‘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

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

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

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

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

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 ‘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

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

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

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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Best Poster Awards – Precision Health

140 researchers came together recently at the EMBL Advanced Training Centre in Heidelberg, Germany, for the EMBO Workshop: Precision Health: Molecular Basis, Technology and Digital Health (13 – 16 November 2019) to present and discuss the promises and challenges of precision health and the molecular insights necessary to enable a maintenance of wellness and prevention of disease.

Out of the posters presented, 4 were awarded a poster prize based on popular vote. Here we present the poster abstracts of four of the winners.

A computational modelling approach to characterizing postprandial glucose responses in individuals
Balazs Erdos from TiFN Wageningen and MaCSBio, Maastricht University, The Netherlands, PHOTO: Balazs Erdos

Balazs Erdos (1), (2)*, Bart van Sloun (1), (2), Shauna O’Donovan (2), Michiel Adriaens (2), Natal van Riel (3), Ellen Blaak (4), Ilja Arts (2)

The large variability in the dynamic properties of the postprandial glucose response curves in individuals suggest that it is not sufficient to use average values or single time point measures of postprandial glycemia in order to characterize individuals’ glycemic control. Instead, approaches that are capable of capturing the dynamic events are necessary. In this study, we develop personalized computational models based on ordinary differential equations, to describe the glucose and insulin dynamics of individuals in response to an oral glucose tolerance test. We observed that these personalized models are capable of capturing a wide range of glucose and insulin dynamics including normal, prediabetic and type 2 diabetic responses as well as responses from intermediate states.

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(1) TiFN, Wageningen, The Netherlands, (2) Maastricht Centre for Systems Biology (MaCSBio), Maastricht University, Maastricht, The Netherlands, (3) Dept. of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands, (4) Dept. of Human Biology, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University, Maastricht, The Netherlands

*E-mail: balazs.erdos@maastrichtuniversity.nl

Predict nephrotoxicity associated with cisplatin-based chemotherapy in testicular cancer patients

Sara Garcia (1), Jakob Lauritsen (2), Zeyu Zhang (3), Mikkel Bandak (2), Marlene Danner Dalgaard (1), Rikke Linnemann Nielsen (1), Gedske Daugaard (2), Ramneek Gupta (1)

In industrialized countries, testicular cancer (TC) is the most common solid tumor in men between 20 and 40 years old and besides being one of the most treatable types of cancer, the long-term side-effects of chemotherapy are worrisome, since they are largely irreversible. Their severity is normally related to the total amount of chemotherapy received, which makes that an important factor to a successful treatment. The standard treatment for TC is 3 cycles of cisplatin, etoposide and bleomycin (BEP), being that the number of cycles can vary between 4-5 or more if the prognosis of the patient is intermediate or poor. Some of the late side-effects include nephrotoxicity, which can be measured by the drop in glomerular filtration rate after the patient follows chemotherapy. Materials and Methods: Integrative machine learning models were built using a dataset of 400 Danish individuals in order to identify clinical and/or genomics features and classify patients at higher risk of developing nephrotoxicity given a treatment of BEP-cycles. Results: First, only clinical features, such as age at the time of treatment, dose of cisplatin, patient’s prognosis, and number of cycles, were considered, and relevant features were selected to use in the classifier (AUC 0.66, SD 0.02). The classifier was then optimized by adding genomics markers, which helped improving the prediction (AUC 0.75, SD 0.02). Conclusions: Therefore, it is proposed a machine learning algorithm which, by helping predicting nephrotoxicity in advance, can benefit to improve chemotherapy efficacy in TC patients. These data driven models can also be applicable to other cancers, such as ovarian, bladder, and lung cancer where more elderly patients are at risk of nephrotoxicity and identification upfront will have direct clinical implications.

Poster currently not available

(1) Technical University of Denmark, Denmark, (2) Copenhagen University Hospital, Denmark, (3) University of Chinese Academy of Sciences, China

Loss of N-glycanase 1 alters transcriptional and translational regulation
Petra Jakob from EMBL Heidelberg, Germany, PHOTO: Petra Jakob

Petra Jakob (1), William Mueller (1), Sandra Clauder-Münster (1), Han Sun (2), Sonja Ghidelli-Disse (3), Diana Ordonez (1), Markus Boesche (3), Markus Bantscheff (3), Paul Collier (1), Bettina Haase (1), Vladimir Benes (1), Malte Paulsen (1), Peter Sehr (1), Joe Lewis (1), Gerard Drewes (3), Lars Steinmetz (1)

N-Glycanase 1 (NGLY1) deficiency is an ultra-rare, complex and devastating neuromuscular disease. Patients display multi-organ symptoms including developmental delays, movement disorders, seizures, constipation and lack of tear production. NGLY1 is a deglycosylating protein involved in the degradation of misfolded proteins retrotranslocated from the endoplasmic reticulum (ER). NGLY1-deficient cells have been reported to exhibit decreased deglycosylation activity and an increased sensitivity to proteasome inhibitors. We show that the loss of NGLY1 causes substantial changes in the RNA and protein landscape of K562 cells and results in downregulation of proteasomal subunits, consistent with its processing of the transcription factor NFE2L1. We employed the CMap database to predict compounds that can modulate NGLY1 activity. Utilizing our robust K562 screening system, we demonstrate that the compound NVP-BEZ235 (Dactosilib) promotes degradation of NGLY1-dependent substrates, concurrent with increased autophagic flux, suggesting that stimulating autophagy may assist in clearing aberrant substrates during NGLY1 deficiency.

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(1) EMBL Heidelberg, Germany, (2) Stanford University, United States of America, (3) Cellzome, Germany

Data integration for prediction of weight loss in clinically controlled dietary trials

Rikke Linnemann Nielsen (1), Marianne Helenius (1), Sara Garcia (1), Henrik Munch Roager (2), Derya Aytan (3), Lea Benedicte Skov Hansen (1), Mads Vendelbo Lind (2), Josef Vogt (1), Marlene Danner Dalgaard (1), Martin I Bahl (3), Cecilia Bang Jensen (1), Rasa Muktupavela (1), Christina Warinner (4), Vincent Appel (5), Rikke Gøbel (5), Mette B Kristensen (2), Hanne Frøkjær (6), Morten H Sparholt (7), Anders F Christensen (7), Henrik Vestergaard (5), Torben Hansen (5), Karsten Kristiansen (6), Susanne Brix Pedersen (1), Thomas Nordahl Petersen (3), Lotte Lauritzen (2), Tine Rask Licht (3), Oluf Pedersen (5), Ramneek Gupta (1)

Diet is a key strategy in weight loss management. Advances in omics technologies research allow analyses of determinants of clinical interventions outcomes. We have previously reported diet-induced weight loss in non-diabetic middle-aged Danes in two clinically controlled dietary trials where the content of whole grain or gluten was changed. However, it remains elusive how predictable weight loss is at the individual level. We here classify weight loss responders and non-responders from the whole grain and gluten trials by integrating multi-omics data (host genetics, gut microbiome, urine metabolome) together with physiology and anthropometrics into random forest models. The most predictive models for weight loss included features of diet, gut microbial species and urine metabolites (ROC-AUC:0.84-0.88, model only with diet type ROC-AUC:0.62). Furthermore, we demonstrate that a model ensemble is robust to missing information of microbiome and metabolome profiles given features of physiology (including postprandial response), host genetics and transit-time (ROC-AUC:0.72).

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(1) Technical University of Denmark, Denmark, (2) University of Copenhagen, National Food Institute, Technical University of Denmark, Denmark, (3) National Food Institute, Technical University of Denmark, Denmark, (4) Harvard University, United States of America, (5) The Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Denmark, (6) University of Copenhagen, Denmark, (7) Bispebjerg University Hospital, Denmark

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Best Poster Awards – EMBO Workshop: Tools for Structural Biology of Membrane Proteins

183 researchers convened at the Centre for Structural Systems Biology (CSSB) in Hamburg, Germany, for the recent EMBO Workshop: Tools for Structural Biology of Membrane Proteins (7 – 9 October 2019) to present and discuss new technologies and approaches applied in studying membrane protein structure, dynamics and functions.

Out of the 82 posters presented, 6 were awarded a poster prize based on popular vote. Here we present the poster abstracts of four of the winners.

Structural insights into the role of the conserved ATPase component, EccC, in the mycobacterial T7SS

Katherine Beckham is a postdoctoral fellow in Matthias Wilmanns’ group at EMBL Hamburg. PHOTO: Katherine Beckham

Authors: Katherine Beckham (1), Luciano Ciccarelli (1), Mandy Rettel (2), Mikhail Savitski (2), Jan Kosinski (1), Annabel
Parret (1), Matthias Wilmanns (1)

Mycobacteria have a unique membrane structure with a complex hydrophobic outer-membrane rich in mycolic acids. To transport substances across this impermeable barrier, mycobacteria rely on a highly specialised translocation machinery – the Type VII secretion system (T7SS). Pathogenic mycobacteria encode up to five distinct T7SSs ESX-1 to 5 [1]. Our previous work characterised the structure of the of the inner-membrane complex of the ESX-5 T7SS from Mycobacterium xenopi using negative stain electron microscopy, revealing a hexameric 1.8 MDa complex comprising the four conserved core components: EccB5, EccC5, EccD5 and EccE5 [2]. The large cytosolic domain of EccC5, an FtsK/SpoE-like ATPase, is absent in our current EM map due to its conformational flexibility, which may be required to accommodate a range of protein substrates. Our current work aims to understand the role of EccC5 in secretion. In isolation this component can oligomerise into a hexameric ring-like conformation, as observed for other ATPases in this family. In addition, chemical cross-linking of the ESX-5 complex coupled with mass spectrometry (XL-MS) supports the oligomerisation of EccC5 in the secretion complex, suggesting that it may form a channel or ‘translocation tunnel’. Thus, we propose that EccC5 may exist in two conformational states: an extended, flexible monomeric state and a more compact hexameric state. Using an integrative structural biology approach, we are combining structures of isolated proteins derived from X-ray crystallography and electron microscopy studies with XL-MS data. Together these data aim to further elucidate the secretion pathway across the mycobacterial cell envelope.

[1] Houben, E. N. G., et al. Take five — Type VII secretion systems of Mycobacteria. Biochim. Biophys. Acta – Mol. Cell Res.1843, 1707–1716 (2014).
[2] Beckham, K. S. H. et al. Structure of the mycobacterial ESX-5 type VII secretion system membrane complex by single-particle analysis. Nat. Microbiol.2, 17047 (2017).

(1) EMBL Hamburg, Germany, (2) EMBL Heidelberg, Germany

Poster currently not available

Dissection of protonation sites for antibacterial recognition and transport in QacA, a multidrug efflux transporter

Puja Majumder is a Ph.D student at the Indian Institute of Science. PHOTO: Puja Majumder

Authors: Puja Majumder (1), Shashank Khare (1), Arunabh Athreya (1), Nazia Hussain (1), Ashutosh Gulati (2), Aravind Penmatsa (1)

Emergence of multidrug-resistance poses serious threat to the society. One of the effective way by which bacteria gain drug resistance is through active efflux of antibiotics and other antibacterial compounds using multidrug efflux transporters. Among the battery of efflux pumps present in pathogenic bacteria, our work is focused on QacA, a drug-proton anitiporter (DHA) with 14-transmembrane helices that provide resistance to methicillin resistant Staphylococcus aureus (MRSA) strain, with homologs present in other pathogenic organisms. QacA is a highly promiscuous transporter, capable of effluxing diverse array of monovalent and divalent cationic antibacterial compounds and dyes. This study using a homology model, dissects the role of six protonatable residues present in the transport vestibule of QacA. Systematic mutagenesis resulted in identification of D34 (TM1) and E407 (TM13) as crucial residues and D323 (TM10) and D411 (TM13) as conditional residues needed for transport process of QacA. Whole cells, inside-out vesicles, substrate-induced proton release and microscale thermophoresis based assays were used to investigate the transport and binding properties of the transporter and its mutants. The activity of purified protein was checked with reconstituted QacA in a proteoliposome using substrate-induced proton transport assay. We identify two sites, D34 and D411 playing vital role in recognition of most of the substrates tested while E407 facilitates substrate efflux as a protonation site. It was also observed that E407 has an additional role as a recognition site for the transport of dequalinium, a divalent quaternary ammonium compound. These observations rationalize the promiscuity at the residue level of QacA for diverse substrates. The study identifies the role of acidic residues in QacA with implications for substrate recognition, promiscuity and processive transport in multidrug efflux transporters, related to QacA.

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(1) Indian Institute of Science, India, (2) Stockholm University, Sweden

Biophysical analysis of circularized MSP nanodiscs for structural studies

Melina Daniilidis is a Ph.D student in Prof. Dr. Franz Hagn’s group at the Bavarian NMR Center (BNMRZ) of the Technical University of Munich. PHOTO: Melina Daniilidis

Authors: Melina Daniilidis (1), Ralf Stehle (1), Franz Hagn (1,2)

Structure and dynamics of membrane proteins are crucial aspects for understanding functional properties of this protein class. Unfortunately, stabilizing them in their isolated form is still difficult. By incorporating membrane proteins into nanodiscs, they can be studied in a native-like
environment using biochemical and structural methods. However, thermal and long-term stability of small nanodiscs limit these studies and make it difficult to carry out nuclear magnetic resonance spectroscopy (NMR) measurements at elevated temperatures. Circularized membrane scaffold proteins (MSPs) produced via split-inteins have been shown to be more stable and homogenous than their linear counterparts. However, their biophysical properties, as well as suitability for membrane protein insertion and structural studies have not yet been assessed in a systematic manner. Thus, we examined circular and linear nanodiscs of varying size using several biophysical methods. An important issue for NMR structural studies is that the size and shape of circular nanodiscs do not expand above the phase transition temperature, increasing their homogeneity and reducing their size as compared to linear nanodiscs at high temperatures. 1H,15N-TROSY experiments could demonstrate that circular MSP1D1 nanodiscs with incorporated VDAC-1 are stable at higher temperatures, making it possible to obtain high-resolution NMR spectra of superior quality. Furthermore, NMR relaxation experiments were carried out to compare rotational correlation times of VDAC-1 in circular and linear nanodiscs, respectively. Despite the higher molecular weight, the circular nanodiscs showed lower rotational correlation times, which corroborated the biophysical results on the temperature-dependecy of the nanodisc diameter and homogeneity. The presented data demonstrate that these very stable circularized MSPs are well applicable to the study of membrane proteins in a lipid environment by NMR, but also other structural methods like electron microscopy.

(1) Technical University of Munich, Germany, (2) Helmholtz Zentrum München, Germany

Poster currently not available

Reconstitution of the activity of RND efflux pumps into proteoliposomes

Dhenesh Puvanendran is a Ph.D student at the Institute of Physical and Chemical Biology in Paris, France. PHOTO: Dhenesh Puvanendran

Authors: Dhenesh Puvanendran, Quentin Cece, Martin Picard, IBPC, France

Efflux pumps are the major systems in bacterial resistance against antibiotics. They are classified by the energy needed to be active (ATP hydrolysis or ion counter-transport). Efflux pumps from the RND (Resistance, Nodulation, and cell Division) family use a proton gradient to be active and are composed of three proteins: a membrane fusion protein (MFP) and a transporter (RND) in the inner membrane, and an Outer Membrane Factor (OMF) localized in the outer membrane. We focus on the MexA-MexB-OprM efflux pump from Pseudomonas aeruginosa.The overall goal of my research is to measure in vitro the velocity of transport by efflux pumps. To that end, we reconstitute MexA and MexB as one population of proteoliposome, and OprM as another population of proteoliposome. The whole tripartite pump forms upon association of the respective populations of liposomes. The proof of concept of this method has already been described, leading to a qualitative monitoring of transport. We now work at defining a reconstitution procedure amenable to now quantify the rate of transport. To do so we take extreme care to precisely determine the efficiency of protein reconstitution and the type of lipids component used to perform liposomes. I will present the roadmap towards the rational, step-by-step, reconstitution of the MexA-MexB-OprM efflux pump as well as the methodologies that are undertaken to measure the velocity of transport, and possible perspectives regarding the screening of efflux pump inhibitors.

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The other award-winning posters are:

Novel antigen-binding chimeric proteins as tolls in crystallography and cryo-EM of membrane proteins presented by Thomasz Uchanski, Vrije Universiteit Brussel, Belgium

Cryo-Electron tomography of synaptic vesicle fusion junctions presented by Lucy Ginger, MRC Laboratory of Molecular Biology, United Kingdom

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Creating is Understanding: Synthetic Biology Masters Complexity – Best Poster Awards

The recent EMBO Workshop: Creating is Understanding: Synthetic Biology Masters Complexity (22 – 25 Sep) covered various themes that are geared toward basic research while being at the forefront of synthetic biology.

110 researchers came together at the EMBL Advanced Training Centre in Heidelberg, Germany for 3,5 days of talks, posters and networking. Here we present the work of 4 scientists who received best poster awards at the conference by popular vote.

Engineering portability of the CcaSR light switch for the control of biofilm formation in Pseudomonas putida

Angeles Hueso-Gil is a PhD researcher at the Spanish National Centre for Biotechnology in Madrid.

Authors: Angeles Hueso-Gil (1), Ákos Nyerges (2), Csaba Pál (2), Belén Calles (1), Victor de Lorenzo (1)

Two of the technical challenges faced by contemporary microbiology involve controlling gene expression using light and regulating bacterial biofilm formation, determined by the intracellular levels of the secondary messenger c-di-GMP. CcaSR system is one of the light switches repeatedly used for transcription induction in Escherichia coli. This two-component system represented a good candidate for its adaptation to Pseudomonas putida. Previous attempts have tried to use this microorganism as chassis for the implementation of new pathways, being biofilm formation an important function to control. To this end, we unified CcaSR components in one single construct and randomly mutagenized their regulatory regions to find a clone with a balanced expression of the system key parts inside P. putida. The combination of this novel mutagenization process with a proper screening, which included a first sorting of the libraries and the later isolation of colonies, lead us to a clone with a much improved induction by green light. The selected variant had a notable capacity in response to green light. Finally, optimized CcaSR was used to control the expression of super-efficient variant of PleD, a diguanylate cyclase of Caulobacter which allowed a tight control of c-di-GMP levels, and therefore, of biofilm production.

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(1) National Centre for Biotechnology, Spain
(2) Biological Research Centre of the Hungarian Academy of Sciences, Hungary

Designer membraneless organelles enable orthogonal translation in eukaryotes

Christopher Reinkemeier is a PhD student at EMBL Heidelberg, JGU Mainz and IMB Mainz, Germany

Christopher Reinkemeier (1,2,3), Gemma Estrada Girona (3), Edward A. Lemke (1,2,3)

Genetic code expansion is a powerful tool to study and control protein function with single-residue precision. It is widely used to e.g. perform labeling for microscopy or to photocontrol proteins. This is achieved by introducing an orthogonal tRNA/synthetase suppressor pair into the host, to recode a stop codon to incorporate a noncanonical amino acid (ncAA) into the nascent chain. This technique is codon-specific, but it cannot select specific mRNAs, so naturally occurring stop codons could be suppressed leading to potential interference with housekeeping translation. Nature avoids cross-talk between cellular processes by confining specific functions into organelles. We aimed to design an organelle dedicated to protein engineering, but as translation is a complex process requiring hundreds of factors to work together, membrane-encapsulation would not be feasible. Inspired by the concept of phase separation we hypothesized that such an organelle could instead be designed membraneless. Phase separation can generate high local concentrations of proteins and RNAs in cells and has recently gained attention owing to its role in the formation of specialized organelles such as nucleoli or stress granules. Despite being membraneless and constantly exchanging with the cytoplasm/nucleoplasm, these organelles still perform complex tasks, such as transcription. We combined phase separating proteins with microtubule motor proteins to generated orthogonally translating organelles in living cells that contain an RNA-targeting system, the stop codon suppression machinery and ribosomes. These large organelles enable site- and mRNA-specific ncAA incorporation, decoding one specific codon exclusively in the mRNA of choice. Our results demonstrate a simple yet effective approach to the generation of semi-synthetic eukaryotic cells containing artificial organelles to harbor two
distinct genetic codes, providing a route towards customized orthogonal translation and protein engineering.

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(1) Johannes Gutenberg University Mainz, Germany
(2) Institute of Molecular Biology, Germany
(3) EMBL Heidelberg, Germany

Metabolic perceptrons for neural computing in biological systems

Paul Soudier is a PhD Student at the French National Institute of Agricultural Research, France

Amir Pandi (1), Mathilde Koch (1), Peter Voyvodic (2), Paul Soudier (1), Jerome Bonnet (2), Manish Kushwaha (1), Jean-Loup Faulon(1)

Synthetic biological circuits are promising tools for developing sophisticated systems for medical, industrial, and environmental applications. So far, circuit implementations commonly rely on gene expression regulation for information processing using digital logic. Here, we present a new approach for biological computation through metabolic circuits designed by computer-aided tools, implemented in both whole-cell and cell-free systems. We first combine metabolic transducers to build an analog adder, a device that sums up the concentrations of multiple input metabolites. Next, we build a weighted adder where the contributions of the different metabolites to the sum can be adjusted. Using a computational model fitted on experimental data, we finally implement two four-input of metabolite combinations by applying model-predicted weights to the metabolic perceptron. The perceptron-mediated neural computing introduced here lays the groundwork for more advanced metabolic circuits for rapid and scalable multiplex sensing.

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(1) French National Institute of Agricultural Research, France
(2) INSERM, France

Programmed uptake of biomacromolecules into protocells

Wiggert Altenburg is a PhD student at the Eindhoven University of Technology, The Netherlands

Wiggert Altenburg, Amy Yewdall, Daan Vervoort, Alex Mason, Jan van Hest

The bottom up recreation of cellular processes into synthetic compartments has, in recent years, emerged as an exciting line of research with which to study biological processes in a controlled environment. However, the interior of a living cell is a difficult milieu to mimic in bottom-up synthetic cells, as it is an environment crowded with high concentrations of many different biomacromolecules. In this work, we describe the development of a powerful new tool to more accurately emulate the cell cytosol in discrete coacervate-based protocells. The coacervate core utilized herein not only provides an inherently crowded and highly charged microenvironment, but has also been chemically modified to interact specifically with recombinantly expressed proteins. Our method leverages the well-established binding of His-tagged proteins to Ni2+-nitrilotriacetic acid, which ensures that macromolecules are taken up in a highly efficient, yet gentle manner, thus preserving biological activity. The straightforward method allowed for both control over the amount taken up and an increased local concentration. Moreover, the engineered uptake of proteins was then employed to study two key aspects: the effect of the Ni-NTA interaction on the diffusivity of incorporated proteins, and the enhancement in activity of an encapsulated two-enzyme cascade. This direct and targeted method of protein uptake into a discrete, membrane bound platform is a significant step forward for synthetic cells, and will enable the engineering of highly complex enzyme and signaling networks with increasingly life-like properties.

Poster currently not available

Eindhoven University of Technology, The Netherlands

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