Cell News Newsletter of the German Society for Cell Biology full electronic version Volume 44, 2 2017
Report from the DGZ Spring Member Meeting in Leipzig Adding efficiency to your fluorescence imaging. ZEISS Celldiscoverer 7 More Information at DGZ Spring Meeting
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Cell News 01/2017 3 PREFACE
DEAR COLLEAGUES
Please look into the present issue of Cell News that highlights the Unfortunately, despite multiple calls to come and discuss the 2017 DGZ prize winners: the Nikon Young Scientist Award winner future of the DGZ and showcasting fantastic science, this member Evgenij Fiskin from Goethe-University Frankfurt, the Binder Inno- meeting with its short format, still did not mobilize the majority of vation Prize winner Sara Wickström from the University of Cologne our member community. The executive board of the DGZ therefore and the Max-Planck-Institute for Biology of Ageing and the Werner decided to go back to our established format of an International Risau Prize prize winner Kerstin Wilhelm from the Max-Planck-In- Meeting as a platform for exchange, to hear cutting-edge cell stitute for Heart and Lung Research in Bad Nauheim. They present biology and to meet our sponsors. The DGZ International Meeting their research on p 8, 14 and 20, respectively. will take place in Leipzig from September 17 to 19, 2018. Moreover, Simone Reber from the Humboldt University, Berlin, will We look back on a scientifically highly successful and inspiring organise a Young Investigator meeting on quantitative Biology DGZ spring meeting that took place on March 1 and 2 in Leipzig. on February 27/28, 2018 targeted at PhD students, postdocs and The former president of the American Society of Cell Biology, Peter early PIs. Please find a preliminary program overview in this Cell Walter (see cover image!), thrilled the audience when giving the news issue. Carl Zeiss Lecture and presenting his ground-breaking work on protein quality control on the first day of our member meeting. Take the chance and convince yourself about the extraordinary The above mentioned prizes awardees were honoured and gave science in the cell biology field when reading this Cell News issue. outstanding presentations. On the second day of the meeting, The DGZ meetings are always worth the trip and we need your the Walther Flemming Award winner, Kikue Tachibana-Konwalski support to make them a success. We are looking forward to seeing joined us and introduced her fascinating research when receiving you at the next DGZ meeting! the award. In addition, young DGZ member PIs, Simone Reber (Berlin), Martin Beck (Heidelberg), Susanne Önel (Marburg) and Carien Niessen Julia Gross (Göttingen) organized 4 fantastic sessions that covered Oliver Gruss a wide range of cell biology and stimulated a lot of discussion. The Thomas M. Magin meeting, as announced, also included a DGZ member forum on Karin Schumacher the future of the DGZ, in which we received very valuable feed- back. The 2017 DGZ Spring meeting was an incredibly stimulating, intense meeting with excellent science and dense communication.
DGZ Young Scientists‘ Forum 2018
In February 2018, the German Society for Cell Biology will hold an international meeting organized by and for junior scientists with a special focus on early independence. The upcoming DGZ Young Scientists’ Forum topics will - amongst others - include maintenance of cell identity, synthetic biology, dynamic cytoskeletal systems, emergent core technologies and other relevant and challenging topics (see page 5). The meeting will take place on February, 26th and 27th in Berlin. Save the date for this highly interactive, scientifically stimulating and fun meeting!
4 Cell News 02/2017 DGZ YOUNG SCIENTISTS FORUM 2018
Deutsche Gesellschaft für Zellbiologie Young Scientists’ Forum 26. & 27.02.2018 Berlin
Homeostasis and Adapta- tion: How Cells and Com- Dynamic Cytoskeletal Cell Shape in Space and Time partments Change whilst Systems Maintaining their Identity hosted by hosted by Alba Diz-Muñoz & Gaia Pigino hosted by Peter Bieling & Alex Bird Robert Ernst & James Saenz
Quantitative Imaging Bottom up Synthetic Biology Science & Society of Development
hosted by hosted by hosted by Dora Tang Simone Reber Stephan Preibisch
APPLICATION & ABSTRACT DEADLINE: 01.12.2017 SCIENTIFIC ORGANIZER: Simone Reber CONTACT: [email protected]
Cell News 01/2017 5 MINUTES OF THE DGZ MEMBER MEETING
Protokoll der DGZ-Mitgliederversammlung am 01.03.2017 in Leipzig HS 8, Universität Leipzig Anwesende: 40 MitgliederInnen
1. Confirmation of the minutes of the last year’s Äußerungen der Mitglieder: DGZ member meeting 2016 Einstimmig angenommen • Themenfokussiertes oder breit angelegtes Meeting: überwäl- tigende Mehrheit für breit angelegtes Meeting, Länge 3 volle 2. The president’s annual report Tage (über 4 Tage verteilt) K. Niessen bittet um Zustimmung, keinen ausführlichen • Wenig Parallel-Sitzungen Bericht vorzulegen, weil seit AM 2016 keine einschneidenden • High profile Sprecher sind wichtig Dinge zu berichten sind: keine Einwände • Möglichst viele Mitglieder sollen vortragen (15 min Vorträge) • Ständiges Organisations-Komitee aus 4-5 Mitgliedern zur 3. Financial and auditors report Organisation von Tagungen; ¼ der Mitglieder sollen jährlich O. Gruss stellt den Finanzbericht vor. Keine Fragen. Bitte um ersetzt werden Entlastung des Vorstands (R. Gräf): einstimmig angenommen • Professioneller Tagungsorganisator soll wieder an Bord sein (MCI oder andere) Neubestellung auditors: Ralph Gräf und Julia Gross stehen • Vorschlag: Royal Society for Microscopy (UK) möchte an ab Kassenjahr 2018 zur Verfügung. Tagung teilnehmen und 1-2 sessions gestalten; einhellige Zustimmung; R. Faix kümmert sich um Kontakte und konkrete 4. C. Niessen stellt folgende Punkte nach kurzer Erläuterung Planung, sobald Ort und Zeitpunkt des 2018 International zur Diskussion: Meetings festliegen a. Our mission • GBM möchte Kooperation mit DGZ stark intensivieren, die b. What do members expect of the DGZ GBM hat momentan ~5.500 Mitglieder. Bei GBM gibt es lokale c. Future Goals of DGZ Junior groups, die sehr erfolgreich sind und für die Reisesti- d. Cooperation with GBM and other related societies pendien etc. verfügbar sind. Bringen viele Jungmitglieder zu e. Means to achieve goals Tagungen. Außerdem gibt es study sections f. Problem: only ~10 % of members attend meeting, unlike in • Optionen für Kooperation: punktuelle Zusammenarbeit, other societies Gründung einer neuen Dachgesellschaft für Life Sciences; ein- helliges Mitgliedervotum für intensive Kooperation als Auftrag an DGZ Vorstand
• Weitere Vorschläge: Kooperation mit GfE, Eurobioimaging, BILANZ 2016 Stammzellinitiative; wird ebenfalls sehr positiv gesehen EINNAHMEN / AUSGABEN • Frage: was hat höchste Priorität für MitgliederInnen:
Einnahmen EUR Ausgaben EUR nach zähem Schweigen: Meeting
Mitgliedsbeiträge 43.680,00 Bankkosten 850,33 • Wer möchte Meetings 2018 und 2019 organisieren? (abzgl. Retouren) Retoure Mitgliedsbeiträge 700,00 Bleibt offen Spenden, Preisgelder 20.000,00 Reisekosten 2.782,66 • Vorschlag zur Erfassung von Mitgliederinteressen und Zinsen 246,30 Spenden, Preisgelder 18.850,00 -meinungen: Online-Fragebogen. Findet große Zustimmung, CellNews, Homepage 9.401,00 (Werbeanzeigen, Firmen-Links) Cell News 1.172,03 Ingrid Hoffmann und Carien Niessen erklären sich bereit, einen
DGZ-Tagungen 17.998,10 DGZ-Tagungen 8.035,53 Bogen zu erarbeiten Überträge 112.617,03 Bürokosten/Gehalt Sekr. Büromaterial, Homepage 50.892,46 • C. Niessen: Ressourcen der Gesellschaft angespannt, Mitglie- Sonstige 5.329,68 Überträge 112.617,03 derbeiträge finanzieren nicht vollständig Sekretariat in Hei-
Sonstige 5.199,61 delberg, verbesserte Personalausstattung und Infrastruktur zur
Umsetzung strategischer Ziele braucht ~100.000 E zusätzlich.
Summe der Einnahmen: 209.272,11 Summe der Ausgaben: 201.099,65 Frage nach Höhe der Mitgliederbeiträge und Tagungsgebühren
Guthaben am 31.12.2015: 211.810,97 Guthaben am 31.12.2016: 219.983,43 zur Finanzierung der Gesellschaft bleibt unbeantwortet
Guthaben DGZ: 167.573,47 Guthaben DGZ: 175.790,42 Werner Risau Preis: 44.237,50 Werner Risau Preis: 44.193,01
Oliver Gruss (Vice President) and Thomas M. Magin (CEO)
Die Einnahmen und Ausgaben wurden am 07.02.2017 in Heidelberg von den beiden Kassenprüfern Marie- Christine Dabauvalle und Hans-Georg Mannherz geprüft und für richtig befunden.
6 Cell News 02/2017 Orcs-V3_GEMINI_179x120_Layout 1 15/09/2016 18:13 Page 1
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Cell News 01/2017 7 NIKON YOUNG SCIENTIST AWARD 2017
Evgenij Fiskin
Regulation of host-pathogen interactions by the ubiquitin system
Institute of Biochemistry II, Goethe University School of Medicine, University Hospital, Frankfurt, Germany
The increased emergence of antibiotics resistance and the dan- and contributes essentially to cellular homeostasis, M1-linked ger it poses to human health prompted the WHO a few months chains, also designated as linear chains, have recently attracted ago to publish a “Global priority list of antibiotic-resistant a lot of attention due to their critical role in the regulation of bacteria to guide research, discovery, and development of new innate and adaptive immune pathways (Rittinger and Ikeda, antibiotics” (WHO, 2017). These recent developments clearly 2017). Hereby, linear Ub chains are essential to control NF-KB illustrate the over-reliance of current therapies on antibiotics signaling downstream of cytokine receptors or upon recognition and call for the establishment of alternative treatment options. of bacteria (Wild et al., 2011). M1-linked Ub chains are formed Accumulated evidence indicates that deciphering pathogenic by a single dedicated E3 ligase termed the linear Ub chain as- virulence and host defense mechanisms during bacterial infec- sembly complex (LUBAC) consisting of heme-oxidized iron-re- tions may be the key to future developments of novel antibac- sponsive element binding protein 2 ubiquitin ligase-1 (HOIL-1L), terial strategies. Being one of the most versatile and prevalent the catalytic subunit HOIL-1L interacting protein (HOIP/RNF31) post-translational modifications in eukaryotic cells, ubiquiti- as well as the third subunit SH3 and multiple ankyrin repeat nation is also a major regulator of host-pathogen interactions domains protein (SHANK)-associated RBCK1 homology (RH)-do- (Hu and Sun, 2016). As such, ubiquitin (Ub) drives a multitude main-interacting protein (SHARPIN) (Kirisako et al., 2006). of microbicidal programs including for example innate immune Importantly, M1-linked Ub chains were demonstrated to harbor receptor signaling as well as selective autophagy (Gomes and important anti-bacterial functions as they are formed on the Dikic, 2014). Given its central role as a regulator of host defense, surface of cytosolic Salmonella enterica (Noad et al., 2017; van ubiquitination is a major target of pathogen-derived virulence Wijk et al., 2012; van Wijk et al., 2017), and are able to recruit factors. In this work, we performed the first systematic analy- autophagy receptors in order to promote lysosomal clearance of sis of the cellular ubiquitin landscape upon bacterial infection cytosolic pathogens (Gomes and Dikic, 2014) to obtain a thorough understanding of molecular mechanisms of host-pathogen interplay required for the rational design of novel anti-bacterial strategies. Systematic analysis of ubiquitin-driven events upon Salmonella Typhimurium infection
Ubiquitination in host defense Whereas previous efforts focused on the study of a small num- ber of ubiquitination events triggered by the detection of iso- The ubiquitination reaction requires the sequential action of at lated bacterial products (e.g. lipopolysaccharide or peptidogly- least three enzymes: an Ub-activating enzyme (E1), an Ub-con- can), it is now well established that certain host inflammatory jugating enzyme (E2) and a Ub ligase (E3) (Hershko et al., 2000). pathways are exclusively stimulated upon live bacterial infection In eukaryotes three distinct classes of Ub ligases mediate the E3 (Sander et al., 2011; Vance et al., 2009). In similar fashion, the step of ubiquitination: (i) RING-type E3s functioning as scaf- subversion of cellular cytoskeleton and membrane trafficking folds, bringing Ub-charged E2s into close proximity of sub- systems upon bacterial invasion were shown to require the ac- strates, thereby facilitating Ub transfer from E2 to a substrate tive and timely secretion of pathogen-derived virulence proteins lysine (ii) HECT-type E3s forming a thioester intermediate with (LaRock et al., 2015). In light of these observations, we sought Ub before transferring it directly to the substrate and (iii) RING- to provide a global view of ubiquitination during live pathogenic between-RING (RBR) ligases, which function as RING-HECT hy- insult and to uncover novel Ub-involving host-pathogen inter- brids (Zheng and Shabek, 2017). Ub itself contains seven lysine actions. In particular, we focused our efforts on the Ub-modified residues (K6, K11, K27, K29, K33, K48, K63) and a methionine proteome of cells exposed to the invasive Gram-negative patho- at position 1 (M1), all of which can be used to form Ub chains gen Salmonella enterica serovar Typhimurium (S. Typhimurium), in vivo (Yau and Rape, 2016). Whereas the best studied role of which can infect a broad range of hosts including humans and ubiquitination concerns protein degradation by the proteasome, elicits severe gastroenteritis representing one of the major which is commonly mediated by K48- or K11-linked Ub chains causes of food and waterborne disease. In order to monitor the
8 Cell News 02/2017 NIKON YOUNG SCIENTIST AWARD 2017
ubiquitinome upon Salmonella infection, we established a quan- implying that LUBAC is stimulated upon bacterial exposure (Fig. titative proteomics platform for the detection of endogenous 2a,b). Moreover, Salmonella-driven M1-ubiquitination was ac- ubiquitinated proteins (Fig. 1a). Importantly, we combined the companied with the downstream activation of NF-KB signaling. use of different workflows to characterize ubiquitinated sub- As previous reports indicated that Salmonella effector proteins strates both on the level of their modified lysine residues as well SopE, SopE2 and SopB contribute to bacterial NF-KB stimulation as attached polyubiquitin chains. To globally map ubiquitination (Bruno et al., 2009), we tested whether these effectors have sites, we performed stable isotope of amino acids in cell culture an impact on LUBAC activity. Indeed, a triple knockout strain (SILAC)-coupled diGly proteomics experiments. Developed in deficient in SopE/SopE2/SopB demonstrated an impaired ability recent years this methodology is based on the immunoprecipita- to induce both linear ubiquitination as well as downstream NF- tion of diGly-remnant containing peptides, which directly result KB activation (Fig. 2b). Notably, treatment of cells with the HOIP from tryptic digestion of ubiquitinated proteins (Ordureau et al., inhibitor Gliotoxin significantly impaired theSalmonella induced 2015). Using this approach, we were able to map multiple thou- pro-inflammatory response supporting the notion that LUBAC sand novel ubiquitination events derived from both host and activity is required for these events (Fig. 2c). Using linear Ub pathogen including previously uncharacterized modifications of specific immunoprecipitation in combination with SILAC based secreted bacterial effectors and outer-membrane proteins. Unbi- quantitative proteomics we were able to identify the complete ased gene ontology enrichment analysis of this data set revealed set of Salmonella-induced LUBAC substrates (Fig. 2d). These several significantly over-represented biological processes included proteins involved in multiple NF-KB activating path- including "regulation of actin cytoskeleton" and "regulation of ways. We identifiedSalmonella induced linear ubiquitination of apoptosis". These categories nicely corroborate previous findings IRAK1 and IRAK4, events previously linked to the activation of that Salmonella infection impacts inflammation and cell archi- TLR signaling (Emmerich et al., 2013). Infection also induced the tecture respectively (Fig. 1b). M1-ubiquitination of RIPK2 indicative of NOD stimulation (Fiil et al., 2013). These findings are consistent with the ability of bacterial products to stimulate these PRRs, as LPS and Flagellin The host inflammatory response toSalmonella can stimulate TLR4 and TLR5 respectively and bacterial peptido- requires the formation of linear ubiquitin chains glycan is sensed via NOD1/2.
Observed changes in the ubiquitination patterns revealed a number of important insights including the identity of multiple Bacterial manipulation of the host ubiquitin system pathogen-stimulated inflammatory pathways establishing a role - discovering host targets of the Salmonella E3 ligase for linear ubiquitin chains in bacteria triggered inflammation. SopA Salmonella infection as such is a complex stimulus expected to trigger multiple different inflammatory pathways unlike the Although prokaryotes lack the canonical Ub-proteasome system, treatment of cells with a single cytokine. Salmonella is there- a wide range of bacterial pathogens acquired strategies to fore able to stimulate a variety of both extra- and intracellular exploit the host Ub machinery in order to support their own life pattern recognition receptors via its surface antigens. Moreover, cycle and to counteract host immune defense programs. One live Salmonella impacts cellular inflammatory signaling addi- example is the injection of bacterial encoded E3 ligases into the tionally via the secretion of metabolites and the translocation host cytosol to modify specific targets and to trigger diverse of virulence factors (Gaudet et al., 2015). Salmonella-induced cellular responses (Maculins et al., 2016). Work over the past gastroenteritis for example requires several virulence factors, years has identified a variety of bacterial E3 ligase effectors and which contribute to the induction of inflammation (Bruno et began to elucidate their impact on host proteins and signaling al., 2009). The analysis of the ubiquitinome upon Salmonel- cascades. Among these, the ligase effector SopA from Salmo- la infection of epithelial cells revealed the clear presence of nella typhimurium constitutes a very striking example of so an inflammation signature within the data (Fig. 1b). Robustly called molecular mimicry. Despite the lack of obvious sequence increased ubiquitination was observed for multiple regulators of homology to eukaryotic HECT E3s, SopA adopts a HECT-like the NF-KB pathway. Importantly, we detected highly increased architecture consisting of N- and C-lobe connected by a flexible ubiquitination sites throughout all subunits of LUBAC (RNF31/ helical element (Diao et al., 2008). While these previous studies HOIP, HOIL, SHARPIN). These data suggested that LUBAC activity have characterized the biochemical activity of SopA and im- might be stimulated upon Salmonella infection. In order to test plicated it in the induction of the enteritis phenotype, the host this hypothesis, we immunoprecipitated linear polyubiquitinated targets and molecular functions of this virulence factor have proteins upon exposure of cells to Salmonella under denaturing remained unknown. In our studies, we established the use of conditions using a previously characterized M1-Ub specific anti- ubiquitin proteomics for the identification of SopA substrates in body (Matsumoto et al., 2012). Our experiments revealed an in- pathogen-infected cells (Fig. 3a). These experiments interesting- crease in linear ubiquitin chains within 15 minutes of infection ly revealed human TRIM56 and TRIM65 as bona fide substrates
Cell News 01/2017 9 NIKON YOUNG SCIENTIST AWARD 2017
of SopA (Fig. 3b). Using biochemical analysis, we elucidated the References substrate recognition mechanism of SopA, which recognizes these two TRIM proteins by binding their catalytic RING domain. Bruno, V.M., Hannemann, S., Lara-Tejero, M., Flavell, R.A., Klein- Despite the presence of multiple hundred RING domain-containing stein, S.H., and Galan, J.E. (2009). Salmonella Typhimurium type proteins in the human proteome, we find SopA to harbor incred- III secretion effectors stimulate innate immune responses in ible specificity for its two targets TRIM56 and TRIM65. By solv- cultured epithelial cells. PLoS Pathog 5, e1000538. ing the crystal structure of SopA in complex with the TRIM56 Diao, J., Zhang, Y., Huibregtse, J.M., Zhou, D., and Chen, J. (2008). RING domain, we addressed the atomic basis of this selectivity Crystal structure of SopA, a Salmonella effector protein mimick- (Fig. 3c). Moreover, our biochemical and structural data indicate ing a eukaryotic ubiquitin ligase. Nat Struct Mol Biol 15, 65-70. that SopA-mediates inhibition of its two targets via a dual Emmerich, C.H., Ordureau, A., Strickson, S., Arthur, J.S., Pedrioli, mechanism. On the one hand SopA binding to the TRIM RING P.G., Komander, D., and Cohen, P. (2013). Activation of the ca- domain dampens TRIM E3 ligase activity by obstructing the nonical IKK complex by K63/M1-linked hybrid ubiquitin chains. RING-E2 binding site, while on the other hand SopA´s HECT-like Proc Natl Acad Sci U S A 110, 15247-15252. activity mediates ubiquitination and proteasomal degradation Fiil, B.K., Damgaard, R.B., Wagner, S.A., Keusekotten, K., Fritsch, of TRIM56 and TRIM65. Previous work has demonstrated the M., Bekker-Jensen, S., Mailand, N., Choudhary, C., Komander, involvement of TRIM56 and TRIM65 in the stimulation of type D., and Gyrd-Hansen, M. (2013). OTULIN restricts Met1-linked I interferon expression upon detection of foreign nucleic acids ubiquitination to control innate immune signaling. Mol Cell 50, (Kamanova et al., 2016). In light of these reports, our identifi- 818-830. cation of bacterial intervention via an inhibitory SopA-TRIM56/ Gaudet, R.G., Sintsova, A., Buckwalter, C.M., Leung, N., Cochrane, TRIM65 relationship suggests an uncharacterized function of A., Li, J., Cox, A.D., Moffat, J., and Gray-Owen, S.D. (2015). IN- interferons in gastroenteritis (Fig. 3d). NATE IMMUNITY. Cytosolic detection of the bacterial metabolite HBP activates TIFA-dependent innate immunity. Science 348, 1251-1255. Conclusions and outlook Gomes, L.C., and Dikic, I. (2014). Autophagy in antimicrobial immunity. Mol Cell 54, 224-233. The presented results illustrate the broad impact that ubiquiti- Hershko, A., Ciechanover, A., and Varshavsky, A. (2000). Basic nation has during an infection with Gram negative bacterial Medical Research Award. The ubiquitin system. Nat Med 6, pathogens. In this context, Ub driven processes range from 1073-1081. the control of invasion and inflammation to the regulation Hu, H., and Sun, S.C. (2016). Ubiquitin signaling in immune of cellular autophagy and endo-lysosomal systems. Our work responses. Cell Res 26, 457-483. identifies a large number of previously unknown host-pathogen Kamanova, J., Sun, H., Lara-Tejero, M., and Galan, J.E. (2016). interaction nodes providing a rich resource for future studies. In The Salmonella Effector Protein SopA Modulates Innate Immune particular, the identification of interferon-regulating TRIM E3 Responses by Targeting TRIM E3 Ligase Family Members. PLoS ligases as host targets of the Salmonella effector ligase SopA Pathog 12, e1005552. suggests a so far unappreciated role for type I interferon in the Kirisako, T., Kamei, K., Murata, S., Kato, M., Fukumoto, H., Kanie, intestinal pathogenesis of S. Typhimurium and opens up multiple M., Sano, S., Tokunaga, F., Tanaka, K., and Iwai, K. (2006). A new lines of investigation. ubiquitin ligase complex assembles linear polyubiquitin chains. EMBO J 25, 4877-4887. LaRock, D.L., Chaudhary, A., and Miller, S.I. (2015). Salmonellae interactions with host processes. Nat Rev Microbiol 13, 191- 205. Maculins, T., Fiskin, E., Bhogaraju, S., and Dikic, I. (2016). Bacte- ria-host relationship: ubiquitin ligases as weapons of invasion. Cell Res 26, 499-510. Matsumoto, M.L., Dong, K.C., Yu, C., Phu, L., Gao, X., Hannoush, R.N., Hymowitz, S.G., Kirkpatrick, D.S., Dixit, V.M., and Kelley, R.F. (2012). Engineering and structural characterization of a linear polyubiquitin-specific antibody. J Mol Biol418 , 134-144. Noad, J., von der Malsburg, A., Pathe, C., Michel, M.A., Komander, D., and Randow, F. (2017). LUBAC-synthesized linear ubiquitin chains restrict cytosol-invading bacteria by activating autopha- gy and NF-kappaB. Nat Microbiol 2, 17063.
10 Cell News 02/2017 NIKON YOUNG SCIENTIST AWARD 2017
Ordureau, A., Munch, C., and Harper, J.W. (2015). Quantifying Acknowledgments ubiquitin signaling. Mol Cell 58, 660-676. Rittinger, K., and Ikeda, F. (2017). Linear ubiquitin chains: en- I am very grateful to Professor Ivan Dikic for his support. I wish zymes, mechanisms and biology. Open Biol 7. to thank all the present and past members of the Dikic and Beh- Sander, L.E., Davis, M.J., Boekschoten, M.V., Amsen, D., Dascher, rends laboratories. I am thankful to all our internal as well as C.C., Ryffel, B., Swanson, J.A., Muller, M., and Blander, J.M. external collaborators for their help with this work. I also thank (2011). Detection of prokaryotic mRNA signifies microbial viabil- the Boehringer Ingelheim Fonds for supporting my graduate ity and promotes immunity. Nature 474, 385-389. work. Finally, I wish to thank Nikon and the DGZ for selecting van Wijk, S.J., Fiskin, E., Putyrski, M., Pampaloni, F., Hou, J., Wild, and honoring our work. P., Kensche, T., Grecco, H.E., Bastiaens, P., and Dikic, I. (2012). Fluorescence-based sensors to monitor localization and func- tions of linear and K63-linked ubiquitin chains in cells. Mol Cell About the author 47, 797-809. van Wijk, S.J.L., Fricke, F., Herhaus, L., Gupta, J., Hotte, K., Pam- Evgenij Fiškin studied biochemistry at the Freie University Berlin paloni, F., Grumati, P., Kaulich, M., Sou, Y.S., Komatsu, M., et al. with support from the German National Academic Founda- (2017). Linear ubiquitination of cytosolic Salmonella Typhimuri- tion. Evgenij did his Diploma thesis in the laboratories of Sven um activates NF-kappaB and restricts bacterial proliferation. Diederichs and Lee Zou at the German Cancer Research Center Nat Microbiol 2, 17066. and Harvard Medical School in Boston. He then moved to the Vance, R.E., Isberg, R.R., and Portnoy, D.A. (2009). Patterns of Goethe University Frankfurt, where in 2016 he received his PhD pathogenesis: discrimination of pathogenic and nonpathogenic working as a Boehringer Ingelheim Fonds fellow in the group of microbes by the innate immune system. Cell Host Microbe 6, Ivan Dikic at the Institute of Biochemistry. His postdoctoral re- 10-21. search is focused on the investigation of novel cytoskeletal and Wild, P., Farhan, H., McEwan, D.G., Wagner, S., Rogov, V.V., Brady, inflammatory regulators during bacterial pathogenesis using a N.R., Richter, B., Korac, J., Waidmann, O., Choudhary, C., et al. combination of high-accuracy mass-spectrometry, cell biological (2011). Phosphorylation of the autophagy receptor optineurin and biochemical approaches. restricts Salmonella growth. Science 333, 228-233. Yau, R., and Rape, M. (2016). The increasing complexity of the ubiquitin code. Nat Cell Biol 18, 579-586. Zheng, N., and Shabek, N. (2017). Ubiquitin Ligases: Structure, Function, and Regulation. Annu Rev Biochem.
Evgenij Fiskin and Frank Schütze (Nikon GmbH)
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a LC-MS SCX LysC / Trypsin digest anti-K-GG IP pH Data analysis
GG-K 4.5 K-GG 5.5 6.5 7.5
K-GG Intensity 8.5
m/z anti-K48 pUb IP LC-MS Trypsin digest Data analysis
Uninfected GG-K Lys0 Arg0 K-GG K48 Intensity K K-GG
m/z S. Typhimurium LC-MS anti-M1 pUb IP Data analysis Lys8 Arg10 Trypsin digest
GG-K M1 K-GG Intensity K K-GG m/z
b GG-K K-GG Log2 (H:L) # of regulated ubiquitination sites
K-GG 1 2 3 4 5 ≥6 -2.5 5 1 MYO1B PFN1 BNIP2 MYO1E NRAS ARHGDIA HSPA6 EPN1 NEDD4L ATP6V0A1 BIRC2 PRKCI SH3RF1 IQGAP1 IQGAP2 WASL FAS ARHGAP1 DAG1 CLTC ATP6V1H PDCD6IP CLINT1 EGFR HSPA8 CAPN1 TRAF6 CDC42 TRAF2 BCAR1 ABI2 RAC1 RELA ARHGAP29 IKBKG STAM TOM1 RAB14 FAM105B EPS15 RAB10 ITGB4 MSN ITGB1 PIP5K1A UBE2L3 RHOG GRTP1 NFKB1 RNF31 COPA ANXA1 STOM TFRC STAM2 STAT3 RDX EZR SHARPIN PIP5K1C PI4KA TRIM25 TNIP1 RALA RBCK1 SNX9 SH3BP4 PPP1R1A2
Regulation of actin cytoskeleton Regulation of apoptosis Internal side of plasma membrane
Figure 1: Global Analysis of the Ubiquitinome in human epithelial cells infected with Salmonella typhimurium (a) Experimental workflows of complementary SILAC-based ubiquitin proteomics methods used to monitor changes in the ubiquitination status of proteins upon Salmonel- la infection. (b) Known protein-protein interactions of protein hits harboring regulated ubiquitination sites (log2 (H:L) ratio >1 or <-1). Hits were clustered based on proces- ses found over-represented in gene ontology enrichment analysis. Node size and color for each protein indicate the number of regulated diGly sites and their mean log2 (H:L) ratio, respectively. Adapted from Fiskin et al., 2016
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a b IP: M1 1.4 Ex1 HCT116 (0.5h) Ex2 ∆SopB/ 1.2 SL1344 WT E/E2 1.0 (min) 0 15 30 0 15 30 ) L : 0.8 245 H
( IB: M1-Ub
180 2 0.6 135 100 Log 0.4 35 IB: IκBα 0.2 Input 48 IB: pJNK 0.0 63 48 IB: Tubulin -0.2 M1 K6 K11 K27 K29 K33 K48 K63 6
R = 0.81 c d 5 TRAF6
4 TRAF2 BIRC2 4 TIFA TNIP1 3 IKBKG IRAK1 Gliotoxin (µM) 0 0.3 0.6 RNF31 IRAK4 BCL10 SL1344 (min) 0 15 30 0 15 30 0 152 30 2 RBCK1 TAB2 35 IB: IκBα TRAF3 ZNRF2 TANK RIPK2 (H:L) Rep2 1 48 IB: pJNK 2 MALT1 N4BP1 0 -4 -3 63 IB: Tubulin Log 48 -5-2 -1 1 2 3 4 5 -1 Ratio H/L normalized Rep2
-2 -2 Log (H:L) Rep1 -3 2 -4 -6
Figure 2: Linear poly-ubiquitination-4 is required-6 for Salmonella-5 -4 -induced-3 -2inflammation-1 0 1 2 3 4 5 6 (a) Salmonella infection induces the formation of linear M1-linked ubiquitin chains. Log2Ratio H/L (H:L) normalized ratios Rep1 of all 8 types of ubiquitin chains are displayed. (b) The formation of linear Ub chains and-5 activation of NF-κB is dependent on the presence of Salmonella virulence factors SopE/E2/B. Lysates from infected epithelial cells were subjected to immunoblotting using the indicated antibodies. (c) Inhibition of LUBAC activity by Gliotoxin dampens the inflammatory response upon bacterial infection. Lysates from infected epithelial cells were subjected to immunoblotting using the indicated antibodies. (d) M1-Ub proteomics identifies bona fide linear polyubiquitinated proteins. Scatter plot displaying log2 (H:L) ratios of linear Ub modified proteins recovered upon Salmonella infection. Adapted from Fiskin et al., 2016
a HCT116 infected d with Salmonella SopA
Light (K0) Heavy (K8) ΔsopA SL1344 WT P P P TRIM56 TRIM65 RIG-I MDA-5 Cell Lysis In-solution digest LysC/Trypsin + + anti-K(ε)-GG-IP Strong cation-exchange chromatography Peptide identification LC-MS/MS MAVS MAVS MAVS MAVS
b SopA-dependent ubiquitination sites 3 TRIM56 2.5
2 TRIM65 2 P 1.5 TBK1 P 1 1 IKKε 0.5 0 -2.5 -2 -1.5 -1 -0.5 0.5 1 1.5 2 2.5 Ratio H/L normalized Rep2
(H:L) Rep2 -0.5 2 -1 -1 P IRF3 Log -1.5
-2 -2 -2.5
-3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 3 Ratio H/L normalized Rep1 Log2(H:L) Rep1 c Type I IFN N
C N
TRIM56 RING β-helix domain
C
N-lobe
Figure 3: The secreted Salmonella E3 ligase effector SopA targets human TRIM56 and TRIM65 ligases to dampen the interferon response upon infection (a) Design and workflow of the SILAC ubiquitin proteomics experiment used to identify targets of Salmonella virulence factor SopA upon infection. (b) Ubiquitin proteomics recovers human TRIM56 and TRIM65 as targets of SopA. Scatter plot of two replicated experiments displaying log2 (H:L) ratios is shown. (c) Crystal structure of SopA (163-425) in complex with TRIM56 RING domain. SopA binding is inhibiting TRIM ligase activity (d) Model for inhibition of host interferon signaling by SopA-mediated targeting and degradation of TRIM56 and TRIM65. Adapted from Fiskin et al., 2017
Cell News 01/2017 13 BINDER INNOVATION PRIZE 2017
Sara A. Wickström
Regulation of epidermal stem cell fate by niche-derived signals and mechanical forces
Paul Gerson Unna Group ‘Skin Homeostasis and Ageing’, Max Planck Institute for Biology of Ageing, Cologne, Germany.
Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Germany
Summary ly to disturbances and to efficiently restore proper functions. Our research aims to understand how complex but stereotyped However, such mechanisms of population-level regulation are tissues are formed, maintained and regenerated through local poorly understood. deformation, growth, differentiation and remodeling. To decipher this fundamental question it is important to understand how SCs reside in spatially distinct microenvironments termed niches single cell behaviors are coordinated on the population level and that consist of neighboring cells, extracellular matrix and signals how population-level dynamics is coupled to tissue architecture. derived from these compartments. Niches integrate signals that Uncovering these regulatory principles will further facilitate de- control the balanced response of SCs to the needs of organisms, velopment of stem cell (SC) therapies and effective treatments prevent SC depletion while at the same time restrict excessive against cancers. SC expansion into the surrounding tissue (Blanpain and Fuchs, 2014; Morrison and Spradling, 2008; Scadden, 2014). Although As a self-renewing organ maintained by multiple distinct SC the critical importance of niches in SC regulation has been populations, the mammalian epidermis represents an outstand- established, the complexity of mammalian SC niches has pre- ing, clinically highly relevant research paradigm to address these vented identification of the precise nature of the niche-derived questions. Epidermal and hair follicle stem cells (HFSCs) fuel signals and hindered mechanistic studies of adult SC regulation. tissue self-renewal, repair epidermal injuries and, when deregu- The mammalian epidermis is a self-renewing organ maintained lated, initiate carcinogenesis. We aim to decipher how these SCs by multiple distinct tissue-resident SC populations. Due to interact with their niches to regulate fate decisions and phe- its exceptional ability to combine constant self-renewal with notypic plasticity. In addition, we strive to understand the role extreme structural robustness, the epidermis represents an of mechanical forces in the regulation of nuclear and genomic excellent, and clinically highly relevant research paradigm to architecture, and thereby in gene expression and SC fate. These study SCs and their interactions with the niche. The mammalian focus areas are pursued by an interdisciplinary research strategy epidermis is composed of a pilosebaceous unit that consists of that builds on mouse genetics and molecular cell biology, com- the hair follicle (HF) and the sebaceous gland, with individu- bined with state-of-the-art biological imaging, biochemistry, al units surrounded by the interfollicular epidermis (IFE). The biophysics and theoretical approaches. various compartments contain distinct SC populations that facilitate the constant renewal of the IFE and HFs during post- natal tissue homeostasis and regeneration (Blanpain and Fuchs, The hair follicle stem cell niche as a paradigm for 2009). The bulge niche of the HF harbors quiescent hair follicle adult stem cell studies stem cells (HFSCs) that fuel cyclical bouts of HF regeneration and represent an outstanding paradigm for uncovering funda- Adult somatic SCs fuel tissue renewal, repair, and remodeling to mental principles of somatic tissue-resident SC regulation and maintain organ structure and function by tuning their prolif- organ self-assembly (Fig. 1). HFSCs are activated in a two-step eration and differentiation rates to match the changing needs process: first, quiescent HFSCs are activated to generate primed of their resident tissues. Given their potency, even incremental HFSCs that in a second step establish a pool of transit-amplify- alterations in SC behavior could lead to substantial chang- ing cells (TACs) (Greco et al., 2009; Hsu et al., 2014b). TACs are es in tissue size and architecture. Yet, these types of effects a transition state between SCs and their differentiated progeny, are strikingly rare, strongly implying that SCs are under tight and their generation is a rate-limiting step in SC differentiation homeostatic regulation that allows the system to react rapid- (Fig. 1). It has recently been demonstrated that TACs coordinate
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the self-renewal of HFSCs during regeneration by signaling back the key molecular components of the niche: a 3-dimensional to the SCs to control their activation (Hsu et al., 2014b). When (3D) ECM microenvironment, FGF-2, VEGF and EGF, that not a subset of HFSCs is activated at the onset of the HF growth only maintain but strikingly also promote stemness ex vivo phase, they leave the niche to generate a new HF that grows (Chacon-Martinez et al., 2016). downward into the dermis (Muller-Rover et al., 2001). Intriguingly, studies in this system have led us to uncover that cultured epidermal cell mixtures self-evolve into a dynamic Interestingly, when restricted by tissue architecture, the specific population equilibrium state of HFSCs and progenitors, as shown SC pools within the skin epidermis remain strictly compartmen- by lineage tracing and transcriptomics analyses (Chacon-Mar- talized, and the pilosebaceous unit is maintained independent tinez et al., 2016). This involves bidirectional signaling crosstalk of the IFE in the absence of wounding (Levy et al., 2005; Levy et mediated by Sonic hedgehog (Shh)- and Bone morphogenetic al., 2007; Nowak et al., 2008). However, upon epidermal injury protein (BMP) pathways, that have been previously implicated in or removal from tissue and subsequent transplantation, the regulating the crosstalk between HFSCs and their TAC progeny specialized SCs exhibit broader potency in their new microen- (Hsu et al., 2014a), highlighting that our culture system faithful- vironment. For example, transplanted HFSCs generate not only ly recapitulates complex signaling networks of the in vivo niche. HFs, but also IFE and sebaceous glands (Morris et al., 2004; Strikingly, we observe that the bidirectional interconversion of Oshima et al., 2001). This strongly indicates that differences HFSCs and progenitor cells drives the system into equilibrium in the molecular composition of the HF and IFE niches tightly proportions in a dynamic, self-organizing process. Moreover, instruct SC lineage progression, but the mechanisms remain HFSCs can be derived completely de novo even from purified poorly understood. populations of epidermal non-HFSCs (Chacon-Martinez et al., Surprisingly, lineage tracing and ablation studies have demon- 2016). Consequently, a stable HFSC – non-HFSC equilibrium strated that HFSCs are dispensable for HF regeneration and that can evolve from a pure population of non-HFSCs (Fig.2). This activated progeny re-populate the ablated SC compartment not only corroborates previous studies showing that activated to sustain hair regeneration (Hsu et al., 2011; Rompolas et al., progeny re-populate an ablated SC niche and subsequently 2013). This indicates that the niche might be able to instruct adopt a SC fate (Hsu et al., 2014a; Rompolas et al., 2013), but committed progenitors to be reprogrammed to a SC state. defines a set of factors that can drive this reprogramming. The Collectively these studies underscore the need to understand the dynamic reprogramming and tunable nature of the HFSC cul- complexity of the signaling circuitry governing HFSC identity tures together with the absence of terminal differentiation also and behavior. distinguishes our system from the classical organoid cultures (Sato and Clevers, 2013).
Mechanisms and functions of stem cell-niche interactions in stem cell fate decisions and Mechanical regulation of SC fate reprogramming The use of SCs in regenerative medicine is being intensively Intestinal SC organoid cultures that recapitulate the prolifera- explored due to their potential to generate or repair tissues in a tive capacity and multipotency of their in vivo counterparts have sustained manner. However, hematopoietic SCs and progenitors been extremely successful in elucidating mechanistic details on that are being used in treatment of hematopoietic disorders intestinal SC biology (Sato and Clevers, 2013). In contrast, the remain the only SC type that has reached the clinics. Although lack of a system that recapitulates the in vivo niche, enabling various SCs can be isolated and studied in vitro, these cells maintenance of HFSCs in the absence of other heterologous generally lose key functions, limiting their potential for tissue cell types, and allowing precise manipulation and monitoring engineering or organogenesis. This limitation had led to exten- of HFSC fate decisions has been one of the major obstacles in sive efforts to identify specific molecules and cellular compo- uncovering fundamental principles of HFSC regulation. nents of SC niches that would promote SC function or retain the We have recently developed an ex vivo culture system that, for stemness state (Shin and Mooney, 2016). Importantly, advances the first time, allows to enrich and maintain HFSCs without loss in biomaterials and in the ability to experimentally address me- of their multipotency (Chacon-Martinez et al., 2016) (Fig.2). chanical aspects of biology have led to a key new paradigm in We have previously deciphered how cells, through their ability SC biology: SCs generate forces and sense physical properties of to generate force at cell-matrix adhesions, remodel their own the matrix through adhesion, which activate signaling cascades extracellular matrix (ECM) microenvironment (Radovanac et to control SC fate and function (Fedorchak et al., 2014; Heisen- al., 2013). We further demonstrated that the precise molecular berg and Bellaiche, 2013). Thus, understanding the mechanisms composition of the ECM within the HFSC niche is critical for that sense physical forces and how they control organ growth maintaining HFSC quiescence (Morgner et al., 2015). Using this and patterning through SC fate and self-organization is a key knowledge as a starting point we were able to further identify unresolved step towards generation of successful SC therapies.
Cell News 01/2017 15 BINDER INNOVATION PRIZE 2017
We have recently uncovered a fundamental molecular mechanism strain. Taken together, our results reveal how mechanical signals for this process in epidermal SCs, where we observe that extrin- integrate transcriptional regulation, chromatin organization and sic forces modulate chromatin compaction and epigenetic gene nuclear architecture to control lineage commitment and nuclear silencing through actin-dependent remodeling of the nuclear mechanoadaptation (Fig. 3). envelope (Le et al., 2016). To decipher how mechanical forces regulate SC identity, we sought to identify pathways that respond to force and establish their functional significance in SC fate Outlook determination. We observed that a mechanosensory complex of emerin (Emd), non-muscle myosin IIA (NMIIA) and actin relays ex- We will continue combining in vivo studies with innovative in vitro trinsic mechanical forces to the nucleus to control gene silencing models and apply scale-bridging technologies, from single mole- and chromatin compaction state (Le et al., 2016). cule-level atomic force microscopy to genome-level analysis and in Functionally, chromatin condensation typically results in gene vivo organismal imaging, to establish quantitative principles of epi- silencing, while allowing the selective access of the transcription dermal tissue maintenance through stem cells and to identify the machinery to some lineage-specific and constitutively expressed key processes of ageing-induced decline. Furthermore, our ongoing genes within euchromatin (Pombo and Dillon, 2015; Tessarz and screening approaches using the 3C SC culture technology will en- Kouzarides, 2014). Yet, such nuclear rearrangements also have able us to identify molecular targets with direct clinical relevance. direct implications for the cellular response to mechanical cues. It has been observed that densely packed heterochromatin and more loosely packed euchromatin have different mechanical proper- Acknowledgements ties. Specifically, the nuclear interior becomes more viscous and deformable upon decondensation of the tightly-packed heteroch- I would like to thank all past and present members of my lab- romatin (Chalut et al., 2012; Spagnol and Dahl, 2016). We observe oratory for their fantastic work and enthusiasm towards their that force-driven enrichment of Emd at the outer nuclear mem- projects. I would also like to thank all the great mentors that brane of epidermal stem cells leads to defective heterochromatin have supported me throughout my scientific career, includ- anchoring to the nuclear lamina, and a switch from H3K9me2,3 ing Jorma Keski-Oja, Kari Alitalo, Reinhard Fässler, Thomas to H3K27me3 occupancy at constitutive heterochromatin (Le Krieg, and Carien Niessen. Research in my laboratory has been et al., 2016). Putting this into the context of the previous work, supported by the Max Planck Society, the Max Planck Förder- we hypothesize that the switch from H3K9me2,3 to H3K27me3 stiftung, the Behrens Weise Foundation, and the Deutsche occupancy could play a role in the nuclear mechanoresponse by Forschungsgemeinschaft through SFB 829 and WI 4177/2. making the nucleus more elastic and thereby more resistant to deformation. A number of recent studies have expanded the role of actin be- About the author yond that of the cytoplasmic polymeric form, and several studies implicate monomeric nuclear as a critical co-factor for a number Sara Wickström studied medicine at the University of Helsinki, Fin- of transcription factors as well as for the RNA polymerases them- land, and obtained her MD in 2001. In 2004 she obtained her PhD selves (Grosse and Vartiainen, 2013; Treisman, 2013; Virtanen and in the group of Jorma Keski-oja. She then moved to the Max Planck Vartiainen, 2017). Interestingly, we found that Emd enrichment at Institute of Biochemistry, Martinsried to do her postdoctoral work the outer nuclear membrane is also accompanied by the recruit- with Reinhard Fässler. Since 2010 she is a Max Planck Research ment of NMIIA to promote local actin polymerization that reduces Group Leader at the Max Planck Institute for Biology of Ageing. nuclear actin levels, which results in attenuation of global RNA polymerase II-mediated transcription. This global transcriptional repression leads to accumulation of H3K27me3 at specific targets of the polycomb repressive complex 2 (PRC2) target genes, which in the case of epidermal SCs are the terminal differentiation genes (Ezhkova et al., 2009; Le et al., 2016). Consequently, tran- scription of these genes is inhibited, leading to attenuated termi- nal differentiation of SCs in the presence of strain. Importantly, restoring nuclear actin levels in the presence of mechanical stress counteracts PRC2-mediated silencing of transcribed genes (Le et al., 2016). The precise molecular mechanism(s) by which nuclear actin regulates transcription remain open for further studies, but this work provides initial evidence for direct coupling of cytoskeletal and transcriptional states in response to mechanical Thomas M. Magin (DGZ), Sara A. Wickström, André Bachmann (BINDER GmbH)
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References Morgner, J., Ghatak, S., Jakobi, T., Dieterich, C., Aumailley, M., and Wickstrom, S.A. (2015). Integrin-linked kinase regulates the Blanpain, C., and Fuchs, E. (2009). Epidermal homeostasis: a niche of quiescent epidermal stem cells. Nature communications balancing act of stem cells in the skin. Nat Rev Mol Cell Biol 10, 6, 8198. 207-217. Morris, R.J., Liu, Y., Marles, L., Yang, Z., Trempus, C., Li, S., Lin, Blanpain, C., and Fuchs, E. (2014). Stem cell plasticity. Plastici- J.S., Sawicki, J.A., and Cotsarelis, G. (2004). Capturing and profil- ty of epithelial stem cells in tissue regeneration. Science 344, ing adult hair follicle stem cells. Nat Biotechnol 22, 411-417. 1242281. Morrison, S.J., and Spradling, A.C. (2008). Stem cells and niches: Chacon-Martinez, C.A., Klose, M., Niemann, C., Glauche, I., and mechanisms that promote stem cell maintenance throughout Wickström, S.A. (2016). Hair follicle stem cell cultures reveal life. Cell 132, 598-611. self-organizing plasticity of stem cells and their progeny. EMBO Muller-Rover, S., Handjiski, B., van der Veen, C., Eichmuller, J. S., Foitzik, K., McKay, I.A., Stenn, K.S., and Paus, R. (2001). A Chalut, K.J., Hopfler, M., Lautenschlager, F., Boyde, L., Chan, C.J., comprehensive guide for the accurate classification of murine Ekpenyong, A., Martinez-Arias, A., and Guck, J. (2012). Chroma- hair follicles in distinct hair cycle stages. J Invest Dermatol 117, tin decondensation and nuclear softening accompany Nanog 3-15. downregulation in embryonic stem cells. Biophys J 103, 2060- Nowak, J.A., Polak, L., Pasolli, H.A., and Fuchs, E. (2008). Hair 2070. follicle stem cells are specified and function in early skin mor- Ezhkova, E., Pasolli, H.A., Parker, J.S., Stokes, N., Su, I.H., Hannon, phogenesis. Cell Stem Cell 3, 33-43. G., Tarakhovsky, A., and Fuchs, E. (2009). Ezh2 orchestrates gene Oshima, H., Rochat, A., Kedzia, C., Kobayashi, K., and Barrandon, expression for the stepwise differentiation of tissue-specific Y. (2001). Morphogenesis and renewal of hair follicles from adult stem cells. Cell 136, 1122-1135. multipotent stem cells. Cell 104, 233-245. Fedorchak, G.R., Kaminski, A., and Lammerding, J. (2014). Cellu- Pombo, A., and Dillon, N. (2015). Three-dimensional genome lar mechanosensing: Getting to the nucleus of it all. Progress in architecture: players and mechanisms. Nat Rev Mol Cell Biol 16, biophysics and molecular biology. 245-257. Greco, V., Chen, T., Rendl, M., Schober, M., Pasolli, H.A., Stokes, Radovanac, K., Morgner, J., Schulz, J.N., Blumbach, K., Patter- N., Dela Cruz-Racelis, J., and Fuchs, E. (2009). A two-step mech- son, C., Geiger, T., Mann, M., Krieg, T., Eckes, B., Fässler, R., et al. anism for stem cell activation during hair regeneration. Cell (2013). Stabilization of integrin-linked kinase by the Hsp90- Stem Cell 4, 155-169. CHIP axis impacts cellular force generation, migration and the Grosse, R., and Vartiainen, M.K. (2013). To be or not to be as- fibrotic response. EMBO J32 , 1409-1424. sembled: progressing into nuclear actin filaments. Nat Rev Mol Rompolas, P., Mesa, K.R., and Greco, V. (2013). Spatial organiza- Cell Biol 14, 693-697. tion within a niche as a determinant of stem-cell fate. Nature Heisenberg, C.P., and Bellaiche, Y. (2013). Forces in tissue mor- 502, 513-518. phogenesis and patterning. Cell 153, 948-962. Sato, T., and Clevers, H. (2013). Growing self-organizing mini- Hsu, Y.C., Li, L., and Fuchs, E. (2014a). Emerging interactions guts from a single intestinal stem cell: mechanism and applica- between skin stem cells and their niches. Nat Med 20, 847-856. tions. Science 340, 1190-1194. Hsu, Y.C., Li, L., and Fuchs, E. (2014b). Transit-amplifying cells Scadden, D.T. (2014). Nice neighborhood: emerging concepts of orchestrate stem cell activity and tissue regeneration. Cell 157, the stem cell niche. Cell 157, 41-50. 935-949. Shin, J.W., and Mooney, D.J. (2016). Improving Stem Cell Thera- Hsu, Y.C., Pasolli, H.A., and Fuchs, E. (2011). Dynamics between peutics with Mechanobiology. Cell Stem Cell 18, 16-19. stem cells, niche, and progeny in the hair follicle. Cell 144, 92- Spagnol, S.T., and Dahl, K.N. (2016). Spatially Resolved Quanti- 105. fication of Chromatin Condensation through Differential Local Le, H.Q., Ghatak, S., Yeung, C.C., Tellkamp, F., Gunschmann, C., Rheology in Cell Nuclei Fluorescence Lifetime Imaging. PLoS One Dieterich, C., Yeroslaviz, A., Habermann, B., Pombo, A., Niessen, 11, e0146244. C.M., et al. (2016). Mechanical regulation of transcription con- Tessarz, P., and Kouzarides, T. (2014). Histone core modifications trols Polycomb-mediated gene silencing during lineage commit- regulating nucleosome structure and dynamics. 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Figure. 1 Schematic illustration of HFSC regulation in their bulge niche
Quiescent HFSCs reside in the bulge niche within hair follicles where they are activated in a two-step process to proliferate, to leave the niche, and to differentiate in order to regenerate the hair follicle.
Figure 2: HFSC cultures reveal dynamic bi-directional plasticity between SCs and progenitors. (A)
Mouse epidermis (Epi d0) cultured under various conditions show that 3C conditions (3-dimentional Matrigel with keratino- cyte growth medium (KGM) supplemented with VEGF (V), bFGF (F), Y2732 (Y)) enrich for HFSCs (CD34+α6+), whereas 3C medi- um in 2-dimentional culture (3C 2D) does not support growth. (B) Cultured HFSCs retain their multipotency as shown by trans- plantation assays into nude mice. (C) HFSCs (CD34+) and non- HFSCs (CD34-) self-evolve into a 50:50 population equilibrium over time. The equilibrium can be achieved from either pure HFSCs or non-HFSCs, or any mixed ratio of the two populations (D) 3C-HFSCs (in orange) express key lineage identity transcription factors and closely resemble bona fide CD34+α6+ HFSCs directly isolated from mouse epidermis (in pink), whereas 3C progenitors (CD34-α6+; in green) express intermediate levels of stem cell genes, when compared to freshly purifiedin vivo progenitors (in blue). Modified from Chacon-Martinez et al., 2016
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Figure 3: Transcriptional and rheological adaptation of the nucleus through regulation of the nuclear lamina and chromatin.
We analyzed the effect of mechanical strain on transcription (A) and observed global transcriptional repression (B) with specific enrichment of polycomb repressive complex 2 (PRC2) target gene downregulation through gain of H3K27me3 (C, D), leading to inhibition of lineage progression gene expression (E). Intriguingly, at the same time lamina-associated domains lost H3K9me3, which was replaced by H3K27me3 to maintain si- lencing of these regions (F). The changes in gene silencing were induced by a mechanosensitive switch at the nuclear lamina: assembly of a stress-dissipating, contractile perinuclear actin ring and remodeling of the emerin-containing stiff nuclear lam- ina meshwork. This was accompanied by large-scale chromatin rearrangements as visualized with whole chromosome FISH for Chromosomes (Chr) 1 and 18 (G). Our work indicates that mechanical strain promotes a switch from a lamina-associated H3K9me3 state of heterochromatin to a state characterized by perinuclear actin and more loosely packed and lamina-disasso- ciated H3K27me3 allowing mechanoadaptation. Modified from Le et al., 2016.
Figure 2: HFSC cultures reveal dynamic bi-directional plasticity between SCs and progenitors. (A)
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Cell News 01/2017 19 WERNER RISAU PRIZE 2017
Kerstin Wilhelm
FOXO1 couples metabolic activity and growth state in the vascular endothelium
Max Planck Institute for Heart and Lung Research, Angiogenesis & Metabolism Laboratory, Bad Nauheim, Germany
Endothelial cells (ECs) line the luminal side of blood vessels. hyperplastic vasculature and resulted in the inability of ECs to When tissues are in need of oxygen and nutrients they secrete extend proper sprouts (Fig. 1b–f). Instead, ECs grew in clusters proangiogenic factors, which trigger ECs to become invasive leading to vessel enlargement and blunting of the angiogenic and protrude filopodia. The so-called tip cells lead the sprouts front (Fig. 1d, f). Strikingly, numerous filopodial bursts were and extend their filopodia towards the source of the angiogenic emanating from the stunted front (Fig. 1c, d), suggesting that signal. Tip cells are followed by stalk cells, which proliferate to FOXO1 deficiency results in uncoordinated vascular growth. As- elongate the sprout. Eventually, tip cells connect with tip cells sessment of 5-bromodeoxyuridine (BrdU) incorporation demon- from adjacent sprouts to establish new vessel circuits. This strated a substantial increase in endothelial proliferation in the process of vessel growth from pre-existing vessels is named Foxo1iEC-KO mutants (Fig. 1g, j), indicating that deregulated pro- angiogenesis. Angiogenesis continues until nutrient and oxygen liferation drives this aberrant vessel phenotype. Because of the supply meets tissue demand, proangiogenic molecules are fact that in the majority of mouse mutants the retinal vascular silenced, and ECs become quiescent again. Compared to quies- phenotypes resolve during later stages of retinal vascular devel- cence active sprouting is highly energy demanding1. While the opment, we analyzed the Foxo1iEC-KO mutants at postnatal day 21 tip cells need an extensive amount of ATP, because they have to (P21) when overall vessel morphogenesis is completed. Impor- remodel their cytoskeleton in order to protrude filopodia, stalk tantly, the vascular defects did not normalize at later stages of cells need to double their biomass in order to be able to divide development, but showed a persistent increase in endothelial and proliferate. This suggests that ECs have to adapt their me- number, density and vessel diameter (Fig. 1h, i). We conclude tabolism when switching from quiescence to vascular growth. that FOXO1 is a suppressor of endothelial growth and prolifera- However, how ECs couple their metabolic activity to growth tion, whose inactivation leads to uncontrolled overgrowth. state is poorly understood at the molecular level. We analyzed the role of FOXO in the endothelium. FOXO is an Next, we determined the consequences of FOXO1 activation effector of the phosphatidylinositol-3-OH kinase (PI(3)K)/AKT in ECs. We used a Cre-inducible gain-of-function allele (Foxo- pathway that links growth and metabolism. PI(3)K signalling 1CA) in which the AKT phosphorylation sites are mutated, thus inhibits FOXOs through AKT-mediated phosphorylation leading rendering FOXO1 constitutively nuclear (Fig. 2b)4. Tie2-cre-me- to their nuclear exclusion. We investigated the role of FOXO1 in diated expression of this IRES-GFP-coexpressing mutant (Fox- ECs, an enriched FOXO family member in the endothelium. To o1EC-CA) was incompatible with embryo survival beyond E10.5 this end, we bred floxedFoxo1 mice (Foxo1fl/fl)2 with a Tie2-cre (Fig. 2a), highlighting the sensitivity of ECs towards changes deleter, which recombines in endothelial and haematopoietic in FOXO1 status. We then used the Pdgfb-creERT2 strain to cells. Tie2-cre-mediated deletion of Foxo1 (Foxo1EC-KO) caused express Foxo1CA in the retinal endothelium (Foxo1iEC-CA). Immu- defective vascular development and embryonic lethality around nofluorescence studies revealed an enriched FOXO1 signal in embryonic day (E)10.53, suggesting that endothelial FOXO1 is endothelial nuclei and confirmed the EC-specific expression of essential for embryo development. Immunofluorescence analysis GFP (Fig. 2b). Forced activation of FOXO1 led to a sparse and of developing blood vessels in the postnatal retina showed high hyperpruned vascular network that contained fewer ECs (Fig. levels of FOXO1 expression in the endothelium (Fig. 1a). Further 2c, d, f–h). These retinal vessels established a lumen but were examination of the subcellular distribution revealed a diffuse thinner (Fig. 2g, h). Staining for phospho-histone H3 (pHH3) nucleocytoplasmic localization of FOXO1 at the angiogenic revealed a reduction in EC proliferation in Foxo1iEC-CA mice while front, where most of the EC proliferation occurs, but a stron- endothelial apoptosis was not altered (Fig. 2e, f, i). Given the ger nuclear pattern in the plexus, where vessels remodel, and fact that different vascular beds might cope variably with forced endothelial proliferation abates (Fig. 1a). This spatial difference FOXO1 expression, we investigated the consequence of FOXO1 in subcellular localization suggests that FOXO1 is important activation in the embryonic hindbrain. Similar phenotypes were for governing endothelial growth. To test this, we assessed observed in the hindbrain vasculature (Fig. 2j, k), indicating that the impact of Foxo1 deletion on retinal angiogenesis using FOXO1 is a critical driver of endothelial quiescence. the tamoxifen-inducible, endothelial-selective Pdgfb-creERT2 line (Foxo1iEC-KO). Endothelial loss of Foxo1 caused a dense and We next assessed whether FOXO1 regulates endothelial metab-
20 Cell News 02/2017 WERNER RISAU PRIZE 2017
olism. Since ECs rely on glycolysis for vessel branching1, we first crease in EC number, proliferation and vessel density (Fig. 4f, g). studied the effects of FOXO1 on this metabolic pathway. Trans- We then combined the MycOE, Foxo1CA and Pdgfb-creERT2 alleles duction of human umbilical vein endothelial cells (HUVECs) with to generate endothelial-specific double mutants. Remarkably, a FOXO1CA-encoding adenovirus (AdFOXO1CA) led to a robust re- re-expression of MYC in ECs of Foxo1iEC-CA mice normalized the duction in glycolysis as evidenced by a reduction in extracellular hypobranched and hypocellular vascular phenotype caused by acidification rate (ECAR), glucose uptake, glycolytic flux and lac- FOXO1 activation (Fig. 4h, i). Moreover, coexpression of MYC tate production (Fig. 3a–d). This metabolic phenotype correlates and FOXO1CA in HUVECs restored glycolysis and mitochondrial with the reduced proliferation in FOXO1CA-expressing ECs and respiration (Fig. 4j, k), indicating that regulation of MYC sig- raises a question as to whether FOXO1 promotes mitochondrial nalling by FOXO1 is critical for the coordination of endothelial oxidative phosphorylation. Surprisingly, FOXO1 did not stimulate metabolism and growth. but instead diminished oxidative metabolism as indicated by a decline in oxygen consumption in AdFOXO1CA-expressing HU- This study identifies FOXO1 as a critical checkpoint of endothe- VECs (Fig. 3e). Moreover, reactive oxygen species (ROS) forma- lial growth that restricts vascular expansion. Our data suggest tion were decreased (Fig. 3f). Importantly, FOXO1 did not induce that FOXO1 promotes endothelial quiescence by antagonizing endothelial apoptosis, senescence, autophagy or energy distress MYC, which leads to a coordinated reduction in the proliferative under the same experimental conditions (Fig. 3g). Together, our and metabolic activity of ECs. The FOXO1-induced deceleration data indicate that FOXO1 adapts metabolic activity to the lower of metabolic activity might not only enforce quiescence but also requirements of the quiescent endothelium. To gain insight into support endothelial function. For instance, by lowering metabo- the underlying mechanisms for this adaptability, we performed lism, ECs will consume less energetic fuel for their homeostatic transcriptome analysis of FOXO1CA- and GFP-transduced HU- needs, thereby ensuring efficient nutrient and oxygen delivery. VECs. Gene set enrichment analysis (GSEA) revealed an enrich- Reducing metabolic activity might also contribute to endothe- ment of the FOXO1 DNA-binding elements in genes induced by lial redox balance. ECs are long-lived cells that need to protect FOXO1, while the MYC DNA-binding motif was highly enriched themselves against oxidative damage exerted by high oxygen in the repressed genes (Fig. 3h). Moreover, MYC target gene levels in the bloodstream. The FOXO1-induced reduction in signatures were downregulated in the FOXO1 transcriptome (Fig. oxidative metabolism might thus be a mechanism to minimize 3i). Since MYC is a powerful driver of glycolysis, mitochondrial the production of mitochondria-derived ROS, thereby conferring metabolism and growth5, FOXO1 might antagonize endothelial protection against the high-oxygen environment. Such a role MYC signalling. In line with this, overexpression of FOXO1CA sup- of FOXO1 in endothelial metabolism aligns with the broader pressed MYC expression and protein levels in HUVECs (Fig. 3j–l). function of FOXOs in mediating oxidative stress resistance11-13, Accordingly, numerous genes that are induced by MYC were and might also explain why ECs are exquisitely sensitive to a downregulated in FOXO1CA-overexpressing HUVECs, including change in FOXO1 status. It will be interesting to determine how genes involved in cell metabolism and cell cycle progression (Fig. endothelial FOXO1 is regulated in vivo and how deregulation 3i, l). This regulation is in line with the repression of MYC by contributes to disease. FOXOs in cancer cells5-7 and points to MYC as a crucial effector of FOXO1 in the coordination of endothelial metabolism and About the author growth. Remarkably, FOXO1 also induced the expression of neg- ative regulators of MYC signalling including MXI1, an antagonist Kerstin Wilhelm (PhD) of MYC transcriptional activity8, and FBXW7, an E3 ubiquitin Max Planck Institute for Heart and Lung Research, Angiogen- ligase that targets MYC for proteasomal degradation8 (Fig. 3l). esis & Metabolism Laboratory, Ludwigstraße 43, 61231 Bad These data suggest that FOXO1 intersects with MYC signalling Nauheim, Germany at different levels. To explore further the role of MYC in ECs, E-mai: [email protected] we analyzed the consequences of MYC inactivation for endo- thelial metabolism. Bioenergetic analysis revealed that MYC 10/2016 to Present: Postdoctoral fellow deficiency attenuated glycolysis and mitochondrial respiration Max-Planck Institute for Heart and Lung Research Bad Nauheim, (Fig. 4a, b). Conditional deletion of Myc (Mycfl/fl)9 in mice using Germany the Pdgfb-creERT2 deleter impaired vascular expansion and led to a thinned and poorly branched vasculature (Fig. 4c–e). These 11/2012 - 09/2016 PhD Student phenotypes resemble the vascular defects in Foxo1iEC-CA mutant Max Planck Institute for Heart and Lung Research Bad Nauheim, mice and imply that MYC is a central component of endothelial Germany FOXO1 signalling. To test this directly, we attempted to rescue 10/2010 - 10/2012 M.Sc. Molecular Medicine the endothelial phenotypes imposed by FOXO1 activation by re- Friedrich Schiller University, Jena, Germany storing MYC signalling with a Cre-inducible Myc overexpressor allele (MycOE)10. Pdgfb-creERT2-induced overexpression of MYC 10/2007 - 09/2010 B.Sc. Biochemistry caused sustained vascular overgrowth and led to a profound in- University of Bayreuth, Bayreuth, Germany
Cell News 01/2017 21 WERNER RISAU PRIZE 2017
Acknowledgement References Though only my name appears on the cover of this work, a great many people have contributed to its production. I owe my 1. De Bock, K., Georgiadou, M. & Carmeliet, P. Role of Endo- gratitude to all those people who have made this publication thelial Cell Metabolism in Vessel Sprouting. Cell Metab. 18, possible. Especially, I would like to express my sincere gratitude 634–647 (2013). to my advisor Michael Potente for his support, motivation and 2. Keller, C. et al. Alveolar rhabdomyosarcomas in conditional enthusiasm. I’m deeply grateful for all his scientific inputs and Pax3:Fkhr mice: cooperativity of Ink4a/ARF and Trp53 loss of discussions, as well as for the excellent environment that I function. Genes Dev. 18, 2614–2626 (2004). experienced at the Max Planck Institute in Bad Nauheim. 3. Sengupta, A., Chakraborty, S., Paik, J., Yutzey, K. E. & Ev- I am also grateful to the Werner Risau Prize committee and the ans-Anderson, H. J. FoxO1 is required in endothelial but not DGZ for recognizing my work. I feel honored being part of the myocardial cell lineages during cardiovascular development. vascular biology community, which Werner Risau pioneered. Dev. Dyn. 241, 803–813 (2012). 4. Stöhr, O. et al. Insulin receptor signaling mediates APP pro- cessing and β-amyloid accumulation without altering survival in a transgenic mouse model of Alzheimer's disease. Age (Dordr) 35, 83–101 (2013). 5. Delpuech, O. et al. Induction of Mxi1-SR alpha by FOXO3a contributes to repression of Myc-dependent gene expression. Mol. Cell. Biol. 27, 4917–4930 (2007). 6. Jensen, K. S. et al. FoxO3A promotes metabolic adaptation to hypoxia by antagonizing Myc function. EMBO J. 30, 4554– 4570 (2011). 7. Ferber, E. C. et al. FOXO3a regulates reactive oxygen metabo- lism by inhibiting mitochondrial gene expression. Cell Death Differ. 19, 968–979 (2012). 8. Adhikary, S. & Eilers, M. Transcriptional regulation and transformation by Myc proteins. Nat. Rev. Mol. Cell Biol. 6, 635–645 (2005). 9. de Alboran, I. M. et al. Analysis of C-MYC function in normal cells via conditional gene-targeted mutation. Immunity 14, 45–55 (2001). 10. Sander, S. et al. Synergy between PI3K signaling and MYC in Burkitt lymphomagenesis. Cancer Cell 22, 167–179 (2012). 11. Kops, G. J. P. L. et al. Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature 419, 316–321 (2002). 12. Tothova, Z. et al. FoxOs are critical mediators of hematopoi- etic stem cell resistance to physiologic oxidative stress. Cell 128, 325–339 (2007). 13. Yeo, H. et al. FoxO3 coordinates metabolic pathways to maintain redox balance in neural stem cells. EMBO J. 32, Kerstin Wilhelm and Barbara Risau 2589–2602 (2013).
22 Cell News 02/2017 WERNER RISAU PRIZE 2017
Figure 1 a Angiogenic front Remodelling plexus b Control Foxo1iEC-KO IB4 VECAD / IB4
V A V A A V A 50 μm 200 μm FOXO1 / c VECAD IB4
FOXO1 / 50 μm 25 μm
d 100 30 50 80 μ m) 40 20 60 30 40 20
FOXO1 10 20 10 Number of filopodia EC area per field (%) 0 diameter ( Vessel 0 0 Control Foxo1iEC-KO e Control Foxo1iEC-KO h Control Foxo1iEC-KO
P21 IB4
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f Merge i COL IB4 ICAM2 P21
100 μm PECAM
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ICAM2 / IB4 COL 50 μm 100 μm g j 140 30 120 60 100 20 80 40 60 -ECs per field -ECs per field -cells per field + + 10 + 40 20 BrdU / IB4 20 BrdU ERG 100 μm 0 0 pHH3 0 Control Foxo1iEC-KO
Figure 1 – Endothelial FOXO1 is an essential regulator of vascular growth. a, Immunofluorescence staining for FOXO1 (red), VE-cadherin (green) stained P5 retinas of control and Foxo1iEC-KO mutant ret- (VECAD; grey) and isolectin-B4 (IB4; green) in a P5 mouse reti- inas showing the hyperplastic growth of Foxo1-deficient blood na. The lower panels depict the isolated FOXO1 signal (grey) of vessels. f, Confocal images of PECAM (magenta) and ERG (green) the boxed area shown in the middle panel. Note the diffuse nu- stained P5 retinas in control and Foxo1iEC-KO mice illustrating the cleo-cytoplasmic localization of FOXO1 at the angiogenic front clustering of ECs at the angiogenic front. g, BrdU (grey) and IB4 (left), while a stronger nuclear pattern is observed in the central (red) labelling of whole-mount control and Foxo1iEC-KO P5 retinas. remodelling plexus (right). Arrowheads point to ECs with weak h,i, Overview (h) and higher magnification (i) confocal images FOXO1 nuclear staining. b,c, Overview (b) and higher magnifica- of ICAM2 (green), IB4 (blue) and collagen IV (COL; red) stained tion (c) confocal images of IB4-stained (grey) retinal vessels of retinas at P21 showing the venous enlargement in Foxo1iEC-KO P5 pups in Foxo1iEC-KO as compared to control (Foxo1flox/flox) mice. mice. A, artery; V, vein. j, Quantifications of ERG/IB4- (n≥ 9), A, artery; V, vein. d, Bar graphs showing the mean endothelial BrdU/IB4- (n ≥ 5) and pHH3/IB4- (n ≥ 7) positive cells showing area (n ≥ 7), branch diameter (n ≥ 7), and number of filopodia increased endothelial proliferation in the hyperplastic retinal per vessel length (n ≥ 5) in Foxo1iEC-KO mutant as compared to vasculature of Foxo1iEC-KO mutant mice. Data represent mean control (Foxo1flox/flox) mice. Data represent mean ± s.d. Two-tailed ± s.d. Two-tailed unpaired t-test. Controls are Cre-negative unpaired t-test. e, Confocal images of IB4 (red) and nuclear ERG littermates. ***P < 0.001; ****P < 0.0001.
Cell News 01/2017 23 WERNER RISAU PRIZE 2017
Figure 2 a Control Foxo1EC-CA b Control Foxo1iEC-CA FOXO1 / PECAM
E10.5 E10.5 50 μm FOXO1 / GFP c Control Foxo1iEC-CA g Control Foxo1iEC-CA IB4
V A A V
200 μm ICAM2 / IB4 COL 100 μm d h COL
ERG / IB4 A V A V
200 μm VECAD / IB4 100 μm e i / IB4 pHH3
100 μm CASP3 / IB4 cleav. 100 μm
f 100 j E11.5 hindbrain 15 80
60 10
IB4
40 -ECs per field -cells per field + + 5 Control 20 100 μm ERG pHH3
0 0 k 50 30 20 40 - 4 + m ) iEC-CA 40
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+ 20 Foxo1 -sleeves
- 30 20 2 10 20 per field / IB4 10 +
cells per field 5 10 1 10 COL Vessel diameter (µ Vessel cleav. CASP3 cleav. EC area per field (%) 0 0 0 Branch points per field 0 0
Figure 2 – Forced activation of FOXO1 restricts endothelial growth and vascular expansion
a, Overview images of freshly dissected E10.5 mouse embryos Foxo1iEC-CA when compared to control. The right half of both showing severe growth retardation in the constitutive Foxo1EC-CA images shows the ICAM2 staining (green) alone. h, Whole- mutants compared to control mice. b, Immunofluorescence mount triple immunofluorescence for VECAD (green), IB4 (blue) staining for FOXO1 (magenta), GFP (green) and PECAM (blue) and COL (red) of P5 control and Foxo1iEC-CA retinas. The number in P5 Foxo1iEC-CA and control mice. Note the enhanced nuclear of empty (COL+, IB4--negative) sleeves (white arrows) in the FOXO1 signal in the GFP+/PECAM+-vessels. The right half of retinal plexus is increased in the Foxo1iEC-CA mutants. i, Confocal both images shows the FOXO1 staining (grey) alone. c, Confocal images of IB4- (magenta) and cleaved Caspase 3 (CASP3; green) images showing the IB4-stained vasculature of P5 retinas in stained P5 retinas showing no difference in endothelial apopto- inducible Foxo1iEC-CA and control mice. A, artery; V, vein. d, Anal- sis between control and Foxo1iEC-CA mutant mice. j, IB4-stained ysis of ERG- (green) and IB4- (red) stained P5 retinas in control (grey) E11.5 hindbrains following 4-OHT injection on E8.5 – and Foxo1iEC-CA mice illustrates a reduced number of ECs in the E10.5 showing reduced vascularization in the ventricular zone vasculature of Foxo1iEC-CA mice when compared to controls. e, of Foxo1iEC-CA mice. k, Quantification of EC area, vessel branch IB4 (red) and pHH3 (cyan) labelling of whole-mount P5 retinas points and mean vessel diameter in control and Foxo1iEC-CA mu- reveals reduced endothelial proliferation in Foxo1iEC-CA ani- tant hindbrains. Controls are littermate animals without cre (n ≥ mals when compared to controls. f, Quantification of vascular 5). Data represent mean ± s.d. Two-tailed unpaired t-test. **P < parameters in the control and mutant retinas as indicated (n 0.01; ***P < 0.001; ****P < 0.0001. ≥ 5). Data represent mean ± s.d. Two-tailed unpaired t-test. g, Whole-mount ICAM2 (green), IB4 (blue) and COL (red) staining of retinas at P5 showing preserved luminal ICAM2 staining in
24 Cell News 02/2017 WERNER RISAU PRIZE 2017
Figure 3 a b c d AdCTL AdCTL AdCTL AdCTL AdFOXO1CA AdFOXO1CA AdFOXO1CA AdFOXO1CA 250 1.5 1.5 5000
200 4000 1.0 1.0 150 3000
100 2000 0.5 0.5 Glycolysis (fold change) 50 (fold change) 1000 ECAR (mpH/min) Lactate production (nmol gluc./h/mg prot.) 0 Glucose (2-DG) uptake 0 0 0 Oligo - + CA AdCTL e Oligo FCCP AA / R f AdFOXO1CA g 1.5 400 AdCTL AdCTL AdCTL AdFOXO1 CA M (K) CQ AdFOXO1 r /min) 2 300 1.0 15 LC3-I LC3-II 200 0.5 75 FLAG 100
OCR (pmol O 50 Relative ROS levels TUB
0 (CM-DCF fluorescence) 0 100 ± 2 110 ± 1ns Ratio (%)
0 20 40 60
Time (min) CA CA h i j AdCTL k AdFOXO1CA AdFOXO1 FOXO1 motif AdCTL MYC CCNB1 TYMS 1.5 AdCTL UBE2C AdFOXO1 ES 0.44 CAD M (K) 0.4 MTHFD1 r TK1 NES 1.52 CDC25A 0.3 MRTO4 CDK4 p < 0.001 CKS2 MYC 0.2 RPL27A PRPS2 50 DKC1 1.0 POLD2 0.1 AKAP1 MSH2 STEAP2 PRKCE 75 FLAG 0 FXN FASN PA2G4 -0.1 CA SHMT1 AdFOXO1 AdCTL EXOSC8 APEX1 0.5 NUP155 PYCR1 50 TUB HSPD1 Fold regulation CCND2 HSPA9 HNRNPA1 RPP30 100 ± 2 44 ± 4**** Ratio (%) EMP1 NCL HSPE1 0 CA TP53 Enriched in AdFOXO1 ElF48 GNL3 RPS6KA2 PPAT CCT5 l FOSL1 MYC motif ENO1 DDX10 CEBPZ AdCTL LTA4H ES -0.45 PSMG1 CA H2AFZ AdFOXO1 0 E2F1 ODC1 4.0 Enrichment Score NES -1.36 EIF2S1 EIF4A1 EIF48 -0.1 p < 0.001 SRM RPL22 TRFC NBN -0.2 PRDX3 RPL13 DDX18 -0.3 EIF4E METTL1 2.0 PHB E2F3
HMGA1 Fold regulation -0.4 CA CBX3 AdFOXO1 AdCTL ELAVL1 TIMM10 LDHA EIF4B RFC2 PTMA NPM1 SNRPB 0
max min Enriched in AdCTL MYC MXI1 CDK4 ENO1PKM2 LDHA LDHB FASN FBXW7 CCND1CCND2 CCNB2
Figure 3 – FOXO1 slows endothelial metabolic activity and suppresses MYC signalling. a, Extracellular acidification rate (ECAR), an indicator of positive control. TUB, Tubulin. Densitometric quantifications 0 glycolytic lactate production, in ECs treated with or without are shown below the lanes (n = 10). Data represent mean ± s.d. oligomycin (Oligo) showing reduced basal and maximal gly- Two-tailed unpaired t-test. h, Gene set enrichment analysis of colytic activity in AdFOXO1CA-transduced ECs when compared the FOXO1- (AAACAA) or MYC- (CACGTG) DNA binding element to control adenovirus- (AdCTL) transduced ECs (n = 6). Data gene sets in AdFOXO1CA- or AdCTL-transduced ECs. Note that represent mean ± s.d. Two-tailed unpaired t-test. b-d, Reduced the FOXO1 motif is enriched in the genes induced by AdFOXO- uptake of the glucose analogue 2-deoxy-D-glucose (2-DG) (n = 1CA, while the MYC motif is enriched in the genes repressed by 13), decreased relative lactate production(n = 10) and lowered FOXO1. Black bars represent individual genes in rank order. ES, glycolytic flux (n = 4) in control and FOXO1CA-expressing ECs. enrichment score, NES, normalized enrichment score. i, Heatmap Data represent mean ± s.d. Two-tailed unpaired t-test. e, Oxy- of downregulated MYC signature genes in FOXO1CA-overexpress- gen consumption rates (OCR) in control and FOXO1CA-overex- ing ECs (n=3). j,k, Analysis of MYC expression by microarray (j) pressing ECs under basal conditions and in response to oligomy- and immunoblot (k) demonstrating the suppression of MYC in cin (Oligo), fluoro-carbonyl cyanide phenylhydrazone (FCCP) or FOXO1CA-Flag-overexpressing endothelium. The densitometric antimycin A (AA) and rotenone (R). (n = 5). Data represent mean quantification of MYC protein levels is shown below the lanes ± s.d. Two-way ANOVA with Bonferroni’s multiple comparison of the immunoblot. (j, n = 6; k, n= 10). Data represent mean ± test. f, Relative ROS levels in ECs 24 hours post transduction s.d. Two-tailed unpaired t-test. l, Quantitative polymerase chain with AdCTL or AdFOXO1CA (n = 7). Data represent mean ± s.d. reaction (qPCR) expression analysis of FOXO1CA-regulated genes Two-tailed unpaired t-test. g, Western blot analysis showing involved in MYC signalling. Relative mRNA levels are shown (n ≥ that overexpression of the Flag-tagged FOXO1CA does not induce 3). Data represent mean ± s.d. Two-tailed unpaired t-test. *P < autophagy in ECs as the LC3-II to LC3-I ratio was not signifi- 0.05; **P < 0.01; ****P < 0.0001; #P < 00.1; ns, not significant. cantly changed. Chloroquine- (CQ) treated EC were used as a
Cell News 01/2017 25 WERNER RISAU PRIZE 2017
Figure 4 siSCR siSCR a b c iEC-KO siMYC siMYC Control Myc 200 200
150 /min) 2 150
100 100
50 50 ECAR (mpH/min) 50 μm OCR (pmol O
0 0 MYC / VECAD PECAM Oligo - + d Control MyciEC-KO f Control MyciEC-OE IB4 IB4
V A V A A V A V A 200 μm 200 μm e g IB4 IB4 / ERG / A V A A V A pHH3 200 μm 100 μm
h Control Foxo1iEC-CA Foxo1iEC-CA;MyciEC-OE MyciEC-OE
100 μm iB4
V A V V A V A V V A V 200 μm
Oligo FCCP AA / R i 100 j k 400 200 AdCTL 80 AdFOXO1CA CA /min) 300
2 AdFOXO1 /MYC 150 60 AdMYC 200 40 100 20 50 100 ECAR (mpH/min) OCR (pmol O EC area per field (%) 0 0 0 Oligo Control FOXO1CA/MYCOE - + 0 20 40 60 FOXO1CA MYC AdCTL AdFOXO1CA/MYC Time (min) AdFOXO1CA AdMYC
Figure 4 – MYC is a critical component of FOXO1 signalling in ECs.
a,b, ECAR (b) and OCR (c) in HUVECs showing a reduced metabolic activity in MYC siRNA- (siMYC) compared to SCR nas. h,i, Confocal images of IB4-stained (grey) P5 retinas siRNA- (siSCR) transfected ECs (ECAR: n = 5; OCR: n = 5). Data in control, Foxo1iEC-CA, MyciEC-OE and Foxo1iEC-CA/MyciEC-OE dou- represent mean ± s.d. Two-tailed unpaired t-test. c, Immuno- ble mutants. Representative images (h) and quantification of fluorescence staining forMYC (red), VECAD (grey) and PECAM endothelial coverage (i) in the respective genotypes are shown. (cyan) in P5 retinas of MyciEC-KO and control mice. d, Confocal Controls are littermate animals without cre expression (n ≥ 6 images of the IB4-stained vasculature in P5 retinas of MyciEC-KO from 3 independent litters). Data represent mean ± s.d. One- and control mice. A, artery; V, vein. e, Confocal images of IB4 way ANOVA with Bonferroni’s multiple comparison post-hoc (red) and nuclear ERG (green) stained P5 retinas of control and test. j,k, ECAR (j) and OCR (k) in AdCTL, AdFOXO1CA, AdFOXO1CA/ MyciEC-KO mutant retinas showing a reduced number of ECs in AdMYC and AdMYC-transduced HUVECs showing the resto- the vasculature of MyciEC-KO mice when compared to controls. f, ration of metabolic activity in FOXO1CA/MYC co-expressing ECs Overview images of IB4-stained P5 retinal vessels in MyciEC-OE (ECAR: n = 8; OCR: n ≥ 3). Data represent mean ± s.d. One-way and control mice. g, Enhanced EC proliferation in MyciEC-OE mice ANOVA with Bonferroni’s multiple comparison post-hoc test was as revealed by IB4 (red) and pHH3 (cyan) co-staining in P5 reti performed in (j). **P < 0.01; ***P < 0.001; ****P < 0.0001.
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16th Workshop „Cell Biology of Viral Infections“, Schöntal, November 8-10, 2017
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Cell News 01/2017 27 MEETING REPORT
Meeting report of the DGZ study group ‘Signal Transduction’ and its participation in the 20th anniversary Joint Meeting "Signal Transduction – Receptors, Mediators and Genes"
Klaudia Giehl, Detlef Neumann, Ottmar Janssen, Katharina Hieke-Kubatzky and Ralf Hass
As in the previous years, the annual Joint Meeting "Signal activation and formin-mediated actin dynamics to subsequently Transduction – Receptors, Mediators and Genes" took place in form non-apoptotic plasma membrane blebbing. Weimar (November 9 to 11, 2016) whereby this special meeting celebrated its 20th anniversary. The conference was organized by Following further mechanisms of cellular movement, Francesca the Signal Transduction Society (STS) together with signaling Odoardi (Göttingen) presented a neurodegenerative multiple study groups of the German Societies for Cell Biology (DGZ), sclerosis model of experimental autoimmune encephalomyelitis for Biochemistry and Molecular Biology (GBM), for Immunology (EAE) to investigate how T cells become brain-associated. In the (DGfI), and for Pharmacology (DGP). Other financial and scientif- workshop “Immune Cell Signaling”, Prof. Odoardi also dis- ic contributors were the Netherlands Societies for Biochemistry cussed potential checkpoints for T cell infiltration of the central and Molecular Biology (NVBMB) and the SFB 854 “Molecular nervous system and appropriate addressing of T cell to different Organisation of Cellular Communication in the Immune System” compartments of the brain. (B. Schraven, Magdeburg). The meeting organisation was per- formed by the STS council together with the chairpersons of the Within the special subtopic on “Molecules in Motion”, Robert study groups and members of the STS Advisory Board. Tampé (Frankfurt) and Irmgard Sinning (Heidelberg) focused on intracellular peptide and protein shuttling to the plasma The special focus of the 2016 meeting ‘Cells and Molecules in membrane. After proteasomal degradation of protein products Motion’ was coordinated by the GBM study group Signal Trans- to small peptides from virally-transformed cells or cancer cells duction with a number of exciting keynote lectures. during immune surveillance, Prof. Tampé discussed the local- ization of these peptides in the ER lumen associated with MHC Jan Faix (Hannover) opened this year’s Joint Meeting by his class I molecules and their subsequent transfer to the plasma keynote talk about actin-based cell migration and associated membrane to allow further recognition by the immune system. molecular mechanisms within the special subtopic “Cells in Prof. Sinning introduced the preparation of moving proteins Motion”. In various fluorescence microscopic videos Prof. Faix to the plasma membrane and proper unfolding by presenting impressively demonstrated different forms of cellular movement mechanisms of the SRP (signal recognition particle) cycle. The including a slower mesenchymal mode of migration which is SRP requires a N-terminal signal sequence for a RNA-protein exhibited by fibroblasts or mesenchymal stroma/stem cells complex formation with subsequent processing and further by strong substrate adherence, prominent stress fibers and remodeling for translocation to appropriate cellular compart- extended formation of protruding lamellipodia/ruffles at the ments. leading edge. Alternatively, a faster amoeboid-like cell migration is utilized e.g. by immune cells and characterized by weaker Guanylate-binding proteins (GBPs) including GTPases can be adhesion, absence of stress-fibers and formation of action-rich induced in macrophages by γ-interferon signaling in part via pseudopods in the cellular fronts paralleled by myosin II-driven JAK2 and possess homo- and hetero-multimerization proper- contractility in the rears. These actomyosin-dependent dynamics ties which was extensively discussed by Klaus Pfeffer (Düssel- were followed during the next keynote talk by Robert Grosse dorf) within the special subtopic “Organelles in Motion”. Prof. (Marburg) with respect to the migratory and invasive potential Pfeffer demonstrated the reversible oligomerization of GMPs in of cancer cells liberated from the primary tumor and subsequent vesicle-like structures to form large supramolecular complex- trans-endothelial migration. Prof. Grosse further focused on es comprising up to several thousand GMP monomers which entosis, a process of cell in cell invasion as a form of membrane can exhibit anti-microbial activity by targeting pathogens in blebbing-associated amoeboid migration of the invading cell. vacuoles and therefore can be considered as effector molecules Entosis-promoting signals involve certain LPA-Rs (Lysophos- of host immunity. phatidic acid receptors), e.g. LPA-R2 coupled with Rho-GTPases
28 Cell News 02/2017 MEETING REPORT
stimulates signaling within the tumor microenvironment, inhib- its invasion and metastasis, activates autophagy, and inhibits the reprogramming of differentiated cells back to stem cells (retrodif- ferentiation) among others.
All keynote lectures were followed by a number of short talks selected from the submitted abstracts. Here, the mixture of pre- sentations given by group leaders, post-doctoral fellows and also a number of PhD students was highly appreciated as a unique feature of the STS meetings.
Travel grants awarded to young scientists at the STS meeting 2016 As one of the traditions during these meetings since 2010, the STS honors an outstanding scientist in the field of signal transduction research to conclude the workshop program with a “Honorary The workshop on “G protein-coupled receptors” was introduced Medal Lecture”. The STS/CCS Honorary Medal was introduced by by Daniel Legler (Kreuzlingen, Switzerland) who demonstrated the STS in cooperation with the open access journal “Cell Commu- the importance of CCR7 (C-C chemokine receptor type 7) and nication and Signaling” (CCS). Following Tony Pawson ( ) in 2010, its ligands, the cytokines CCL19 and CCL21 for migration of Tony Hunter in 2011, Carl-Henrik Heldin in 2012, Klaus Rajewsky in adaptive immune cells. Following CCR7-CCL19/CCL21-mediated 2013, the Nobel prize winner Jules Hoffmann in 2014, Mina Bissell signaling pathways, Prof. Legler pointed out that imbalanced in 2015, it was Tak Wah Mak from Toronto, Canada, who received regulations of these complexes contribute to pathophysiolog- the 2016 STS/CCS Honorary medal. The laudatio was given by Klaus ical developments such as chronic inflammation, autoimmune Pfeffer (Düsseldorf), one of Prof. Mak’s former postdocs. diseases, and tumorigenesis. Prof. Mak was honored with the STS/CCS medal for his seminal Reinhold Förster (Hannover) addressed his keynote talk in the and pioneering immunological and cell biological research work. workshop “Signaling in Infection and Inflammation”. He focused He was the lead scientist of the group that cloned the genes of on homing of immune cells to the lymph node. Following in- the human T cell receptor (TCR), and thus unraveled the molecular tra-lymphatic vessel injection in vivo, Prof. Förster demonstrated basis of cellular immunity. In order to address individual signaling T cell migration in afferent lymphatic vessels to enter the outer steps in lymphocyte activation and differentiation, his laboratory lymph node sinus and relocate from the sub-capsular sinus to generated a whole set of mouse strains lacking various surface the lymph node parenchyma which also depends on functional receptors or kinases, phosphatases or adapter proteins involved in G protein-coupled receptor signaling in T cells. signal transduction through the TCR complex. A second major focus of Prof. Mak’s work is the biology of programmed cell death. His Tobias Dansen (Utrecht, The Netherlands) presented a keynote laboratory significantly contributed to our present understanding of talk in the workshop „Receptor and Redox Signaling“ where- the bifunctionality of death receptors and the control of apoptot- by he introduced the pathways of reversible oxidation upon ic pathways in different cellular entities by caspases or adapter bridging and disrupting specific thiol side chains in appropriate proteins. More recently, Prof. Mak has devoted his research to receptors and proteins. For example, the formation of reversible investigating the pathogenesis of cancer. As a new and promising disulfide-dependent protein-protein interactions and accompa- concept to identify potential targets for novel cancer therapeutics, nying conformational changes serve as a signaling pathway in he focuses on understanding mechanisms of “metabolic transfor- addition or alternative to phosphorylation cascades. mation”. This Honorary Medal Lecture was very much appreciated by the audience. In the workshop “Differentiation, Stress and Death” Ute Moll Another important aspect of the STS joint meeting has always (New York/Göttingen) focused on new functional aspects of p53 been the support of young scientists. At the occasion of the family proteins including the structural homologues p63 and 20th anniversary meeting, the STS grant committee selected 15 p73. In her keynote lecture Prof. Moll highlighted effects on p53 Bachelor/Master or MD/PhD students to receive travel grants activation during cellular stress such as hypoxia, oxidative stress of 3,750.-- € in total to support their meeting attendance. and generation of reactive oxygen species (ROS), telomere attri- Ten travel grants were provided by the STS and additional five tion, replicative senescence, or DNA damage. Activation of p53 travel awards were sponsored by industrial partners. The 2016 occurs after disruption of MDM2 and MDM4 interactions and STS Science Award of 1,000.-- €, sponsored again by BIOMOL a paralleled Ser15 and Ser20 phosphorylation of p53 via ATM/ GmbH was received by Melanie Brinkmann (Braunschweig), who ATR and CHK1/CHK2 kinases, respectively. Regulatory responses presented data on the murine cytomegalovirus protein M35 as a of activated p53 include cell cycle arrest with subsequent DNA novel negative regulator of type I interferon response. Moreover, repair, senescence and apoptosis. Moreover, p53 activation all participants selected by the chair people for poster presentation
Cell News 01/2017 29 MEETING REPORT
had the opportunity to present their poster work during the well-known ‘one minute – one transparency’ session to attract the audience to the following extensive poster discussion in a casual atmosphere. Five poster prizes were selected from about 60 poster presentations and rewarded with a total of 750.-- € of prize money.
Finally, the ‘Signal Transduction’ Meeting 2016 was extensively celebrated with the live band “Paolo Macho” contributing to a great 20th anniversary party. Now, the preparations for the 21st Joint meeting have already started whereby the special focus for 2017 will be covered by an immunological topic related to the clinic. This will certainly attract also many translational scientists and physicians from all areas of signalling research by bridging basic science with clinical applications. The up-coming next STS joint meeting is scheduled for November 8th to 10th, 2017 and will again take place at the Leonardo Hotel in the his- Poster discussion with Professor Tak Wah Mak torical city of Weimar. Details and updated meeting information will be available at http://www.sigtrans.de.
30 Cell News 02/2017 Cell News 01/2017 31 MEMBERS / INTERN
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