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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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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Best Poster Flash Talk winners of the Mobile Genome

This year, the virtual EMBO Workshop: “the Mobile Genome: Genetic and Physiological Impacts of Transposable Elements” centered around the broad impacts of TEs on organismal biology. It turned out to be a diverse meeting with interesting cross-disciplinary discussions, assembling experts from diverse fields including genomics, epigenetics, structural biology, developmental biology, immunology, cancer biology and neurobiology.

Over 90 posters were available on the virtual platform and three presenters stood out with their poster flash talks. An extraordinary accomplishment in the virtual format. With no further ado, we are introducing the winners and their research!

The repressive SCAN zinc-finger protein family targets transposable elements

Presenter: Wayo Matsushima

Wayo Matsushima, École Polytechnique Fédérale de Lausanne, Switzerland 
Wayo Matsushima, École Polytechnique Fédérale de Lausanne, Switzerland

Abstract

Transposable elements (TEs) contribute to genome innovations through insertions of coding and non-coding elements. KRAB zinc-finger proteins (KZFPs) function as sequence-specific repressors by recruiting KAP1/TRIM28, a scaffold protein of a heterochromatin-inducing complex, to TE-derived sequences. An ancestral KZFP gave rise to another family of transcription factors through the exonisation of the gag gene from the Gmr1-like transposon family as the SCAN domain. This SCAN zinc-finger protein (SZFP) family experienced multiple rounds of segmental duplication events, resulting in the presence of ~300 family members in diverse amniote genomes. Despite its abundance and evolutionary proximity to KZFPs, the functions of the SZFP family are still not well understood.

To analyse evolutionary conservation of SZFPs, we compared the DNA-contacting zinc-finger amino acid sequences of the human SZFPs to those of 65 other amniote species. This revealed that the zinc finger signatures of SZFPs are under rapid lineage-specific selection, and that several human SZFPs harbour primate-specific zinc finger sequences.

To characterise the genomic targets of all of the 55 human SZFPs, we performed ChIP-seq on 293T cell lines, each expressing one of the SZFPs with an HA-tag. We found that the binding sites of a number of SZFPs significantly overlap with specific TE subfamilies, and further observed that the evolutionary ages of SZFPs and their TE targets often matched, suggesting the TE-driven positive selection of this transcription factor family.

We next performed luciferase assays to study the regulatory function of SZFPs. We discovered that SZFPs act as transcriptional repressors owing to their SCAN domain with its conserved C-flanking 15 amino acid residues. Similar levels of repression were obtained in KAP1/TRIM28 knockout cells, demonstrating that, in contrast to KZFPs, SZFPs act independently of this master corepressor.

Together, this study identifies SZFPs as putative controllers of the regulatory potential of TEs, through their lineage-specific zinc finger repertoire and the repressive domain derived from the co-opted retroviral gene. Future work will explore the potential impact of SZFPs and their TE targets on species-specific gene regulatory networks.

The poster contains unpublished data and can therefore not be published. Follow Wayo Matsushima on Twitter for more information on his projects.

A natural transposon affects gene regulation and fitness related traits depending on the developmental stage and environmental conditions in D. melanogaster

Presenter: Miriam Merenciano

Miriam Merenciano, Institute of Evolutionary Biology (CSIC-UPF), Spain

Abstract

TEs have been considered a genome-wide source of regulatory elements capable of regulating nearby gene expression. In Drosophila melanogaster, the FBti0019985 natural TE insertion has been previously reported to add a transcription start site to the Lime transcription factor. In this work, we performed invivo enhancer assays and gene expression analysis with CRISPR/Cas9 mutants and natural populations to explore the effects of FBti0019985 on Lime expression under different stress conditions and different developmental stages. We found that this insertion acts as an enhancer in the adult stage under immune-stress conditions. Indeed, the deletion of predicted immune-related binding sites in the TE significantly reduces its enhancer activity in infected conditions, confirming that it harbors functional cis-regulatory elements. We also found that the TE upregulates Lime in embryos, however, in this case we could not pinpoint the molecular mechanism. Finally, we found that TE-induced Lime upregulation was associated with tolerance to bacterial infection and with increased egg-to-adult viability. Our results suggest that different developmental stages and environmental conditions should be tested in order to fully characterize the molecular and functional effects of a genetic variant.

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An ATPase filament bridge: How a transposon and CRISPR stick together

Presenter: Irma Querques

Irma Querques, University of Zurich, Switzerland

Since the discovery of bacterial adaptive immunity, CRISPR-Cas systems have been mainly regarded as a mechanism to counteract horizontal transfer of mobile genetic elements including transposons in prokaryotic genomes. Conversely, a distinct family of Tn7-like elements co-opted CRISPR-Cas RNA-guided machineries to direct transposon insertion into specific target sites. In type V CRISPR-associated transposons, RNA-directed transposition relies on the cross-talk between the pseudonuclease Cas12k, the transposase TnsB, the zinc-finger protein TniQ and the ATPase TnsC. Yet, the molecular mechanisms underpinning this interplay have remained unknown. Here we present a cryo-electron microscopy structure of DNA-associated TnsC in its ATP-bound state. The structure reveals that the AAA+ ATPase forms an ATP-dependent helical filament that encloses and remodels the underlying target DNA. One strand only of the duplex is tracked by consecutive TnsC protomers with an unexpected two-nucleotide periodicity, resulting in a DNA helix with 12 base pairs per turn. Biochemical studies show that TnsC polymerization is a critical aspect of the system that enables the coupling of RNA-guided target recognition by Cas12k with the downstream recruitment of TnsB by direct protein interactions. In turn, TnsB triggers filament disassembly upon ATP hydrolysis, establishing target immunity. We also show that TniQ directly interacts with TnsC, restricting its polymerization. A crystal structure of TniQ reveals structural diversity within the TniQ protein superfamily, further suggesting a role in TnsC regulation rather than DNA targeting. Together, our data point to a mechanistic model whereby TnsC oligomers bridge between the RNA-guided target selector Cas12k and the transpososome, promoting target DNA remodeling and ultimately transposon integration. This work discloses first mechanistic insights into targeting and regulation of type V CRISPR-associated elements and will guide the rational design of these systems as programmable, site-specific gene insertion tools.

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The EMBO Workshop: “The Mobile Genome: Genetic and Physiological Impacts of Transposable Elements” took place from 29 August – 1 September 2021. 

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Seeing is Believing Best Poster Awards

From its beginning in 2011, Seeing is Believing has embraced novel imaging technologies that open new windows for biological discovery, including single-molecule and super-resolution, light sheet and correlative light electron microscopy. This year, the EMBO| EMBL Symposium was held virtually for the first time, and it did not disappoint! We had around 640 participants, with over 130 posters presented during the digital poster sessions. We are excited to share with you the research from the five best poster prize winners, who were also given the opportunity to share their work with a talk at the end of the meeting!

 

A novel view on protein-protein interaction: Investigating protein-complex formation using correlative dual-color single particle tracking
Presenter: Tim Abel

Tim Abel, Max Delbrück Center for Molecular Medicine in the Helmholtz Association

Abstract

Characterization of protein-protein interactions is one of the central aspects, when investigating cellular mechanisms. Most of the established biochemical techniques to describe protein-interactions rely on the isolation and purification of the proteins of interest. This becomes especially problematic, when analyzing membrane-localized complexes or transient and dynamic interactions, which are difficult to isolate or easily disrupted. In this project, we use dual-color single particle tracking to investigate the formation of the ER-membrane resident multi-protein complex Hrd1 in living cells.

The Hrd1-complex is one of the central ubiquitin ligase complexes in endoplasmic reticulum-associated degradation (ERAD). During ERAD misfolded or otherwise faulty proteins are transported from the ER to the cytosol and targeted for degradation, which both involves the name-giving subunit of the Hrd1-complex Hrd1p. Biochemical analysis indicates that this protein forms homo-oligomers but the stoichiometry within the functional complex and the dynamics of its formation remains disputed.

To assess Hrd1p-oligomerization using live-cell imaging we labeled endogenous Hrd1p with either the SNAP- or the Halo-tag using CRISPR/Cas9 mediated gene integration. Both variants remained fully functional and enabled labeling using dyes compatible with SM-imaging. TIRF microscopy revealed single diffraction limited spots. When labeling competitively with differently colored halo-ligand-conjugates, Hrd1p-punctae exhibited strong correlated movement between both colors indicative of homo-oligomerization. Its degree of interaction was quantified by directly correlating single-steps extracted from the SM-trajectories, which proved to be a robust way to analyze protein-protein interactions. Additionally, PALM-imaging of a Halo-labeled Hrd1-substrate was able to directly show its interaction with Hrd1p-SNAP by correlated movement on the SM-level. In the future we will use this correlative SM-approach in combination with genetic and inhibitor based interventions to interrogate factors required for Hrd1p-complex assembly, its formation dynamics and how substrates are recruited.

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HaloTag9: An engineered protein tag for fluorescence lifetime multiplexing
Presenter: Michelle Frei

Michelle Frei, Max Planck Institute for Medical Research, Heidelberg, Germany and EPFL, Switzerland

Abstract

Self-labeling protein tags have become important tools in fluorescence microscopy. Their use in combination with fluorogenic fluorophores, which only become fluorescent when bound to their protein target, makes them particularly suitable for live-cell applications. The fluorogenic turn-on observed upon labeling as well as the photophysical properties of the fluorophore are mainly determined by the protein surface near the fluorophore binding site. However, up to now, most efforts have been invested in the development of new fluorophores and only little attention has been paid to the engineering of the self-labeling protein tag.

Here we report on the engineering of HaloTag7 to modulate the brightness and fluorescence lifetime of bound rhodamines. Specifically, we developed HaloTag9, which showed up to 40% higher brightness in cellulo and 20% higher fluorescence lifetime than HaloTag7 upon labeling with rhodamines. This makes it an ideal tag for imaging techniques such as confocal microscopy or stimulated emission depletion microscopy. In addition, combining HaloTag7 and HaloTag9 enabled us to perform live-cell fluorescence lifetime multiplexing using a single fluorophore. The difference in fluorescence lifetime was further exploited to generate a chemigenetic fluorescence lifetime based biosensor to monitor cell cycle progression. Overall, our work highlights that the combination of protein engineering and chemical synthesis can generate imaging tools with outstanding properties. We expect HaloTag9 to be beneficial for a multitude of live-cell microscopy applications.

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Asymmetric nuclear division generates sibling nuclei with different identities
Presenter: Chantal Roubinet

Chantal Roubinet, MRC-LMCB, University College London, UK and MRC-LMB, Cambridge, UK

Abstract

Although nuclei are defining features of eukaryotes, we still do not fully understand how the nuclear compartment is duplicated and partitioned during division. This is important, as the mechanism of nuclear division differs profoundly across systems. In studying this process in Drosophila neural stem cells, we recently found that the nuclear compartment persists during mitosis due to the maintenance of a mitotic nuclear lamina. This bounding spindle envelope is then asymmetrically remodelled and partitioned at division, giving rise to two daughter nuclei that profoundly differ in size (physical asymmetry) and envelope composition (molecular asymmetry). The asymmetry in the size of daughter nuclei following division results from: i) an asymmetric nuclear envelope resealing at mitotic exit that depends on the central spindle, and ii) a differential nuclear growth in early G1 that depends on the availability of ER/nuclear membranes reservoir in the cytoplasm. Furthermore, my data show that asymmetric nuclear division in this system is associated with a different chromatin organization between the two daughter nuclei as well as with an asymmetric distribution of several histone marks, before the cortical release of cell fate determinants. This suggests that the asymmetric remodelling of the nuclear envelope has profound functional consequences for these stem cell divisions. Taken together, these data make clear the importance of considering the path of nuclear re-modelling when investigating how a stem cell division generates two distinct sibling cells with different identities/fates.

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A nanoscopic reconstruction of clathrin coat remodeling during mammalian endocytosis
Presenter: Aline Tschanz
Aline Tschanz, EMBL Heidelberg, Germany

Abstract

Clathrin Mediated Endocytosis (CME) is one of the most trafficked endocytic pathways in a cell and is involved in a multitude of processes including cell signaling, nutrient uptake and membrane homeostasis. During CME, extracellular material becomes progressively engulfed by the plasma membrane (PM) until a vesicle is formed and released into the cytosol.

Whilst many endocytic components have been extensively studied, the underlying mechanism by which protein assembly drives membrane invagination is not entirely understood. This is in parts due to technical limitations in the visualization of the endocytic machinery in-situ.

Here, we use 3D superresolution microscopy to resolve clathrin coated pits (CCP) in fixed mammalian cells. We are able to resolve thousands of CCPs, each representing a distinct snapshot of the overall endocytic process. By applying a spherical model fit to thousands of individual CCPs, we can quantitatively describe and compare the population of endocytic sites. We further use the extracted parameters to sort individual CCPs along their endocytic progression and reconstruct the dynamic remodeling of the clathrin coat.

We find that both the curvature as well as the surface area of these structures increase during CME. This motivates a refinement of the currently proposed models used to approximate the process of membrane bending. Our findings further allow for the formulation of new physical models describing the underlying mechanism of force generation by protein assembly at the PM.

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Correlative imaging of high-speed atomic force microscopy and fluorescence microscopy revealed asymmetric closing process of endocytosis
Presenter: Yiming Yu

Yiming Yu, Kyoto University, Japan

Abstract

High-speed atomic force microscopy (HS-AFM) has been a powerful tool for visualizing various biological processes at a single-molecule level. Our group had developed a unique type of correlative imaging system by combining HS-AFM for live-cell imaging and confocal laser scanning microscopy to simultaneously visualize dynamic morphological changes of the cell membrane and protein dynamics in living cells. By using this system, we analyzed the molecular process of clathrin-mediated endocytosis (CME), in which more than 60 different proteins assemble on the plasma membrane to produce a clathrin-coated pit (CCP) with a diameter of ~100 nm by inducing a series of morphological change of the plasma membrane. A unique membrane morphology was frequently observed at the closing step of the CME; a small bulge of the plasma membrane grew besides the CCP and finally closed the pit in an asymmetric manner, which is distinct from the constricting motion induced by dynamin. After a screening of siRNA against CME-related proteins and inhibitors for actin-related proteins, we found that a strong self-assembly of a BAR-domain containing protein near the CCP recruited actin and promoted its polymerization and branching. Such an asymmetric growing of actin filaments near the CCP promoted the closing step of the CME by generating lateral force against the pit. These results demonstrated a clear advantage of the correlative imaging system of HS-AFM and fluorescence microscopy in analyzing nano-scale events on the plasma membrane.

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Multiomics to Mechanisms Best Poster Awards

The EMBO|EMBL Symposium: Multiomics to Mechanisms: Challenges in Data Integration took place virtually 15 – 17 September 2021. With over 400 participants, this was the biggest multi-omics conference since it began in 2017. We had 96 posters presented virtually, and are excited to share the research from the three best poster prize winners. 

Identification of transcription factor signaling molecules by coupling gene expression and metabolomics

A portrait picture of Daniela Ledezma‑Tejeida
Daniela Ledezma‑Tejeida, ETH Zurich, Switzerland (Photo credit: Stefania Laddage)

Abstract

Bacteria need to adapt to changes in their environment in order to survive. Transcription factors (TFs) bind metabolites that signal such changes and in turn alter gene expression. Escherichia coli has the best characterized transcriptional regulatory network involving 300 predicted TFs, of which ~75% have a metabolite‑binding domain. However, the binding partners of only 95 TFs have been identified due to low-throughput of common in vitro identification methods. Here, we combined metabolomics and gene expression data obtained in vivo across several growth conditions to identify TF‑metabolite interactions of four TFs without a known binding partner: CdaR, CsgD, FlhDC and GadX. We have validated our method by accurately predicting the known binding partners of ArgR, TyrR and CysB, three highly studied TFs. The in vivo nature of our approach can not only identify new TF‑metabolite interactions but also provide insight into the most functionally relevant.

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Towards topology‑based multi‑omics pathway enrichment and its application in toxicology

A portrait picture of Sebastian Canzler, Helmholtz‑Centre for Environmental Research
Sebastian Canzler, Helmholtz‑Centre for Environmental Research ‑ UFZ, Germany

Abstract

The call for an application of (multi‑)omics data in toxicology became highly prominent in recent years, since omics experiments are intended to generate comprehensive information on molecular changes in cells and tissues more quickly, more accurately, and with fewer resources than ever before. The associated hopes explicitly include the reduction of live animal testing and an increased number of analyzed substances that can be tested. Therein, multi‑omics data are essential to comprehensively infer mechanistic knowledge on molecular response pathways to subsequently guide and aid chemical risk assessment. However, currently available multi‑omics pathway enrichment methods struggle to cope with different aspects hampering their application in computational toxicology, e.g., the utilization of insufficient enrichment methods, missing support for time‑ and concentration resolved data, and restrictions on the pathway sources. Most approaches utilize a sequential data integration and thereby completely ignore the connections between different omics layers. With ToPaFC, we present the first step towards a consistent and simultaneous multi-omics-based pathway enrichment that accounts for those obstacles and explicitly takes the underlying pathway topology into account. Right now, we can deal with up to eight different pathway databases and two omics layers (trans/meta or prot/meta). The pathway topology is reflected in two different ways: i) the importance of a node (omics feature) is measured based on its connections and its relative localization within the pathway and ii) the influence of each node on the network is specified by the weight of its outgoing edges, whether they are inhibiting, neutral, or activating. With this integration of edge information along the pathway, our method inherently accounts for consistent molecular changes of the features. The derived node‑centered pathway representation is combined with measured multi‑omics features to calculate a topology‑based pathway fold change that accounts for consistent changes within the molecular response.

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Computational approaches to scrutinize results from spatial proteomics of operable pancreatic cancer and neighboring tissue

A portrait picture of Ábel Szkalisity, University of Helsinki, Finland
Ábel Szkalisity, University of Helsinki, Finland

Abstract

The advance of laser‑microdissection technologies coupled with proteomics enables unprecedented insights into tissue proteomes. However, the limited availability of patient materials coupled with the high dimensional output of proteomics necessitates data integration across studies to safeguard the reliability of the results. We microdissected morphologically benign and neoplastic pancreas and surrounding stromal areas from 14 patients with early pancreatic ductal adenocarcinoma and analyzed their protein compositions with nLC‑MS/MS. The results indicated downregulated digestive functions in the malignant exocrine tissue and lower metabolic activity in the stroma vs. exocrine pancreas. Intriguingly, the majority of the most significant proteins for survival originated from the morphologically benign exocrine regions, suggesting that these areas may harbor early, predisposing changes. To scrutinize this idea, we compared their proteomes to proteomics data of 12 healthy control pancreatic samples obtained from publications. The protein identification and quantification pipeline from the raw mass spectrometer files were standardized to minimize variation introduced by search engines or protein sequence databases. Altogether, we identified 7,099 proteins in 67 samples involving 5 tissue types from 2 experiments and 5 batches. We investigated two independent strategies for rendering the values comparable. First, batch effects within experiments were corrected for with ComBat and the abundances across experiments were aligned with housekeeping protein normalization. However, this approach required full observations, removing over 90% of the identified proteins from the analysis. Hence, our second approach involved applying Group Factor Analysis to directly extract factors that reveal relationships between the tissue types in our study without compromising the protein coverage. These approaches not only showed that our main results are independent of the data analysis pipeline but also implicated changes in the mRNA splicing machinery as important players in pancreatic cancer. By surveying 165 patients from The Cancer Genome Atlas we revealed that increased transcriptional complexity indeed associates with poor survival in this disease.

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