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Over 65 posters were presented at the virtual EMBL Conference ‘Cancer genomics’, covering cancer genome projects, cancer functional genomics, systems biology, cancer immunogenomics and epigenomics, cancer mouse models and the translation and clinical impact of obtained scientific results. We are pleased to be able to share with you the research from the winner of the best poster prize, a €200 voucher sponsored by Molecular Oncology.
Characterization of mutational hotspots across cancer genomes
Thanks to the advent of whole genome sequencing into cancer research, the landscape to explore cancer drivers and the mutational processes generating somatic mutations has expanded. As a consequence, it has become clear that these analyses are tightly linked. A paradigmatic case of this is found in the interpretation of the recurrency of somatic mutations in the same nucleotide (hotspot), which can either be a signal of positive selection or a localized mutational process that does not provide selective advantage to the cells. To the best of our knowledge, a comprehensive characterization of mutational hotspots focusing on both coding and non-coding genomic regions is missing. In the present work, we aim to characterize the distribution of hotspots of somatic mutations to study the mutational processes that play a role in their formation and improve the prioritization of non-coding driver hotspots. With this objective, we have developed a new method, HotspotFinder, to identify, filter, and annotate hotspots of somatic single nucleotide variants (SNVs), multiple nucleotide variants (MNVs) and short indels across the genome. We have run HotspotFinder over 7,553 whole genomes from 81 publicly available cancer cohorts comprising 37 different cancer types. We have identified more than 700,000 hotspots pan-cancer. Across cancer types, we observe large variability in the number and mutation type of hotspots: cutaneous melanomas show the highest hotspot rate with the majority of hotspots arising from SNVs. On the contrary, most hotspots in colorectal cancers, non-small cell lung cancers, and breast cancers arise from insertions and deletions. These differences point to the diversity of mutational processes acting on cancer genomes. Ongoing analyses are focused on characterizing the contribution of different mutational signatures and genome covariates to the formation of hotspots across cancers. Finally, we are working towards prioritizing new candidate driver hotspots in non-coding regions.
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
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.
HaloTag9: An engineered protein tag for fluorescence lifetime multiplexing Presenter: Michelle Frei
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.
Asymmetric nuclear division generates sibling nuclei with different identities Presenter: Chantal Roubinet
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.
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.
Correlative imaging of high-speed atomic force microscopy and fluorescence microscopy revealed asymmetric closing process of endocytosis Presenter: Yiming Yu
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.
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
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.
Towards topology‑based multi‑omics pathway enrichment and its application in toxicology
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.
Computational approaches to scrutinize results from spatial proteomics of operable pancreatic cancer and neighboring tissue
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.
For those of us who like data, end of year statistics are like the holidays all over again. It’s a few days combining the external training data from all six EMBL sites, then playing with excel sheets and pivot tables galore. The point of it all? We want to know how many scientists we reach each year, and our goal is to train as many scientists as possible – after all, sharing is caring, and training is one of EMBL’s core missions.
External training activities at EMBL focus largely on the events in the Course and Conference Programme , which saw 7,148 people come through EMBL’s doors in 2018, around 500 more than in 2017. We had 59 courses and 26 conferences across our sites in Heidelberg, Hinxton, Hamburg and Grenoble. For us here in Heidelberg, there was never a dull moment, from the intimate meetings with 50 participants, to the conferences that filled the Klaus Tschira Auditorium to capacity or the course participants chatting away during the coffee breaks. In addition, 345 delegates received financial assistance to attend one of our events, thanks to the EMBL Corporate Partnership Programme and EMBO, as well as Boehringer Ingelheim Fonds who provided support for various practical courses.
EMBL’s external training activities also comprise online training in bioinformatics and wet lab techniques, where we reached almost 500,000 users – and this doesn’t even count all the YouTube views. On- and off-site training activities carried out by EMBL faculty via lectures, workshops, conference exhibitions, etc are where we reach the most people. In total, EMBL staff reached more than 75,000 people – equal to about ½ the population of Heidelberg! The EMBL Scientific Visitor Programme, which allows scientists to come to EMBL through internships or collaborate on specific research projects had 688 students take part from all around the world.
As you can see, 2018 was a very busy year for all of us involved in external training. Whether it be group leaders, scientific organisers or teams of event organisers, it takes a lot of people to deliver the extensive and high quality training that EMBL has to offer – so we would like to give a big THANKS to everyone involved!