Author: Katarzyna Nowacka

Poster prizes at ‘Defining and defeating metastasis’ – meet the winners!

We are excited to present the poster prizes awarded at the recent EMBO | EMBL Symposium  ‘Defining and defeating metastasis’,  hosted at the Advanced Training Centre in Heidelberg and bringing together researchers from diverse fields to enhance our understanding of the dissemination and metastatic colonisation of tumour cells. It provided a unique opportunity for interdisciplinary exchange on current approaches and future collaborations on metastasis and its therapeutic challenges. As with most events this year, for many participants this was the first onsite meeting that they attended since early 2020 which made it very special. It was a fantastic opportunity to meet in person for the three days full of exciting science, exchanging ideas, presenting latest research, catching up with old friends and making new ones. There were two live poster sessions during which the presenters could discuss their research– their work was then voted for by other attendees and speakers. We are pleased to be able to share with you the research from four out of six winners of the conference prizes: congratulations to all!

Jagged-1 promotes breast cancer metastasis through the lymphatic system

Presenter: Benjamin Gordon

Benjamin Gordon – University of Illinois at Chicago College of Medicine, USA
Abstract

While early detection of breast cancer (BC) has improved prognoses, there is an urgent need to improve outcomes for patients with distant metastatic disease. Higher expression of the Notch ligand JAG1 in primary BC tumors is strongly associated with lymph node metastasis and patient mortality, but causality is unclear. We show that JAG1 expression is higher in metastatic BC cells colonizing lymph nodes than in primary tumors, suggesting that tumor cells with high JAG1 are preferentially able to metastasize to lymph nodes. JAG1 expression is higher in a derivative of BC line MDA MB 231 selected for tropism to lymph nodes (MDA231 LN) than in the parental line or derivatives with other tropisms. To determine the mechanism(s) of JAG1 mediated metastasis, we generated clonal JAG1 knockout cell lines from MDA231 LN cells with corresponding JAG1 rescue lines. We investigated the role of JAG1 in spontaneous metastasis under clinically relevant conditions by orthotopically implanting JAG1 knockout and expressing cells, resecting the primary tumor, and following long term metastatic spread in a mouse model. Quantification of tumor cells in blood showed that survival, metastatic burden, and JAG1 expression did not correlate with the number of circulating tumor cells. Conversely, JAG1 expression drove an increase in lymph node and body wide metastatic burden and a trend toward decreased survival. In this model, metastatic cells were abundant throughout lymph vessels, suggesting lymphatics are the primarily route of dissemination. Preliminary transcriptional analysis suggests that JAG1 alters interactions with lymphatic endothelial cells (LEC), leading us to examine downstream events in co cultures of LEC with lymphatically invasive BC lines. Deciphering tumor lymphatic endothelial signaling events may open new avenues to target BC metastasis.

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Poster Prize from the EMBO Journal

Biphasic contribution of platelets to metastasis

Presenter: María J. García-León

Maria J. Garcia-Leon – INSERM UMR_S1109, Tumor Biomechanics, Université de Strasbourg, France
Abstract

Metastasis still remains elusive to treatment, with an overwhelming mortality rate of 90%. Accumulating evidence indicates that metastatic potential of circulating tumor cells (CTCs) can be tuned by intravascular components, including platelets. Platelet depletion impairs metastasis, which can be rescued upon platelet transfusion. Mechanistically, CTCs rapidly bind, activate and aggregate platelets, events that are crucial for the arrest, survival, and extravasation of the former. The current dogma states that platelets tune metastasis by impacting CTCs behaviour at early stages of metastatic seeding. However, whether platelets can regulate metastasis at later stage and which receptors may be involved remains unknown. In this study, we first documented the efficiency of platelet binding to a large panel of metastatic TCs and observed that not all recruit or aggregate platelets with the same efficiency. Interestingly, such binding impacts their intravascular fate by favoring their arrest, as observed in a combination of experimental metastasis models in thrombocytopenic (TCP) mice and zebrafish embryo. Using longitudinal imaging of metastatic seeding and growth in TCP mice at unprecedented spatial and temporal resolution, we demonstrated that binding and aggregation correlates with their metastatic potential in vivo. Additionally, by the dynamic in vivo tracking of TCs in the lungs of fully TCP mice, and the quantification of platelets depositions around arrested CTCs at seeding and late metastatic outgrowth, we showed that early platelet binding, aggregation, clot formation, and the subsequent increased adhesion and survival at lung microvessels, are capital but not exclusive factors increasing TC metastatic fitness. We observed that platelets contribute to late steps of metastatic outgrowth by experimentally interfering with platelet counts in animals already carrying metastatic foci. Doing so, we observed that platelets tune the growth of established foci, independently of their early intravascular interaction with CTCs. Finally, we have identified the platelet collagen receptor GPVI as key in this late modulation of metastatic outgrowth, suggesting its targeting in specific cancer types as a promising adjuvant therapy in oncologic patients to stop the metastatic progression.

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Poster Prize from EMBO Molecular Medicine

Colonic fibroblasts in tissue homeostasis and cancer

Presenter: Michael Brügger

Michael Brügger – University of Zürich, Switzerland
Abstract

Colorectal cancer (CRC) is among the most prevalent cancers in Switzerland (2nd in women 3rd in men, BFS statistics 2013 2017) and worldwide (3rd in women and men). More than half of the patients diagnosed with CRC either harbour metastases or will develop metastatic disease, which is the primary cause of death for CRC patients. There is therefore a dire need for new therapies. These must be guided by a better understanding of the metastatic process. We are only now starting to appreciate the contribution of not only tumour cells themselves, but also the non tumour stromal cells of the tumour microenvironment (TME) to tumour growth, progression and metastasis. To understand how non tumour stromal cells are changed in CRC it is integral to first characterize their identity and functions during colonic homeostasis.
To describe the stromal cell populations in an unbiased manner, we carried out a single cell transcriptome analysis of the adult murine colon, producing a high quality atlas of matched colonic epithelium and mesenchyme. We identify two crypt associated colonic fibroblast populations that are demarcated by different strengths of platelet derived growth factor receptor A (Pdgfra) expression. Crypt bottom fibroblasts (CBFs), close to the intestinal stem cells, express low levels of Pdgfra and secrete canonical Wnt ligands, Wnt potentiators, and bone morphogenetic protein (Bmp) inhibitors. Crypt top fibroblasts (CTFs) exhibit high Pdgfra levels and secrete noncanonical Wnts and Bmp ligands. While the Pdgfralow cells maintain intestinal stem cell proliferation, the Pdgfrahigh cells induce differentiation of the epithelial cells. Notably, these cell populations are conserved in the human colon.
Recently, we established a murine model of metastatic colorectal cancer, based on the orthotopic endoscopy guided injection of cancer organoids (colonic organoids harbouring mutations in APC, Kras, Tp53 and Smad4). In this context we study how the abovementioned fibroblast populations are affected by the primary tumour and how they in turn affect tumour progression.

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Single cell transcriptomic profiling of brain metastatic founders in small cell lung cancer patient derived models to identify potential vulnerabilities

Presenter: Maria Peiris-Pagès

Maria Peiris Pages – Cancer Research UK Manchester Institute, UK
Abstract

Background: Brain metastasis is a major cause of patient morbidity and mortality in small cell lung cancer (SCLC) with an ~80% incidence during disease progression, contributing to the dismal 5 year survival rate of <7%. Mechanisms underpinning SCLC brain metastasis are understudied due to scarcity of brain biopsies and preclinical models. We have developed a biobank of >60 circulating tumour cell (CTC) derived patient explant models of SCLC in immunodeficient mice (CDX) where brain metastasis is routinely observed upon resection of the subcutaneous (S.C) tumour
Methods: We developed an in vivo S.C tumour resection workflow in brain tropic CDX3P to isolate single CTCs, early brain founder tumour cells and subsequent established brain metastases. Following FACS of CDX cells from dissociated mouse brain (using a human CD147 antibody) we performed single cell RNA sequencing (scRNAseq) to reveal potential molecular regulators hypothesised to support brain metastatic founding and subsequent colonisation
Results: Brain metastases were detectable in CDX3P on average 195 days after S.C implantation (study length 174 230 days). We analysed 58 single CTCs (n=6 mice, 191 230 days) and 214 brain metastatic founder cells (n=2 mice, 205 218 days) by scRNAseq. Bioinformatics analyses defined transcriptomic features underpinning single cell heterogeneity and identified sub populations within CTCs and metastatic founders indicative of brain tropic CTC sub clones. We also characterised molecular features unique to brain founders as candidates that could serve as therapy targets
Future Tissue expression of candidate genes of brain metastatic founding will be validated in CDX and patient samples. Genetic manipulation of CDX cells ex vivo combined with pharmacological approaches will be used to explore their roles in metastatic seeding and to identify potential vulnerabilities. Transcriptomic analysis of cells from established brain metastases obtained from the above in vivo protocol will be conducted to explore molecular programs of brain colonisation. Combined, these data will contribute to our long term goal of identifying novel therapeutic strategies that may ultimately improve the quality of life for the significant number of patients with SCLC who present with or subsequently develop brain lesions.

Due to the confidentiality of the unpublished data, we cannot share the poster.

Effective treatment of colorectal peritoneal metastases by exploiting a molecular subtype specific vulnerability

Presenter: Sanne Bootsma

Sanne Bootsma, Amsterdam UMC, The Netherlands
Abstract

In colorectal cancer, peritoneal metastases (PMs) associate with severe morbidity and dismal prognosis. Given the incidence of this disease and the lack of adequate treatments currently available, PMs pose a large unmet clinical need. Although PMs can be accompanied by more widespread metastatic disease, it often occurs as the only sign of dissemination. This implies that the route of metastatic spread to the peritoneum differs from that to distant organs. PMs are thought to result from cancer cells that spill into the abdominal cavity, and are able to attach to the peritoneal lining and form tumor deposits. This cascade places specific demands on the cancer cells.
Here, we report that colorectal cancers that present with PMs almost universally classify as consensus molecular subtype 4 (CMS4). This previously recognized disease entity is characterized by mesenchymal features, poor prognosis, and resistance to therapies currently used against peritoneal metastases, which explains their limited efficacy. By leveraging disease models that capture CMS4 specific features, including the ability to form PMs in vivo, we identified elesclomol as a highly effective agent. Elesclomol kills cancer cells in a copper dependent fashion by targeting the oxidative phosphorylation machinery, which we found to be a specific vulnerability of CMS4 cancers. Elesclomol Cu2+ was effective following only minutes of exposure to CMS4 cell lines and organoids, supporting its use in intra abdominal treatment procedures. It is therefore a promising candidate for the local treatment of peritoneal metastases of colorectal cancer.

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Poster Prize from Metastasis Research Society

 

The remaining prize was:

Short talk Prize from Metastasis Research Society: Eric Rahrmann – University of Cambridge, UK

 

Congratulations to all six winners!

The EMBO | EMBL Symposium ‘Defining and defeating metastasis’ took place from 19 – 22 June 2022 at EMBL Heidelberg and was streamed online for virtual participants.

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‘Building networks: engineering in vascular biology’ – check out the awarded posters!

There were 5 poster prizes awarded at the recent EMBO Workshop ‘Building networks: engineering in vascular biology’,  hosted by EMBL Barcelona at the Barcelona Biomedical Research Park (PRBB) and bringing together vascular biologists and bioengineering researchers from across Europe and beyond, who are pioneering new tools towards understanding vascular biology in health and disease. For many participants this was the first on-site meeting that they attended since early 2020 and the spirits were high during the three days full of exciting science, exchanging ideas, presenting latest research, catching up with old friends and making new ones.
We also didn’t have any complaints regarding outdoor coffee breaks on a sunlit deck overlooking the sea! There were two live poster sessions during which the presenters could discuss their research (over snacks and drinks!) – their work was then voted for by other attendees and speakers. We are pleased to be able to share with you the research from the five winners of the best poster prizes: congratulations to Claire, Akinola, Irene, Nensi and Anjali!

Contact guidance of vascular endothelial cells on microgrooved substrates: influence of groove dimensions and cell density

Presenter: Claire Leclech

Claire Leclech, LadHyX, CNRS, École Polytechnique, Institut Polytechnique de Paris, France
Abstract

In healthy arteries, endothelial cells (ECs) exhibit different morphologies: elongated and aligned in the direction of blood flow or more cuboidal in regions of arterial branches and bifurcations. Factors that regulate EC morphology and alignment are of interest, particularly in light of the observation that atherosclerotic lesions preferentially form in regions where ECs are less aligned and elongated. In vivo, the basement membrane to which ECs adhere is a patterned and topographic surface. We are interested in how this substrate topography may regulate EC shape and alignment and are exploring these questions in vitro using microfabricated surfaces.
When cultured on substrates composed of parallel arrays of microgrooves, ECs align and elongate in the direction of the grooves, a process called contact guidance. We show that we can control the extent of this contact guidance by modulating the groove dimensions (spacing, width, and depth). In particular, we demonstrate that increasing groove depth (from 1 to 6 μm) leads to the most pronounced cell elongation and alignment. We also investigate the influence of cell density on the response to microgrooves by comparing the response of individual cells to monolayers of low or high density. Interestingly, we observe progressive loss of cell alignment and elongation on microgrooves for increasing cell density/culture time, associated with remodeling of the actin cytoskeleton and focal adhesions (FAs).
We are investigating the mechanisms underlying this depth- and density-dependent response of ECs to the microgrooves and propose that a competition between cell-substrate and cell-cell adhesion may explain the existence of different mechanisms. In individual cells, the depth-dependent response predominates, driven by FA clustering and protrusion dynamics, while in highly confluent monolayers, ECs respond primarily to the secreted basement membrane and lose the response to substrate topography.
Beyond highlighting fundamental mechanisms of shape modulation and contact guidance in ECs, the results of this study can also prove useful in the field of implantable endovascular devices where surface topographic functionalization may constitute a promising strategy for improving device efficacy.

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Engineering 3D vascularized cardiac microtissues on-chip

Presenter: Akinola Akinbote

Akinola Akinbote, EMBL Barcelona, Spain
Abstract

Coronary microvessels are implicated in many cardiovascular diseases (CVDs) and their dysfunction is associated with adverse clinical outcomes. These outcomes vary by biological sex and are hypothesized to differ based on estrogen’s cardioprotective effects. However, endothelial-dependent contributions to CVDs and sex-based differences are still largely unexplored, in part due to inadequate models of the cardiac microvasculature. The advent of iPSC-derived Cardiomyocytes (CMs) has resulted in a growth in cardiac models. 3-dimensional cultures and co-cultures with non-myocyte populations, such as cardiac fibroblasts and endothelial cells (ECs), have also been shown to improve cardiomyocyte maturation in vitro (marked by improved calcium handling and metabolic maturation); yet the impact of beating cardiomyocytes on cardiac microvascular remodelling and barrier function is not well understood. By employing microfluidic models of microvessels and iPSC-derived cardiac spheroids we can explore these complex CM-non-myocyte interactions in a controlled and quantifiable manner. We are generating adult-derived cardiac microvessels integrated with cardiac organoids to reveal the contribution of 1) non-myocytes (cardiac fibroblasts and endothelium) to cardiomyocyte function and 2). the effect of beating cardiac organoids on local microvascular remodelling. This unique vascularized cardiac model will be useful for understanding complex vascular-myocyte interactions and may provide clues to the role of sex hormones in promoting both endothelial and cardiac function.

Due to the confidentiality of the unpublished data, we cannot share the poster.

Temporal adaptation of vascular patterning

Presenter: Irene M. Aspalter

Irene Aspalter, Cell Adaptation Laboratory, The Francis Crick Institute, UK
Abstract

Sprouting angiogenesis is highly dependent on effective decision making between endothelial cells (ECs). The feedback between Vegf/Dll4/Notch is well established during the collective selection of tip cells of new vessel sprouts.
Our prior work demonstrates that additional signals (e.g. sema3E-plexinD1) can alter the tip cell selection speed by acting as time-keepers during Dll4/Notch signalling. Changes to the timing of this process alters the vascular network density.
It was believed that tip cells form filopodia post selection, aiding the migration towards Vegf. However, our recent in silico models, validated in vivo, show that ECs form filopodia first, irrespectively of whether they are selected as tip. Our simulations predict that filopodia speed up tip cell selection by moving Vegf-receptors towards the Vegf source, creating a sensory-motor-feedback that speeds up Dll4 production. Indeed, my preliminary in vitro data shows Vegf-receptors at the tip of filopodia. This suggests a vital role of filopodia as time-keepers during tip cell selection.
We aim to better understand the role of filopodia and other time-keepers to fine tune vascular patterning and network topologies.
Using microcontact printing I am developing a method to closely investigate the tip cell selection timing while modulating the involvement of filopodia and other pathways. Thin printed lines of extra cellular matrix allow ECs to interact, but not to swap positions, prohibiting disruption of Notch patterns in order to observe when stable patterns establish.
pERK has been previously shown as suitable tip cell marker in zebrafish and is also a useful marker in my system. Using pERK as readout, my preliminary data shows different selection patterns of tip/stalk cells in the presence or absence of Notch inhibitors, and we are currently developing an analysis pipeline for robust quantification.
This system will be used to carefully dissect the mechanism by which filopodia influence tip/stalk cell selection, with the help of molecular manipulation (growth factors/inhibitors) and micro manipulation (photo-activatable probes).
Our work will shed new light on the tip cell selection process and will offer new targets for therapeutic approaches targeting temporal regulation of vascular patterning, network topology and branching density.

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Resolving vascular endothelial junctions with correlative fluorescent light microscopy and cryo-electron tomography (cryo-ET)

Presenter: Nensi Alivodej

Nensi Alivodej, Max Planck Institute for Brain Research / Goethe University Frankfurt, Germany
Abstract

Endothelial cells (ECs) form the inner lining of blood vessels, where they adhere to one another via junctional complexes, namely adherens and tight junctions, to regulate the integrity and permeability of the vascular barrier. These junctions are critical for tissue development and homeostasis and are structurally different across organs. For example, ECs of the brain possess strong tight junctions that are central to the formation of the blood-brain-barrier while ECs of the lung rely on adherens junctions to maintain vascular permeability at the interface of the blood-air barrier.
While the molecular composition of adherens and tight junctions is well studied, less is known about the assembly of junctional proteins in their native environment and their interaction with the cytoskeleton. Past cryo-electron tomography (cryo-ET) studies have been unsuccessful in resolving the structure of these junctions in situ owing to the difficulty in identifying them under cryo-electron microscopy. To circumvent this limitation, we have established a pipeline employing state-of-the-art correlative fluorescent light microscopy and cryo-ET in order to resolve junctions in ECs. We have isolated ECs from different organs, including the lung, brain and aorta, in mice expressing an EGFP-tagged VE-cadherin knock-in protein and have successfully grown them on electron microscopy (EM) grids. After cryo-fixation via plunge-freezing, we identified areas of interest expressing the fluorescently-tagged VE-cadherin using light microscopy and correlated those areas to EM images for localizing junctions. The areas were then processed to obtain a series of transmission EM images at multiple angles in order to reconstruct the three-dimensional organization of the junctions. This pipeline has allowed us to obtain the very first EM images of in situ adherens junctions from wholly preserved lung ECs, where individual VE-cadherin proteins, auxiliary adapter proteins and underlying cytoskeleton can be visualized with a pixel size of 0.22 nm. We are optimizing our workflow to also resolve tight junctions of the blood-brain-barrier. Altogether, we have established a scalable pipeline to characterize and compare the tissue-specific structural organization of vascular junctions with unprecedented sub-nanometer resolution.

Due to the confidentiality of the unpublished data, we cannot share the poster.

Geometry of self-assembled DNA nanostructures influences in-vitro angiogenesis in HUVECs

Presenter: Anjali Rajwar

Anjali Rajwar, Indian Institute of Technology Gandhinagar, India
Abstract

DNA nanotechnology involves fabricating small strands of DNA to design nano-objects in 1D, 2D and 3D with precise control of shape and size that have been utilized in many applications.
DNA nanostructures have been investigated for their ability to influence cellular behaviour and functions. Recently, new emergent functionalities of DNA nanodevices as a class of biomaterials with immense capacity to interface with biological systems and vast potential in disease diagnosis and therapeutics have emerged. DNA nanostructures, which are chemically robust and biocompatible in nature, have been surface modified and structurally fine-tuned to find emerging applications in stem cell therapy and tissue regeneration. DNA nanostructures can be used for therapeutic angiogenesis, which involves the formation of new blood vessels, and can be used to treat ischemic diseases such as stroke or heart failure. This study looks at how the structural topology of DNA nanostructures affects their ability to stimulate endothelial cell angiogenesis.
We examined the potential of four different DNA nanostructure geometries on the differentiation of human umbilical vein endothelial cells (HUVECs). While different DNA nanostructure geometries successfully induced angiogenesis and cell migration in HUVECs, tetrahedral DNA cages demonstrated the greatest uptake and angiogenesis potential, indicating that not only the composition of materials, but also the 3D arrangement of ligands may play a role in stimulating the angiogenesis process.
Taken together, this research can lay the groundwork for future studies involving DNA nanocages for biological and biomedical applications, explicitly applying their surface topologies in bioimaging, drug delivery, immune activation, and tissue engineering.

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Congratulations to all five winners!

The EMBO Workshop ‘Building networks: engineering in vascular biology’ took place from 9 – 11 May 2022 at EMBL Barcelona.

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Best poster prizes at ‘Inter-organ communication in physiology and disease’

Almost 40 posters were presented at the virtual EMBO | EMBL Symposium ‘Inter-organ communication in physiology and disease’, showcasing how studies in model organisms are revealing novel inter-organ signals that contribute to whole-organism homeostasis. There were two live poster sessions and the presenters could also be contacted via chat, message or video call throughout the conference – their work was then voted for by other attendees and speakers. We are pleased to be able to share with you the research from the three winners of the best poster prizes: congratulations to Emily, Wilson and Maxim!

1st prize: Sex and reproductive differences in gut tumours

Presenter: Emily Strachan

Due to data protection regulations, we cannot publish the poster.

2nd prize: A protective neuro-adipose tissue axis against malaria

Presenter: Temitope Wilson Ademolue

Temitope Wilson Ademolue
Temitope Wilson Ademolue, Instituto Gulbenkian de Ciência, Portugal
Abstract

Infections lead to the development of host sickness behavior. This evolutionarily conserved response includes the withdrawal of the infected host from food, known as anorexia. This behavior limits the exogenous supply of metabolic substrates; and in the absence of a countervailing metabolic response, anorexia can lead to starvation and death. We reasoned that the infected host relies on the mobilization of stored metabolic substrates to minimize the deleterious effects of anorexia. Presumably, this is required to sustain vital metabolic processes compatible with survival. Using a non lethal rodent model of malaria, we show that Plasmodium infected mice progressively developed anorexia of infection. This was coupled to a profound loss of white adipose tissue via adipose tissue triglyceride lipase (Atgl) driven lipolysis; and was associated with the mobilization of free fatty acids, induction of ketogenesis, and triglyceride synthesis. The Inhibition of adipose tissue lipolysis via Atgl deletion specifically in adipocytes increased malaria mortality. This was associated with compromised thermoregulation and the collapse of organismal energy expenditure, indicating that mobilization of fat stores via lipolysis is essential to survive Plasmodium infection. Mechanistically, malaria induced lipolysis is controlled by the central nervous system as demonstrated using chemo genetically sympathectomized mice, lacking the peripheral arm of the sympathetic nervous system. In the absence of sympathetic outflow, Plasmodium infected mice failed to mobilize adipose tissue depots and to maintain energy expenditure, succumbing to malaria. In conclusion, we show that survival from malaria relies on metabolic response and adaptation that is coordinated by the sympathetic nervous system.

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3rd prize: Skeletal muscle-derived IL-6 orchestrates physiological adaptations in the context of inflammation

Presenter: Maxim Nosenko

Maxim Nosenko, Trinity College Dublin, Ireland
Abstract

Survival during infections relies on multiple resistance and tolerance mechanisms and requires the most adaptive response being launched based on the disease setting. While many immunological pathways, distinguishing pathogen type and localization, have been discovered, less is known about the mechanisms, sensing different stages of the infection, especially transition from local to systemic disease. The latter can be life threatening in situations such as sepsis or severe COVID 19, and thus tolerance mechanisms become crucial. Inflammatory cytokines, including IL 6, are believed to govern both immune response and adaptation in the context of infection. However, the mechanisms of tolerance induction are still poorly investigated.
To address this question, we employed a mouse model of LPS induced sepsis and analysed physiological response of the animals as well as proinflammatory cytokines production. Administration of LPS resulted in fatigue, hypoglycaemia, and hypothermia, that were associated with predominant accumulation of IL 6 in the blood. To address the role of IL 6 we employed knock out mice and observed a significant suppression of fatigue, increased blood glucose level and core temperature. Surprisingly, conditional inactivation of IL 6 in myeloid cells did not affect systemic cytokine accumulation in response to LPS. We next performed screening across tissues and found only skeletal muscles showing high expression of IL 6 gene in response to LPS, independently of IL 6 production by myeloid cells. Further investigation revealed a key role of IL 6 in reprogramming of glycogen metabolism in the liver upon LPS challenge, resulting in hypoglycaemia. Recent studies indicated the positive effect of hypoglycaemia during bacterial sepsis, suggesting that IL 6 mediates adaptation to this condition.
Altogether, we hypothesize that skeletal muscles are key systemic inflammation sensing organs, responsible for accumulation of IL 6 and induction of physiological adaptations. Further investigation of the role of IL 6 in the context of local and systemic inflammation could bring novel therapeutic opportunities for induction of the tolerance in life threatening infectious diseases.

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Congratulations to all three winners!

The EMBO | EMBL Symposium ‘Inter-organ communication in physiology and disease’ took place from 21 – 23 March 2022.

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Best poster prizes at ‘Recent advances in structural biology of membrane proteins’

Over 60 posters were presented at the virtual EMBO Workshop ‘Recent advances in structural biology of membrane proteins’, highlighting the importance of lipids in MP research and how they can be preserved. There were two live poster sessions and the presenters could also be contacted via chat, message or video call throughout the conference – their work was then voted for by other attendees and speakers. We are pleased to be able to share with you the research from the three winners of the best poster prizes: congratulations to Kevin, Natalie and Karthik!

Cryo-EM Snapshots of Nanodisc-Embedded Native Eukaryotic Membrane Proteins

Presenter: Kevin Janson

Kevin Janson, Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Germany
Abstract

New membrane complex purification technologies in combination with cryo electron microscopy (cryoEM) recently allowed the exploration of near native membrane protein complex architectures. Polymer nanodiscs in particular provide the basis to study overexpressed membrane proteins at high resolution while retaining protein—protein and protein—lipid interactions. However, how the majority of endogenous membrane proteins are organized remains elusive, mainly due to the inherent complexities that a hydrophobic environment poses to biochemical preparations.
In this work, we combined biochemical enrichment protocols for native membrane complexes together with amphiphilic polymers to increase the quality of recovered endogenous membrane complexes. The derived protein encapsulated nanodiscs were identified by mass spectrometry and imaged with cryoEM. This set of technologies is applied to Chaetomium thermophilum, a thermophilic fungus, that confers additional advantages for protein structure determination due to the increased thermal stability of its biomolecular assemblies.
Our results show a highly efficient recovery of protein encapsulating nanodiscs, amenable to structural and biophysical characterization with a multitude of methods. Initial mass spectrometry results reveal ~1300 proteins while multiple 2D class averages from cryoEM data show prominent nanodisc embedded structural signatures. This combined methodological approach to isolate multiple endogenous membrane protein complexes provides unprecedented opportunities for a deeper understanding of the membrane proteome of a eukaryote.

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Structural studies and inhibition of essential pathogenic secondary active transporters with synthetic nanobodies and solid supported membrane electrophysiology

Presenter: Natalie Bärland

Natalie Bärland, University of Basel, Switzerland
Abstract

Single domain antibodies (nanobodies) have been extensively used in mechanistic and structural studies of membrane proteins. Additionally, to their traditional use in aiding structural elucidation they harbour a great potential as tools for clinical therapies. Nanobodies are specifically selected to target the protein of interest with high affinities and are capable of trapping specific states with inhibitory potential. A high throughput identification of inhibitory nanobodies is essential for subsequent studies and often challenging if a technique consumes large amounts of sample or if the required labelling for their characterisation is costly. Solid supported membrane (SSM) electrophysiology has been a fast way to screen and characterise electrogenic transport of compounds and can be expanded for the identification of putative nanobody inhibitors. Here we show a combination of structural studies assisted by synthetic nanobodies (sybodies), selected by using SSM electrophysiology to screen and identify inhibitors. As our model protein we used the secondary active transporter LicB, which is a choline importer essential for the survival of the pathogen Streptococcus pneumoniae in the host. S. pneumoniae is a highly invasive pathogen that can exhibit multiple resistances to antimicrobials and remains to be a burden for society. The methods described here can be applied for the characterization of any electrogenic transporter. It can help to screen and select with a high throughput putative inhibitory sybodies and help in the development of novel drugs and drug targets.

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Structural and functional properties of a magnesium transporter of the SLC11/NRAMP family

Presenter: Karthik Ramanadane

Karthik Ramanadane, University of Zurich, Switzerland
Abstract

Divalent transition metals are essential nutrients whose uptake is mediated by membrane transporters of the SLC11/NRAMP family. Over the last 25 years, family members have been characterized as proton coupled divalent transition metal symporters, a property, which was later supported in structural and functional studies of prokaryotic homologues. In this study, we have combined the phylogenetic analysis of SLC11 proteins with their biochemical characterization to show that the transition metal ion transporters only represent a fraction of the SLC11/NRAMP family. After extensive screening, we have identified a family member with diverging properties, which was successfully purified and reconstituted. In transport studies, we were able to classify this protein as a prokaryotic NRAMP related magnesium transporter from Eggerthella lenta (ElenNRMT). Unlike known members of the SLC11/NRAMP family, ElenNRMT does not co transport protons along with its primary substrate. These observations are partly contradicting previous studies on the substrate selectivity within this family. To gain insight into the structural basis of the distinct substrate selectivity, ElenNRMT was characterized by single particle cryoEM in absence and presence of Mg2+ at respective resolutions of 3.5 Å and 4.1 Å. Using X ray crystallography, we were able to confirm the location of the bound substrate using the anomalous scattering properties of Mn2+. In all structures, ElenNRMT adopts an inward facing conformation revealing an ion binding site that differs from classical NRAMP transporters in its volume and the distinct residues mediating ion interactions. Together, these results define the determinants of the diverse selectivity in the SLC11/NRAMP family and provide insight into its evolution.

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Congratulations to all three winners!

The EMBO Workshop ‘Recent advances in structural biology of membrane proteins’ took place from 29 November – 1 December 2021.

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