of RhoA-signalling in adult neurons remain largely unidentified. We have previously shown that activation of the RhoA ortholog (RHO-1) in C. elegans cholinergic motor neurons triggers hyperactivity of these neurons and loopy locomotion with exaggerated body bends. This is achieved in part through increased diacylglycerol (DAG) levels and the recruitment of the synaptic vesicle protein UNC-13 to synaptic release sites, however other pathways remain to be identified. Dopamine, which is negatively regulated by the dopamine re-uptake transporter (DAT), has a central role in modulating locomotion in both humans and C. elegans. In this study we identify a new pathway in which RHO-1 regulates locomotory behaviour by repressing dopamine signalling, via DAT-1, linking these two pathways together. We observed an upregulation of dat-1 expression when RHO-1 is activated and show that loss of DAT-1 inhibits the loopy locomotion phenotype caused by RHO-1 activation. Reducing dopamine signalling in dat-1 mutants through mutations in genes involved in dopamine synthesis or in the dopamine receptor DOP-1 restores the ability of RHO-1 to trigger loopy locomotion in dat-1 mutants. Taken together, we show that negative regulation of dopamine signalling via DAT-1 is necessary for the neuronal RHO-1 pathway to regulate locomotion.

Three-dimensional behavioural phenotyping of freely moving C. elegans using quantitative light field microscopy

Behavioural phenotyping of model organisms is widely used to investigate fundamental aspects of organism biology, from the functioning of the nervous system to the effects of genetic mutations, as well as for screening new drug compounds. However, our capacity to observe and quantify the full range and complexity of behavioural responses is limited by the inability of conventional microscopy techniques to capture volumetric image information at sufficient speed. In this article we describe how combining light field microscopy with computational depth estimation provides a new method for fast, quantitative assessment of 3D posture and movement of the model organism Caenorhabditis elegans (C. elegans). We apply this technique to compare the behaviour of cuticle collagen mutants, finding significant differences in 3D posture and locomotion. We demonstrate the ability of quantitative light field microscopy to provide new fundamental insights into C. elegans locomotion by analysing the 3D postural modes of a freely swimming worm. Finally, we consider relative merits of the method and its broader application for phenotypic imaging of other organisms and for other volumetric bioimaging applications.

Atomic force stiffness imaging: capturing differences in mechanical properties to identify and localize areas of prostate cancer tissue.

Prostate cancer is now the most commonly diagnosed cancer in men in western countries. Due to the difficulty for early detection, there are an estimated 10000 deaths a year in the UK from prostate cancer alone; whereby the only curative option is interventional treatment that aims to excise all diseased cells while preserving the neurovascular bundle. To date, several studies have shown that the mechanical properties of cancer cells and tissues i.e. adhesion, stiffness, roughness and viscoelasticity are significantly different from benign cells and regions of tissue that are healthy. Building upon these results, we believe novel methods of imaging the mechanical properties of prostate cancer samples can provide new surgical intervention opportunities beyond what is possible through vision alone. In this paper, we used an Atomic Force Microscope (AFM) to measure the stiffness and topography variations correlating to regions of prostate cancer at the surface of an excised sample at a cellular level. Preliminary results show that by using an AFM we can detect structural differences in non-homogeneous tissue samples, confirming previous results that cancerous tissues appear stiffer than benign areas. Through these results, we aim to develop a stiffness imaging protocol to aid the early detection of prostate cancer, in addition to force sensing surgical tools.