Tag: poster prize

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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Meet the poster prize winners of ‘Cellular mechanisms driven by phase separation’

For many attendees of the EMBO | EMBL Symposium ‘Cellular mechanisms driven by phase separation’, it was the first in-person event after a long while. The excitement was tangible all around the Advanced Training Centre, but especially along the helices where the posters were being displayed. With 108 posters to choose from, the participants had lots of fascinating science to discover and could vote for their favorite until the third day of the event. The fourteen poster presenters who received the most votes had the chance to talk to the scientific organisers who then decided on the final poster prize winners.

Congratulations to the winners: Gea, Alberto, Tom, and Daxiao!

A conserved mechanism regulates reversible amyloids of pyruvate kinase in yeast and human cells

Presenter: Gea Cereghetti, ETH Zürich, Switzerland

Abstract

Amyloids were long viewed as irreversible, pathological aggregates, often associated with neurodegenerative diseases. However, recent insights challenge this view, providing evidence that reversible amyloids can form upon stress conditions and fulfil crucial physiological functions. Yet, the molecular mechanisms regulating functional amyloids and the differences to their pathological counterparts remain poorly understood.

Here, we investigate the conserved principles underlying amyloid reversibility by studying the essential ATP‑producing enzyme pyruvate kinase (PK) in vitro, in yeast, and in human cells. We demonstrate that PK forms stress‑dependent reversible amyloids through a pH‑sensitive amyloid core. Stress‑induced cytosolic acidification promotes PK amyloid formation via protonation of specific glutamate (in yeast) or histidine (in human) residues within the amyloid core. Upon aggregation, PK becomes inactive and is protected from stress‑induced degradation. After stress release, re‑solubilization of yeast PK is essential to restore ATP production, disassemble stress granules (SGs), and restart cell growth. Mechanistically, we demonstrate that yeats PK re‑solubilization is initiated by the glycolytic metabolite fructose‑1,6‑bisphosphate, which directly binds PK amyloids, allowing Hsp104 and Ssa2 chaperone recruitment and aggregate re‑solubilization.

In summary, our work unravels a conserved and potentially widespread molecular mechanism underlying amyloid functionality and reversibility, and highlights the important physiological implications of regulated protein aggregation.

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From Surfactant to glue – how Ki-67 regulates chromosome surface properties

Presenter: Alberto Hernández Armendáriz, EMBL Heidelberg, Germany

Abstract

Compartmentalization into functional units is a key principle of cellular life. In addition to membrane‑bound organelles, eukaryotic cells utilize membrane‑less biomolecular condensates to locally concentrate proteins and nucleic acids. While we are beginning to understand how membrane‑less condensates assemble and disassemble, we know very little about the biological processes that take place at the surface of such condensates. The surface of the largest membrane‑less cellular assembly, the mitotic chromosome, is covered by the intrinsically disordered protein Ki‑67. Our previous studies have revealed that Ki‑67 has dual functionality. In early mitosis, Ki‑67 functions as a surfactant to prevent chromosomes from collapsing into a single chromatin mass, whereas it actively promotes chromosome clustering during exit from mitosis. How Ki‑67 switches between these two opposing processes – chromosome dispersal and chromosome clustering – has remained unknown. Here, we demonstrate that Ki‑67’s biophysical properties radically change during anaphase onset, when all chromosomes merge into a single cluster. Ki‑67’s amphiphilic character is lost as its molecular brush structure collapses and the soluble pool of the protein forms condensates. Our study uncovers a cell‑cycle‑regulated mechanism that controls individualization and coalescence of chromosomes during mitosis.

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

Resolving molecular ageing processes of nuclear pore proteins using a microfluidic droplet assay

Presenter: Tom Scheidt, University of Mainz, Germany

Abstract

The physiological permeability barrier for molecular traffic between the nucleus and the cytosol is filled with intrinsically disorder proteins (IDPs) and assembled by the nuclear pore complex (NPC). These highly enriched disordered nuclear proteins contain domains with high amounts of phenylalanine and glycine (FG‑Nups). In order to understand the physico‑chemical properties of such molecular “gatekeeper”, we made use of a microfluidic device capable of controlled protein condensation, combined with fast and parallelised data acquisition. Our microfluidic device permits studying phase separation on the seconds time scale (due to diffusive mixing and laminar flow), coupled with rapid optical inspection of permeability barrier properties. This time resolution is challenging to achieve by conventional benchtop experiments such as coverslip assays. Our experiments show a rapid aging of FG‑Nups into different material states (liquid, gel, solid etc.) within minutes under physiologically relevant concentrations. Already early droplets show typical properties of a liquid state and resemble a barrier and cargo delivery properties found for physiological nuclear transport. This includes formation of a natural barrier for cargoes larger than ~4 nm, unless accompanied by nuclear transport receptors (NTRs). For a better understanding of the evolution of supramolecular structures as well as the mechanical properties of FG‑rich droplets, we combine our microfluidic system together with coherent anti‑Stokes Raman spectroscopy (CARS) and particle tracking microrheology (PTM). This interdisciplinary approach provides a coherent picture to explain how the balance between homo‑ and heterotypic interactions in FG/NTR mixtures modulates the phase behavior and how this relates to the permeability barrier function of the condensed liquid state. The microfluidic platform described above can work as a general tool to study LLPS of phase separating proteins, particularly those that undergo rapid maturation to gel or amyloid like states, such as e.g. FUS, Tau and alpha‑synuclein or other proteins associated to neurodegeneration.

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Reconstitution of tight junction like networks via ZO1 surface condensation and local actin polymerization

Presenter: Daxiao Sun, Max Planck Institute, Germany

Abstract

Tight junctions are adhesion structures of cell-cell contact in epithelial and endothelial. Biochemical and structural analysis revealed that tight junctions are composed of densely packed proteins forming a continuous membrane associated network at sub-apical, and are involved in adhesion, barrier, polarity, development and mechanotransduction functions, although the molecular basis underlying the assembly and positioning of tight junction network is not clear. Here we combined a bottom-up reconstitution approach on SLBs and computational simulation to investigate the mechanism behind tight junction network formation. We found that tight junction scaffold protein zona occludens1 (ZO1) undergoes surface phase separation with receptor proteins on SLBs and forms membrane condensates under a physiological concentration which is far below its 3D saturation concentration. We also showed that this process depends on receptor valency, receptor density, and ZO1 concentration. With AFM, we found that ZO1 membrane condensates are 2D structures with a height of one-layer molecules. Moreover, these 2D membrane condensates are sufficient to recruit other tight junction related components, like ZO2, ZO3, afadin, cingulin and especially actin. The enrichment of actin to ZO1 membrane condensates promotes actin polymerization and actin bundle formation. ZO1 membrane condensates deform on actin bundles simultaneously, and eventually form a continuous receptor-ZO1-actin network on SLBs together. Applying computational simulation, we showed surface phase separation with the presence of specific surface binding under saturation concentration, and the dependence on receptor valency, receptor density and ZO1 bulk concentration. Thus, combining in vitro reconstitution and computational simulation, our results suggest that surface phase separation and local actin polymerization underlies tight junction network formation. And this approach and mechanism could be applied to investigate and explain other membrane associated compartments formation.

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

The EMBO | EMBL Symposium ‘Cellular mechanisms driven by phase separation’ took place from 9 – 12 May 2022.

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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.

View Tim’s Poster

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.

View Michelle’s Poster

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.

View Chantal’s Poster

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.

View Aline’s Poster

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.

View Yiming’s Poster

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Best Poster Awards – The Identity and Evolution of Cell Types

The second edition of the EMBO | EMBL Symposium: The Identity and Evolution of Cell Types brought together an increased number of researchers from this growing community. 315 scientists joined the virtual meeting and enjoyed four days of talks and poster presentations streamed live. A total of 72 posters were presented at the two live poster sessions out of which three were selected as the best posters by popular vote. Take a look at the winners and their work.

Molecular fingerprinting sea anemones and jellyfish: A transcriptomic approach to characterize Cnidarian cell types
image of Alison Cole
Alison Cole, University of Vienna, Austria

Presenter: Alison Cole, University of Vienna, Austria

Abstract

Animals typically consist of hundreds of different cell types, yet the evolutionary mechanisms underlying the emergence of new cell types are unclear. Cnidarians offer advantages to studies of metazoan cell type evolution, as they are the sister group to the Bilateria and yet comprise an extremely diverse set of lineages that exhibit variable life history strategies, life spans, regenerative properties, animal-defining cell types (ie. muscles and neurons), as well as clade-specific cell types (i.e. cnidocytes). Advances in single cell RNA sequencing have opened the frontiers for molecular profiling of cell types at a genome-wide scale. Application of these technologies for comparisons across species remains in its infancy, and is largely, but not exclusively, restricted to closely related species with well-defined orthologous gene sets. Here we present a large single cell dataset derived from the anthozoan polyp Nematostella vectensis (comprising both developmental and tissue-derived samples),the scyphozoan moon jelly (Aurelia aurita; comprising all life history stages as well as medusa tissue-derived samples), and the hydrozoan Clytia hemispherica (young medusa only). The same cell complement that is identifiable from species-specific genome-wide analyses is recoverable using only a set of 1:1:1 orthologous genes in all three species. Analyses of the reduced gene matrix combining all three species robustly identifies putatively homologous cell types amongst the neurosecretory derivatives, as well as cell populations with clear species-specific transcriptomic profiles. Interpretations of these data in the light of specific cell types will be discussedin order to demonstrate that the combination of unbiased single cell transcriptomes and gene-directed validations can permit the identification of novel and conserved cell types.

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Stylophora pistillata cell atlas illuminates stony coral symbiosis, calcification and immunity
Anamaria Elek, Centre for Genomic Regulation, Spain

Presenter: Anamaria Elek, Centre for Genomic Regulation, Spain

Abstract

Stony corals are colonial cnidarians that sustain the most biodiverse marine ecosystems on Earth: coral reefs. Life cycle of these animals involves a swimming larva that settles and metamorphoses into a sessile polyp, which in turn develops into the adult stage, depositing in the process a protein rich organic matrix and extracellular calcium carbonate crystals to form a stony skeleton. Despite their ecological importance, little is known about the cell types and molecular pathways that underpin the biology of reef-building corals. Using single-cell RNA sequencing, we have defined over 40 cell types across the three life stages of a stony coral Stylophora pistillata. Among others, we characterized previously unknown coral immune cells, endosymbiont alga-hosting cells, and calicoblasts responsible for calcium-carbonate skeleton formation in both settling polyp and the adult coral. Apart from these specialized coral cell types, we identified evolutionary conserved cell types by phylogenetic integration of our S. pistillata cell atlas with three other available cnidarian species. These evolutionary conservations include neuronal and gland cell types, cnidaria-specific cnidocytes, and others. Overall, this study reveals the molecular and cellular basis of stony coral biology, and addresses the evolution of cell type programs in three major cnidarian lineages separated by 500 million years of evolution.

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Gene family evolution underlies cell type diversification in the hypothalamus of teleosts*
Maxwell Shafer, Biozentrum, University of Basel, Switzerland

Presenter: Maxwell Shafer, Biozentrum, University of Basel, Switzerland

Abstract

Hundreds of cell types form the vertebrate brain, but it is largely unknown how similar these cellular repertoires are between or within species, or how cell type diversity evolves. To examine cell type diversity across and within species, we performed single-cell RNA sequencing of ~130,000 hypothalamic cells from zebrafish (Danio rerio) and surface- and cave-morphs of Mexican tetra (Astyanax mexicanus). We found that over 75% of cell types were shared between zebrafish and Mexican tetra, which last shared a common ancestor over 150 million years ago. Orthologous cell types displayed differential paralogue expression that was generated by sub-functionalization after genome duplication. Expression of terminal effector genes, such as neuropeptides, was more conserved than the expression of their associated transcriptional regulators. Species-specific cell types were enriched for the expression of species-specific genes, and characterized by the neo-functionalization of members of recently expanded or contracted gene families. Within species comparisons revealed differences in immune repertoires and transcriptional changes in neuropeptidergic cell types associated with genomic differences between surface- and cave-morphs. The single-cell atlases presented here are a powerful resource to explore hypothalamic cell types, and reveal how gene family evolution and the neo- and sub-functionalization of paralogs contribute to cellular diversity.

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*https://doi.org/10.1101/2020.12.13.414557 

Working on your own conference poster? Then check out these 8 tips for preparing a digital poster that stands out from the crowd.

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Best Poster Awards: Friend or Foe — Transcription and RNA Meet DNA Replication and Repair

During the second virtual EMBO | EMBL Symposium of the year three scientists were awarded a prize for their scientific poster. In this blog, we present the winners and their research.

Friend or Foe attracted 336 participants worldwide, discussing transcription and RNA and DNA replication and repair in live sessions and panel discussions. Three poster session rounds gave the opportunity for participants to view 72 digital posters and interact with the poster presenters.

After the sessions, a voting round followed and three presenters were distinguished with a best poster award by popular vote.

  1. Gianluca Sigismondo of the German Cancer Research Center in Heidelberg, Germany.
  2. Tycho Mevissen, Howard Hughes Medical Institute and Harvard Medical School, USA
  3. Sara Luzzi, Newcastle University, UK

Read our blog on how to create a prize-winning digital poster.

Chromatin dynamics during DNA repair investigated via chromatin-directed proteomics

A portrait picture of scientist Gianluca Sigismondo
Gianluca Sigismondo, German Cancer Research Center, Germany. PHOTO: Gianluca Sigismondo

Poster presenter: Gianluca Sigismondo

Authors: Gianluca Sigismondo, Lavinia Arseni, Jeroen Krijgsveld

DNA lesions predispose to genomic instability, a hallmark of cancer; therefore cells have evolved repair pathways to solve those harmful insults.

Double-strand breaks (DSBs) represent the most lethal DNA damage first marked by the phosphorylation of the histone H2A.X (γH2A.X) which triggers the recruitment of sensor proteins belonging to either the error-prone non-homologous end joining (NHEJ) or the efficient homologous recombination (HR) pathway.

It is now established that chromatin has an active role also in DNA repair, thus its characterization at DSB repair foci is essential to better understand the coordinate action of the repair mechanisms and to identify novel players participating in tumor-associated apoptotic resistance and cell survival.

Here we dissect chromatin changes upon exposure to ionizing radiations through multiple proteomics-based approaches. We applied the Selective Isolation of Chromatin-Associated Protein strategy (ChIP-SICAP; Rafiee, 2016) to investigate the interactors of core NHEJ, HR proteins and γH2A.X while bound to the DNA or in the chromatin soluble fraction.

Through a click chemistry-assisted procedure we profiled the configuration of DNA-bound proteins during DSBs repair; finally we analyzed the histone post-translational modifications (hPTMs) cross-talk at mono-nucleosomes marked by γH2A.X.

Our integrated analysis identified the dynamics of expected chromatin determinants during the DNA repair and interestingly suggested the role for new candidates specifically enriched upon DSB formation.

Validation experiments based on monitoring of DSB foci formation and resolution in AID-DIvA cells proficient or knock-down cells provided evidence of a role for novel candidates in DNA repair. FACS-based analysis of Traffic-light Reporter (TLR) isogenic cells upon silencing of proteins identified by MS characterized their functional role in NHEJ, HR or pathway choice. Furthermore, we defined hPTMs associated with γH2A.X-marked mono-nucleosomes and their dynamics during DSB resolution.

This analysis corroborated expected enrichments (e.g. H4K20me1/me2) and provided insights on new modifications specifically enriched at γH2A.X-nucleosomes.

Chromatin dynamics during DNA repair investigated via chromatin-directed proteomics

Towards transcription-coupled DNA repair in Xenopus egg extract

Poster presenter: Tycho Mevissen

A portrait of scientist Tycho Mevissen
Tycho Mevissen, Harvard Medical School, USA. PHOTO: Tycho Mevissen

This poster and abstract contain unpublished data and are not available at this moment.

Tycho Mevissen is a postdoctoral research fellow in Johannes Walter’s lab at Harvard Medical School. He had completed his PhD with David Komander at the MRC Laboratory of Molecular Biology in Cambridge, UK, where he used structural and biochemical tools to elucidate the intricate mechanisms of enzymes in the ubiquitin system, in particular deubiquitinases (DUBs).

His current research interests in the Walter lab revolve around molecular mechanisms at the intersection of DNA transcription, replication and repair.

In particular, he is interested in understanding how elongating RNA polymerase II deals with various types of obstacles – including different DNA lesions – during transcription elongation. To study this, he uses Xenopus egg extract, which is a powerful cell-free system that has been successfully used to recapitulate a wide range of cellular DNA repair pathways.


RBMX enables productive RNA processing of ultra long exons important for genome stability

A portrait picture of scientist Sara Luzzi
Sara Luzzi, Newcastle University, UK. PHOTO: Sara Luzzi

Poster presenter: Sara Luzzi

Authors: Sara Luzzi, Gerald Hysenaj, Chileleko Siachisumo, Kathleen Cheung, Matthew Gazzara, Katherine James, Caroline Dalgliesh, Mahsa Kheirollahi Chadegani, Ingrid Ehrmann, Graham R Smith, Simon J Cockell, Jennifer Munkley, Yoseph Barash, and David J Elliott.

The nuclear RNA binding protein RBMX has a direct role in genome repair and is required for expression of the tumour suppressor BRCA2. Here we report that RBMX controls RNA processing of key genes involved in genome maintenance in breast cancer cells.

Our data demonstrate that RBMX represses a premature polyadenylation site that would truncate BRCA2 protein, and is essential for full-length mRNA expression from other genes important for genome stability. These include ETAA1, which encodes for a key replication fork protein, where RBMX and its protein interaction partner Tra2ß efficiently suppress a weak splice site to enable ETAA1 protein expression.

More generally, we propose that RBMX facilitates correct inclusion of unusually long exons within mature mRNAs by repressing cryptic RNA processing. Our data provide new molecular insights explaining the role of RBMX in DNA repair and genome maintenance.

Poster RBMX enables productive RNA processing of ultra-long exons important for genome stability

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