2021 Wellcome Four Year PhD Programme in Integrative Cell Mechanisms

Training the next generation of Molecular Cell Biologists

Background and Aims of Programme The Wellcome Four Year PhD Programme in Integrative Cell Mechanisms (iCM) is closely associated with the Wellcome Centre for Cell Biology and trains the next generation of cell and molecular biologists in the application of quantitative methods to understand the inner workings of distinct cell types in different settings.

A detailed understanding of normal cellular function is required to investigate the molecular cause of disease and design future treatments. However, data generated by biological research requires increasingly complex analysis with technological advances in sequencing, mass spectrometry/proteomics, super-resolution microscopy, Wellcome Centre for Cell Biology 2021 synthetic and structural biology generating increasingly large, complex datasets. In addition, innovations in computer sciences and informatics are transforming data acquisition and analysis and breakthroughs in physics, chemistry and engineering allow the development of devices, molecules and instruments that drive the biological data revolution. Exploiting technological advances to transform our understanding of cellular mechanisms will require scientists who have been trained across the distinct disciplines of natural sciences, engineering, informatics and mathematics.

To address this training need, iCM PhD projects are cross-disciplinary involving two primary supervisors with complementary expertise. Supervisor partnerships pair quantitative scientists with cell biologists ensuring that students develop pioneering cross-disciplinary collaborative projects to uncover cellular mechanisms relevant to health and disease. We aim to recruit students with a variety of backgrounds across the biological and physical sciences, including Biochemistry, Biomedical Science, Cell Biology, Chemistry, Computational Data Sciences, Engineering, Genetics, Mathematics, Molecular Biology and Physics. Students are trained to adapt, broaden and apply their skill set to the understanding of cellular mechanisms of biomedical importance.

The next deadline for applications will be early December 2021- and will be posted at: https://www.wcb.ed.ac.uk/how-apply Historical Background Content

The expansion of research in cell biology was planned in 1992 as a result of the vision of Professor Sir Kenneth Murray, who was at the time Biogen Professor at the Institute of Cell and Molecular Biology. A seed contribution of £2.5 million from the Darwin Trust was followed by financial commitments from The Wolfson Foundation, the University and the Wellcome Trust, allowing construction of the Michael Swann Building. The majority of research space was earmarked for Wellcome Trust-funded research. Recruitment, based on research excellence at all levels in the area of cell biology, began in earnest in 1993, mostly but not exclusively, through the award of Research Fellowships from the Wellcome Trust. The Swann Building was first occupied by new arrivals in January 1996 and became “The Wellcome Trust Centre for Cell Biology” from October 2001. Core funding for the Centre from the Wellcome Trust was renewed in 2006, 2011, 2016 and 2021. 04 16 28 40 Director’s Report Bill Earnshaw Kenneth E Sawin List of Groups

06 18 30 46 Robin Allshire Patrick Heun David Tollervey Centre Publications

08 20 32 57 A. Jeyaprakash Adele Marston Philipp Voigt Emeritus Centre Arulanandam Member 22 34 10 Dónal O'Carroll International Scientific Julie Welburn Adrian Bird Advisory Board 24 36 12 Hiro Ohkura Marcus D Wilson Swann Building opening 1996 Dhanya Cheerambathur 26 38 Margarete Heck teaches Prince Phillip the incredible 14 Juri Rappsilber Public Engagement power of Drosophila genetics. Atlanta Cook 2 3 Director’s Report

The Wellcome Centre for Cell Biology (WCB) is one of fifteen UK-based, Wellcome-funded research centres. The WCB Centre staff have also managed to deliver an excellent programme of public engagement, despite the necessity of research groups, and our associated research facilities, occupy the Michael Swann Building on the King’s Buildings switching to an entirely online delivery. Particular thanks to Sarah-Jane Judge, who took on the role of Public Engagement Campus of the University of Edinburgh. Constructed in the mid 1990s, the Michael Swann Building was purpose built as a Manager just as we went into the first lockdown; making for a challenging start. Thanks to everyone who gave up their time centre for research in molecular cell biology. and brought their enthusiasm to share our work with the broader community in Edinburgh. Let me also welcome Karen May in her new role as Centre Manager, which she stepped into at the start of this year. WCB has benefitted from Wellcome Centre status since 2001, and this has allowed us to bring together outstanding scientists in a dynamic, supportive environment, where their work is enhanced by access to a set of world-class core In the absence of a normal seminar programme, we have established a weekly “Renowned Seminar Speaker Programme”. facilities. Such a situation is usually found only in research institutes, but WCB also benefits from being embedded in a This is proceeding well, so thanks to Robin Allshire for getting the ball rolling on this. The first cohort has joined our major university. This setting offers unrivalled opportunities for interaction with researchers across the range of disciplines Wellcome 4-year PhD programme in “Integrative Cell Mechanisms (iCM)”, again under difficult circumstances. Thanks to in science, medicine and social science, as well as the arts. everyone who participated; particularly Vasso Makrantoni who organised the start-up of the programme, before leaving to start her own independent career. This exciting, cross-disciplinary partnership brings cell biologists together with physical This is my last report as Centre Director; after 10 years in the post, I will be passing on the role to Prof. Adèle Marston, and data science experts across Edinburgh. who is currently Deputy Director. A key event for the Centre in the past year was the extension of our Core Grant. Adèle successfully led this bid and will take over as Director at the start of the new funding period – in December 2021. Centre members were also successful in obtaining prestigious posts elsewhere and deserve our warmest congratulations, even though we are very sorry to see them leave. Tony Ly moved to a new position in Dundee University. Vasso Makrantoni It has, of course, been an unusual and difficult year. For most of us, life has changed substantially since the time I sat (Marston lab) was appointed as a lecturer at the Zhejiang University - University of Edinburgh Institute. Clémentine Delan- down to write the previous report, in March 2020. Then, the term of lockdown was strange and new, but still seemed to Forino (Tollervey lab) obtained a prestigious CNRS post in the Institut de Microbiologie de la Méditerranée in Marseille, be something that happened to people elsewhere – although getting ominously closer. During the past year, I have been France. Marie-Luise Winz (Tollervey lab) was appointed as a Junior Professor, in the Johannes Gutenberg University in hugely impressed by the fortitude and resilience shown by the members of WCB. I must also express my deep gratitude to Mainz, Germany. Our best wishes to them all in their future careers. The continuing success of Centre alumni is a great all of the support staff in Swann and elsewhere in King’s Buildings, many of whom have continued to work through all of the source of pride. lockdowns. Prof. Jean Beggs, who was a founding member of WCB, took retirement last year, after a hugely successful and influential This brochure presents a very brief overview of our research and highlights some of the exciting results reported during the research career. She is greatly missed as a colleague in WCB and throughout the RNA community. Happily, however, the past year. I am delighted that, despite the ongoing challenges posed by lockdown, which almost halted our research, and WCB group leaders unanimously voted to create the position of Emeritus Centre Member, with Jean as first recipient of this continuing greatly reduced building occupancy, WCB groups have again made excellent progress, reporting innovative, honour. Jean has therefore contributed an entry in the newly-established Emeritus Centre Member section of the brochure. world-class research, as reflected in our many notable publications. It is a great pleasure to see her contributions resume.

In addition, I would like to congratulate individual WCB members on grant funding successes during the year. Bill Earnshaw Let me end, as usual, by reiterating my sincere thanks to all of our talented and dedicated researchers and support renewed his Wellcome Principal Research Fellowship while, in addition to leading the centre renewal, Adèle Marston staff, and congratulate them on their excellent work over the year. Your efforts underpin all of the continuing received a Wellcome Investigator Award, and Julie Welburn was the recipient of a COVID Reboot grant. Notable awards successes of the WCB. were given as marks of esteem in recognition of outstanding work by WCB PIs: Dónal O'Carroll was awarded the lifetime honour of EMBO (European Molecular Biology Organisation) Membership. Robin Allshire was elected as a Fellow of the Academy of Medical Sciences. Adrian Bird was announced as the joint winner of the Brain Prize – the most valuable research prize for neuroscience. In addition, SBS Achievement Awards were presented to Christine Struthers and Carolyn Fleming for their sterling work in keeping WCB and the iCM PhD programme running during this very difficult period.

4 55 Robin Allshire Epigenetic inheritance: establishment and transmission Co-workers: Tatsiana Auchynnikava, Roberta Carloni, Tadhg Devlin, Andreas Fellas, Elisabeth Gaberdiel, of specialized chromatin domains Dominik Hoelper, Marcel Lafos, Nitobe London, Sunil Nahata, Alison Pidoux, Severina Pociunaite, Desislava Staneva, Manu Shukla, Sito Torres-Garcia, Sharon White, Weifang Wu, Imtiyaz Yaseen, Rebecca Yeboah

Chromosomal DNA wraps around nucleosomes containing core histones (H3/H4/H2A/H2B). At centromeres however, a AA ChIP-seq: H3K9me2 DD FISH DNA a ChIP-seq: H3K9me2 Chr I Chr II Chr III specific histone H3 variant, CENP-A, replaces histone H3 to form specialized CENP-A nucleosomes. CENP-A chromatin tel1L cen1 tel1R tel2L cen2 tel2R tel3L cen3 tel3R 125 wt 0 is critical for chromosome segregation machinery assembly (kinetochores) at these specific chromosomal locations. UR-1 UR-1 C Kinetochores are flanked by histone H3 lysine 9 methylation (H3K9me)-dependent heterochromatin. UR-2 UR-2 UR-3 UR-3 Our goal is to decipher conserved mechanisms that establish, maintain and regulate heterochromatin and CENP-A Mouse FISH DNA

chromatin domain assembly. Heterochromatin is required for CENP-A chromatin establishment on centromere DNA. H3K9me2 enrichment 20 hba1 locus UR-1 30 ncRNA.394 locus UR-2 chromosome Fission yeast wt wt One objective is to provide further insight into mechanisms that promote heterochromatin formation on pericentromeric 10 DNA insert repeats. Heterochromatin might also silence genes throughout the genome; we therefore also investigate how 10 15 heterochromatin formation is regulated and whether such mechanisms influence phenotype. We endeavour to determine

0 0 how heterochromatin, spatial nuclear organisation and non-coding RNAPII transcription combine to mediate CENP-A SPBC17G9.13c eno101 pmt3 kin17 arp5 grp2 hba1 alp4 rpl1102 ncRNA.394 cut2 tim44 fma2 incorporation at centromeres. ish1 uge1 pyr1 prl68 der1 ncRNA.1524 SPBC17G9.12c Our main questions are: tmf1 ncRNA.1523 ncRNA.135 ncRNA.393 Chr II 2,510 2,520 kb Chrchr II 2,195 2,200 kb 1. How do DNA, RNA and chromatin signatures instigate the assembly of specialized chromatin domains? 60 ppr4 locus UR-3 80 grt1 locus UR-4D 2. How does chromatin architecture and subnuclear compartmentalization affect specialized chromatin domains? B N/S +CAF +CLT +TEB +FLC 3. How does heterochromatin influence gene expression? B a N/S +CAF +CLZ +TEZ +FLZ wt

H3K9me-dependent heterochromatin renders embedded genes transcriptionally silent. Fission yeast KEDу (Schizosaccharomyces pombe) H3K9me-dependent heterochromatin can be transmitted through cell division provided the UR-1 Mb counteracting demethylase Epe1 is absent. Can heterochromatin heritability allow wild-type cells under certain conditions UR-2 to acquire epimutations, which influence phenotype through unstable gene silencing rather than DNA mutation? We have C discovered that heterochromatin-dependent epimutants resistant to caffeine can arise in fission yeast (Torres-Garciaet E Narrower C Wild-type Dead cells E chromosome al. 2020). Isolates with unstable resistance have distinct heterochromatin islands with reduced expression of underlying cells Lethal Insult width insult Survivors removal Mutant genes, including some whose mutation confers caffeine resistance (Figure A). Our analyses suggest that epigenetic phenotype processes promote phenotypic plasticity, allowing wild-type cells to adapt to unfavourable environments without genetic Higher condensin alteration. Isolates with unstable caffeine resistance show cross-resistance to fungicides (Figure B), suggesting that Resistant Wild-type to chromatin ratio Resistant epimutant phenotype related heterochromatin-dependent processes may contribute to resistance in plant and human fungal pathogens. Such cells Resistant mutant epimutations provide a bet-hedging strategy allowing cells to adapt transiently to insults while remaining genetically wild- Less chromatin type (Figure C). per unit length It is not known why heterochromatin has a distinct architecture on mitotic chromosomes. We find that large regions of fission yeast DNA inserted into mammalian chromosomes assemble H3K9me-dependent heterochromatin which adopts A. Unstable caffeine resistant epimutants UR-1 and UR-2 exhibit novel islands of H3K9me-dependent heterochromatin compared a mitotic organisation distinct from surrounding host chromosome regions (Fitz-James et al. 2020). Heterochromatin to wild-type cells (wt). assembled on this inserted fission yeast DNA alters chromatin loop size relative to flanking euchromatic host chromatin B. UR-1 and UR-2 caffeine resistant (CAF) epimutants are cross-resistant to clinical (CLT, Clotrimazole; FLC, Fluconazole) and agricultural (TEB, Tebuconazole) fungicides used to treat human and crop-plant fungal infections, respectively. (Figure D). Thus, altered loop size likely contributes to the distinct appearance of heterochromatin (Figure E). C. Model. Resistant isolates arise after lethal insult exposure. Resistance can be mediated by changes in DNA (resistant mutants) or reversible, heterochromatin-based epimutations (resistant epimutants). Upon withdrawal of insult epimutants lose ectopic Selected Publications heterochromatin islands, resistance, reverting to wild-type (sensitive phenotype). In contrast genetic mutants continue to exhibit Singh, P.P., Shukla, M., White, S.A., Lafos, M., Tong, P., Auchynnikava, T., Spanos, C., Rappsilber, J., Pidoux, A.L., and Allshire, R.C. the mutant resistance phenotype. (2020). Hap2-Ino80-facilitated transcription promotes de novo establishment of CENP-A chromatin. Genes Dev 34, 226–238. D. Large inserts of fission yeast DNA in mammalian chromosomes exhibit an unusual mitotic chromosome structure associated Fitz-James, M.H., Tong, P., Pidoux, A.L., Ozadam, H., Yang, L., White, S.A., Dekker, J., Allshire, R.C. (2020). Large domains of with H3K9me-dependent heterochromatin formation. Hi-C analyses demonstrate more frequent contacts per kb over the fission heterochromatin direct the formation of short mitotic chromosome loops. Elife 9, e57212. doi: 10.7554/eLife.57212. yeast insert compare to flanking mouse chromatin. Torres-Garcia, S., Yaseen, I., Shukla, M., Audergon, P.N.C.B., White, S.A., Pidoux, A.L., Allshire, R.C. (2020). Epigenetic gene silencing by E. Model. A higher ratio of condensin to chromatin over fission yeast DNA assembled in heterochromatin results in more contacts 6 heterochromatin primes fungal resistance. Nature , 453-458. doi: 10.1038/s41586-020-2706-x. and less chromatin per unit length and consequently, reduced mitotic chromosome width. 77 A. Jeyaprakash Arulanandam Structural Biology of Cell Division Co-workers: Maria Alba Abad Fernandaz, Asma Al-Murtadha, Lana Buzuk, Carla Chiodi, Thomas Davies, Lorenza Di Pompeo, Bethan Medina-Pritchard, Paula Sotelo Parrilla, Ignacio Jiménez Sánchez, Pragya Srivastava, Reshma Thamkachy

Cell division is an essential biological process that ensures genome integrity by equally and identically distributing chromosomes to the daughter cells. Errors in cell division often result in daughter cells with inappropriate chromosome numbers, a condition associated with cancers and birth defects. Key events that determine the accuracy of cell division include centromere specification, kinetochore assembly, physical attachment of kinetochores to spindle microtubules and successful completion of cytokinesis. These cellular events are regulated by a number of mitotic molecular assemblies (including the Chromosomal Passenger Complex (CPC), KMN (Knl1-Mis12-Ndc80) network, the Ska complex, Spindle Assembly Checkpoint and the Anaphase Promoting Complex) involving an extensive network of protein-protein interactions. Although much is known about the basic mechanisms of cell division, structural level mechanistic details of pathways regulating error free chromosome segregation are still emerging. In particular, a high-resolution understanding of centromere inheritance and how kinetochores employ dynamic protein interaction to harness the forces generated by spindle microtubules to drive chromosome segregation is yet to be obtained. To address these important questions requires an approach that integrates structural and functional methods capable of dissecting and probing individual roles of protein interactions mediated at varying timescale. We use molecular biology and biochemical approaches to characterize protein interactions in vitro, X-ray crystallography, Cross-linking/Mass spectrometry, Small Angle X-ray Scattering (SAXS) and Electron Microscopy for structural analysis and a combination of in vitro and cell-based in vivo functional assays using structure-guided mutations for functional characterization. The specific questions that we aim to address currently are i) What is the molecular basis for the establishment and maintenance of CENP-A nucleosomes at centromeres? ii) How do the outer kinetochore microtubule binding components cooperate to facilitate spindle driven chromosome segregation? and iii) How CPC, a key player required for eliminating incorrect kinetochore-microtubule attachment is targeted to the kinetochore? We address these questions by characterizing protein complexes involved in centromere maintenance (Mis18 and Mis18-associated), physically coupling chromosomes to kinetochores (the Ska complexes and other outer kinetochore microtubule binding factors) and error- correction (CPC and its centromere/kinetochore receptors). The structural and functional insights from these studies will also provide new avenues for targeting specific protein-interactions to fight mitosis related human health disorders. Our recent crystal structures of Cal1 bound to CENP-A/H4 heterodimer and CENP-C provided a mechanistic understanding of how Cal1 targets CENP-A/H4 to centromeres to maintain centromere identity. Our work revealed that Cal1 combines functions of human Mis18 complex and HJURP, through evolutionarily conserved and adaptive interactions, to target CENP-A/H4 to centromeres in a self-sufficient manner. (Medina-Pritchard et al., 2020).

Selected Publications Medina-Pritchard, B., Lazou, V., Zou, J., Byron, O., Abad, M. A., Rappsilber, J., Heun, P and Jeyaprakash, A. A. (2020) Structural Basis for Centromere Maintenance by Drosophila CENP-A Chaperone Cal1. EMBO J e103234. Doi:10.15252/embj.2019103234 Abad, M. A., Ruppert, J. G*., Buzuk, L*., Wear, M. A., Zou, J., Webb, K. M., Kelly, D. A., Voigt, P., Rappsilber, J., Earnshaw, W. C and Top panel: Overview of cell cycle-controlled CENP-A dilution and enrichment at centromeres. Jeyaprakash, A. A. (2019) Direct Nucleosome Binding of Borealin Secures Chromosome Association and Function of the CPC. J Cell Biol Bottom Panel: Cartoon summarising our recent structural work on Cal1-mediated centromere maintenance in flies: Cal1 targets 218, 3912-3925. CENP-A/H4 to centromeres by directly binding both CENP-A/H4 and centromere receptor CENP-C (Abad et al., 2019). Spiller, F*., Medina-Pritchard, M*., Abad, M. A*., Wear, M. A., Molina, O., Earnshaw, W. C and Jeyaprakash, A. A. (2017) Molecular Basis for Cdk1 Regulated Timing of Mis18 Complex Assembly and CENP-A Deposition. EMBO Rep 18, 894-905. Doi:10.15252/embr.201643564 8 99 Adrian Bird Understanding proteins that interpret the genome to Co-workers: Kashyap Chhatbar, John Connelly, Laura Fitzpatrick, Sara Giuliani, Jacky Guy, stabilise cell states Beatrice Alexander-Howden, Matthew Lyst, Baisakhi Mondal, Raphael Pantier, Katie Paton, Verdiana Steccanella, Christine Struthers

Regulation of gene expression in eukaryotes is complex, but probably ultimately depends on proteins that recognise specific DNA sequences. Our recent work emphasises this relationship by showing an unexpected dependence of gene activity on proteins that recognise short, frequent base sequence motifs (Figure 1). This points to a broad-brush category of control whereby genes can be up- or down-regulated based on the DNA sequence properties of their genomic location. Our studies of this phenomenon have centred on two proteins that are implicated in human disease: MeCP2 and SALL4. MeCP2 contains a DNA binding domain that requires DNA methylation in either a CG or a CAC context. This protein is highly expressed in mature neurons and its deficiency causes the profound neurological disorder Rett syndrome. We showed previously that MeCP2 restrains gene expression by recruiting a corepressor to the genome. Recently, we questioned the importance of the unusual methyl-CAC target site, which is abundant only in neurons. To do this, we replaced the DNA binding domain of MeCP2 with one that can bind mCG but not mCAC. Mice dependent on this protein had Rett syndrome-like phenotypes, indicating that mCAC binding is essential for the function of MeCP2. These experiments also allowed us to identify a subset of de-regulated genes that are mis-expressed in other neurological disorders and may be implicated in Rett syndrome. Future work will explore this possibility further. The second protein under study is SALL4, a multi-zinc-finger protein that plays an important role in development and disease. For example, SALL4 is highly expressed in many cancers with poor prognosis. We speculated that it might interpret DNA base composition by recognising AT-rich DNA and, in agreement with this notion, found that a zinc finger cluster specifically targets short A/T-rich motifs in the genome and recruits a partner corepressor. In embryonic stem cells, this represses a set of AT-rich differentiation genes, thereby prolonging the pluripotent state. Prevention of AT-binding activates these genes, leading to precocious differentiation. It has been recognised for decades that the mammalian genome is organised in long domains with distinct, evolutionarily-conserved base compositions. Our SALL4 study provides the first evidence that base composition can be read as a biological signal to regulate gene expression. Despite major differences between MeCP2 and SALL4 in protein sequence, pattern of expression and the physiological Similarities and differences between SALL4 and MeCP2 function. Both mediate the effects of short, frequent DNA sequence consequences of mutation, they share striking similarities. Both recognise short DNA sequence motifs that are frequent in motifs on gene expression by recruiting corepressors containing histone deacetylases (HDACs). However, they recognise different base sequences (methylcytosine-containing versus AT-rich) and recruit different corepressor complexes (NCoR versus the genome and both recruit corepressor complexes leading to widespread modulation of gene expression. Future work NuRD) associated with distinct HDACs (HDAC3 versus HDAC1/2). will seek a comprehensive mechanistic picture of the ways in which mechanisms of this kind help to define and stabilise cell states, and how defects in this system lead to disease.

Selected Publications Tillotson, R., Cholewa-Waclaw, J., Chhatbar, K., Connelly, J., Kirschner, S.A., Webb, S., Koerner, M.V., Selfridge, J., Kelly, D., De Sousa, D., Brown, K., Lyst, M.J., Kriaucionis, S. and Bird, A. (2021). Neuronal non-CG methylation is an essential target for MeCP2 function. Mol Cell 81, 1-16. https://www.cell.com/molecular-cell/pdfExtended/S1097-2765(21)00011-3 PMID: 33561390 Pantier, R., Chhatbar, K., Quante, T., Skourti-Stathaki, K., Cholewa-Waclaw, J., Alston, G., Alexander-Howden, B., Lee, H.Y., Cook, A.G., Spruijt, C.G., Vermeulen, M., Selfridge, J. and Bird, A. (2020). SALL4 controls cell fate in response to DNA base composition. Mol Cell 81(4): 1-16 https://doi.org/10.1016/j.molcel.2020.11.046 PMID: 33406384 Bird, A. (2020). The Selfishness of Law-Abiding Genes. Trends Genet 36, 8-13. 10.1016/j.tig.2019.10.002 PMID: 31662191 10 1111 Dhanya Cheerambathur Role of microtubule cytoskeleton in building and regenerating Co-workers: Mattie Green, Cameron Finlayson, Vasilis Ouzounidis, Emmanuel Fiagbedzi, the neural connectome Charlotte de Ceuninck

The central nervous system is a complex network of neurons and supporting cells that form the information relaying unit of A B C an organism. During neural development, pioneer neurons extend axons in response to guidance cues from other neurons and non-neuronal cells to establish the framework that build the neural circuits. The assembly of this circuit is a highly orchestrated event that involves neurite outgrowth, fasciculation (axon bundling) and synapse formation to generate a functional nervous system. How these organizational features emerge during development is poorly understood. Microtubules are critical for neuron formation and function. As neurons develop, microtubules are organized and sculpted by the cell machinery to form the axons, dendrites and the neural network. Several human neurodevelopmental disorders are linked to mutations in microtubule cytoskeleton-related proteins. Despite the central role of the microtubule, little is known about how the microtubule cytoskeleton contributes to the assembly of the neural circuit. We aim to understand how the microtubule cytoskeleton uses distinct molecular machinery to build and regenerate 3 dimensional neuronal circuits using the simple multicellular organism C. elegans as a model. During my post-doc, I discovered an unexpected role for kinetochore, the chromosome segregation machinery, in D E developing neurons of C. elegans. Our work showed that the evolutionarily conserved 10 subunit KMN (Knl1-Mis12- Ndc80) network, the microtubule coupler within the kinetochore, acts post-mitotically in developing neurons. A similar function for kinetochores proteins has also been described in Drosophila and rat hippocampal cultures. KMN proteins are enriched in the dendritic and axonal outgrowth during neurodevelopment. Removal of KMN components post-mitotically from developing neurons resulted in a disorganized nerve ring, a network of 181 axons and synapses, considered as the “brain” of C. elegans. We hypothesize that the kinetochore proteins facilitate nerve ring assembly by promoting the proper formation of axon bundles. Starting from this unique angle, we aim to understand how the microtubule cytoskeleton integrates distinct molecular machinery to build and regenerate 3 dimensional neuronal circuits in C. elegans. Our goal is to 1) define the function of the kinetochore proteins in building the nerve ring; 2) build a functional map of microtubule cytoskeleton during nerve ring assembly by addressing the function of non-kinetochore microtubule factors; 3) investigate how kinetochore proteins build and maintain neuronal network by addressing its role in dendritic branching and regeneration.

Nerve ring assembly in C.elegans A. The C.elegans head nervous system in L1 larvae (PH marks the membrane and histone the cell body). The axon bundle in the nerve ring is between white arrowheads (scale 10 mm). B. Schematic of KMN network: Mis-12 interface (red) with the centromere, Ndc80 (purple) binds the microtubule and Knl1 (blue) Selected Publications functions as a scaffold. Cheerambathur, D.K., Prevo, B., Chow, T.-L., Hattersley, N., Wang, S., Zhao, Z., Kim, T., Gerson-Gurwitz, A., Oegema, K., Green, R., et al. C. Structure of C.elegans nerve ring in control and after post-mitotic degradation of KNL-1 in the neurons. Axon defasciculation (2019). The Kinetochore-Microtubule Coupling Machinery Is Repurposed in Sensory Nervous System Morphogenesis. Dev. Cell. defect (white arrowhead, scale 5 mm). Cheerambathur, D.K., Prevo, B., Hattersley, N., Lewellyn, L., Corbett, K.D., Oegema, K., and Desai, A. (2017). Dephosphorylation of the D. Fluorescence image and cartoon of the developing nerve ring in C.elegans embryo (pioneer neurons (PN) in purple, amphid Ndc80 Tail Stabilizes Kinetochore-Microtubule Attachments via the Ska Complex. Dev. Cell 41, 424–437.e424. sensory neurons (ASN) in blue). Note that the ASNs have already extended their dendrites (scale 2.5 mm). Cheerambathur, D.K., Gassmann, R., Cook, B., Oegema, K., and Desai, A. (2013). Crosstalk between microtubule attachment complexes E. Schematic representing the initial stages of nerve ring formation. Insets show the extension and bridging of bilaterally ensures accurate chromosome segregation. Science 342, 1239–1242. symmetrical PN axons (scale 1 mm).

12 1313 Atlanta Cook Structural biology of macromolecular complexes in RNA Co-workers: Uma Jayachandran, Ola Kasprowicz, Alexander Will, Mickey Oliver, James Le Cornu (iCM metabolism and transcriptional silencing student), Atika Al Haisani, Laura Croenen

The expression of individual genes is controlled at the levels of mRNA transcription and also post-transcriptionally, by processes such as splicing, localization, modification or editing, and degradation. To gain a mechanistic understanding of these processes it is important to understand the interactions between the individual players, including both protein and nucleic acid components, at the molecular level. We have used structural approaches to tackle mechanistic questions about how protein-RNA interactions can control RNA maturation and RNA editing and how transcriptional repressors are recruited to methylated DNA. By combining structural studies with biochemical, biophysical and cell-based functional assays we can gain powerful insights into these molecular processes. We have contributed to the understanding of the molecular basis for the neurological disease known as Rett syndrome, which is caused by mutations in the DNA binding protein MeCP2. In collaboration with Adrian Bird’s lab, we showed that a cluster of Rett syndrome associated missense mutations on MeCP2 are part of a motif that binds to a component of a nuclear co-repressor complex. This interaction supports a model where MeCP2 acts a bridge between methylated DNA and complexes that drive gene repression. Recently, we solved a crystal structure of a yeast RNA binding protein, Ssd1, that is important in cell wall biogenesis. It is thought that Ssd1 functions by repressing translation of cognate transcripts. Using CRAC, we found that Ssd1 binds to specific sequences in the 5’UTRs of a small set of transcripts, several of which encode proteins required for cell wall biogenesis. The structure of Ssd1 shows that it has a classical fold of an RNase II family nuclease. However, RNA degradation activity has been lost by two mechanisms. First, the catalytic residues have been altered during evolution. Second, a channel that, in active enzymes, allows RNA substrates to funnel into the active site has been blocked. We propose that Ssd1 has evolved a new RNA interacting surface.

A domain overview of Ssd1 is shown along with the crystal structure with domains marked in blue, cyan, green and pink. The Ssd1-specific insert is shown in the domain overview and structure in orange. The yellow lollipops are phosphorylation sites. Two Selected Publications segments of the Ssd1 structure are shown in black – these block the active site funnel, as can be seen by comparison with the Pantier R., Chhatbar K., Quante T., Skourti-Stathaki K., Cholewa-Waclaw J., Alston G., Alexander-Howden B., Lee H.Y., Cook A.G., C structure of DIS3L2 (left), where RNA is bound. Spruijt C.G., Vermeulen M., Selfridge J., and Bird A. (2021) SALL4 controls cell fate in response to DNA base composition. Mol Cell 81:845-858.e8. Ballou E.R., Cook A.G. and Wallace E.W.J. (2020) Repeated evolution of inactive pseudonucleases in a fungal branch of the Dis3/RNase II family of nucleases. Mol. Biol. Evol. doi:10.1093/molbev/msaa324 Zoch, A., Auchynnikava T., Berrens R.V., Kabayama Y., Schöpp T., Heep M., Vasiliauskaite L., Pérez-Rico Y.A., Cook A.G., Shkumatava A., Rappsilber J., Allshire R.C. and O'Carroll D. (2020) SPOCD1 is an essential executor of piRNA-directed de novo DNA methylation Nature 584:635-639.

14 1515 Bill Earnshaw The role of non-histone proteins in chromosome structure Co-workers: Mar Carmena, Fernanda Cisneros, Lorenza Di Pompeo, Moonmoon Deb, and function during mitosis Natalia Kochanova, Emma Peat, Elisa Pesenti, Bram Prevo, Caitlin Reid, Lucy Remnant, Itaru Samejima, Kumiko Samejima

In 2020/21, our research focused on structural transformations during mitotic chromosome formation, epigenetic regulation A of kinetochore assembly/stability and the role of the chromosomal passenger complex (CPC) in regulating cell division. We spent most of 2020/21 at home analysing data, working on manuscripts, chewing fingernails and attending Zoom Journal Club and Lab Meetings (other brands are available, but we don’t use them). The Zoom format may persist after restrictions ease, as we have become quite comfortable with it. Bill’s PRF renewal application was submitted 1 week before Scotland went into lockdown. Painstaking practice for written responses to the grants panel and subsequent negotiations with Wellcome about the final award were ultimately rewarded with success, providing a glimmer of light in this dark period. Bill and Elisa also spent many hours preparing for our return to the lab in July. For the foreseeable future we are limited to 5 people in the main lab at any one time. Thus, progress is impeded, but not completely blocked. Underpinning most of our studies is Kumiko’s chemical-genetic system for obtaining highly synchronous entry of cultured cells into mitosis. We can now do biochemical analysis on processes that could only previously be studied by live-cell microscopy. • We are writing up a comprehensive study of protein-DNA transactions during mitotic entry. This paper focuses on disassembly of the nucleus during very early prophase. This turned out to be unexpectedly interesting, in part because dramatic changes occur in the nucleolus long before any condensation of the DNA can be seen (see Figure). • We are also preparing to publish our multidisciplinary study of interactions between major chromosome scaffold proteins during mitotic chromosome formation, a collaboration with the groups of Job Dekker, Leonid Mirny and Anton B Goloborodko. We do the genetics, cell biology and imaging. They do Hi-C and polymer modelling, respectively. • We continue to study the enigmatic and perplexing protein Ki-67 and the RNA/protein-rich mitotic chromosome periphery compartment (MCPC). Preliminary studies reveal that Ki-67 still has surprises in store for us and indicate that components of the mysterious MCPC may be essential for accurate chromosome segregation. • Our work on mitotic chromosome segregation focuses on the formation of human artificial chromosomes (HACs). We discovered that major genome scrambling similar to chromothripsis often occurs at an early stage in HAC formation. Ongoing experiments aim to understand these early steps in HAC formation, and hopefully minimize the rearrangements. This is critical for planned efforts to construct synthetic human chromosomes which would be futile if chromosomes assembled at great effort and cost were scrambled when introduced into human cells. Our work is supported by a Wellcome Principal Research Fellowship and by the Centre for Mammalian Synthetic Biology.

Selected Publications Papini, D., X. Fant, H. Ogawa, N. Desban, K. Samejima, O. Feizbakhsh, B. Askin, T. Ly, W.C. Earnshaw† & S. Ruchaud†. (2019). †equal corresponding authors. Plk1-dependent cell cycle-independent furrowing triggered by phosphomimetic mutations of the INCENP STD motif. J. CELL SCI. 132: jcs234401 PMID: 31601613; DOI: 10.1242/jcs.234401. Martins*, N.M.C., F. Cisneros-Soberanis*, E. Pesenti*, N.Y. Kochanova, W.-H. Shang, T. Hori, T. Nagase, H. Kimura, V. Larionov, H. A. tSNE map showing protein clusters that move away from DNA at various times during early mitosis. Masumoto, T. Fukagawa & W.C. Earnshaw. (2020). H3K9me3 maintenance on a Human Artificial Chromosome is required for segregation B. The order of movement of the 6 clusters shown here. The left-most two clusters are highly enriched for proteins involved in but not centromere epigenetic memory. J. CELL SCI. 133: JCS242610: PMID: 32576667; PMC7390644; DOI: 10.1242/jcs.242610. pre-rRNA processing and leave the DNA before chromatin condensation is visible. Many of these proteins end up on the mitotic Pesenti,E., M. Liskovykh, K. Okazaki, A. Mallozzi, C. Reid, M.A. Abad, A.A. Jeyaprakash, N. Kouprina, V. Larionov, H. Masumoto, W.C. chromosome periphery. Nuclear pore proteins and proteins of the inner nuclear envelope are also early to move away from DNA. Earnshaw. (2020) Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation. ACS SYNTH BIOL. 9:3267-3287. PMID: 33289546; PMC7754191; DOI: 10.1021/acssynbio.0c00326. 16 1717 Patrick Heun Establishment and maintenance of chromatin identity Co-workers: Meghan Frederick, Mathilde Fabe, Meena Krishnan, Alessandro Stirpe, Emöke Gerocz, George Yankson, Hwei Ling Tan Figure 1

Our lab is interested in the organisation, establishment and maintenance of specialised chromatin states. Epigenetic 1 Mitosis transmission of centromere identity through many cell generations is required for proper centromere function and when perturbed can lead to genome instability and disease. We use Drosophila and human tissue culture cells as model new organisms to address the following questions: A What is the role of transcription at the centromere? Transcription Loading of CENP-A at the centromere occurs outside of S-phase and requires the removal of H3 “placeholder" H3 nucleosomes. Transcription at centromeres has been linked to the deposition of new CENP-A, although the molecular maintenance Spt6 eviction mechanism is not understood. Using fast acting transcriptional inhibitors we demonstrate that centromeric transcription A H3 is required for loading of new dCENP-A and promoting dCENP-A transition from chromatin association to nucleosome old old incorporation (Bobkov et al., 2018). Unlike placeholder nucleosomes, previously deposited CENP-A is specifically retained Centromeres Spt6 by Spt6 both in human and Drosophila cells, identifying Spt6 as a CENP-A maintenance factor that ensures the stability of Figure 1: Model for the role of transcription at centromeres: Transcription remodels epigenetic centromere identity (Figure 1). We are currently investigating the molecular mechanism how some histones like centromere chromatin and evicts H3-nucleosomes (green) to allow new CENP-A (orange) CENP-A are maintained while others like H3.3 placeholders are evicted to preserve epigenetic centromere identity. 2 loading.A Evicted old CENP-A (red) is maintained byC the transcription elongation factor Spt6. How is the centromeric chromatin fiber organised? To map centromere proteins on the linear centromeric chromatin fiber, we have recently developed a novel approach where proteins-of-interest fused to ascorbate peroxidase (APEX) or Turbo ID leave a “footprint” on the underlying nucleosomes through proximity-biotinylation. With this methodology we have described novel localization patterns of a subset of centromere proteins at human centromeres (Kyriacou and Heun, 2018). We are extending this approach to proteins localising to all layers of the centromere, including the inner centromere, CENP-A loading factors and the outer kinetochore. B How does Su(var)2-10/PIAS contribute to heterochromatin organisation? The Su(var)2-10 gene has been originally identified in position-effect-variegation (PEV) assays designed to uncover proteins involved in heterochromatin formation. Cloning of the gene revealed its homology to the protein family SUMO E3-ligase PIAS (Protein Inhibitor of activated STAT), but how sumoylation promotes heterochromatin formation remains unknown. While PIAS does not localise to pericentric heterochromatin in somatic cells, it is enriched next to centromeres in early fly embryogenesis, suggesting a role in heterochromatin establishment. Specific depletion of PIAS as this point of development will shed light on the link between PIAS’ SUMO targets and chromatin organisation. Figure 1. Model for the role of transcription at centromeres: Transcription remodels centromere chromatin and evicts H3- nucleosomes (green) to allow new CENP-A (orange) loading. Evicted old CENP-A (red) is maintained by the transcription Selected Publications elongation factor Spt6. Bobkov, G.O.M., Huang, A., van den Berg, S.J.W., Mitra, S., Anselm, E., Lazou, V., Schunter, S., Feederle, R., Imhof, A., Lusser, A., Jansen, L.E.T., and Heun, P. (2020). Spt6 is a maintenance factor for centromeric CENP-A. Nature communications 11, 2919. Figure 2. The SUMO E3 Ligase PIAS is required for heterochromatin formation. Medina-Pritchard, B., Lazou, V., Zou, J., Byron, O., Abad, M.A., Rappsilber, J., Heun, P., and Jeyaprakash, A.A. (2020). Structural basis for A. Embryonic cycle 1-14 (image William Sullivan) centromere maintenance by Drosophila CENP-A chaperone CAL1. EMBO J 39, e103234. PMC7110144 B. Fixed cycle 13 embryos showing αPIAS and heterochromatin marker αH3K9me3 in apical heterochromatin in wildtype and Roure, V., Medina-Pritchard, B., Lazou, V., Rago, L., Anselm, E., Venegas, D., Jeyaprakash, A.A., and Heun, P. (2019). Reconstituting PIAS RNAi. ­­ Drosophila Centromere Identity in Human Cells. Cell Rep 29, 464-479 e465. C. Proposed role of PIAS in chromosome organisation. 18 1919 Adele Marston Orienting Chromosomes during Mitosis and Meiosis Co-workers: Weronika Borek, Chuanli Huang, Dilara Kocakaplan, Lori Koch, Melanie Lim, Vasso Makrantoni , Lucia Massari, Bettina Mihalas, Anuradha Mukherjee, Meg Peyton-Jones, Gerard Pieper, Ola Pompa, Xue (Bessie) Su, Aparna Vinod, Menglu (Lily) Wang

Specialization of chromosome segregation mechanisms in meiosis A B Meiosis generates gametes with half the parental genome through two consecutive chromosome segregation events, meiosis I and meiosis II. Meiotic errors are prevalent in humans, accounting for frequent miscarriages, birth defects and infertility. Our vision is to elucidate the molecular basis of the adaptations that sort chromosomes into gametes during meiosis. We use budding and fission yeast as general discovery tools, and Xenopus and mouse oocytes to uncover meiotic mechanisms in vertebrates. Using patient-donated oocytes and ovarian tissue, we address the relevance of our findings for human fertility. Structural and functional organisation of pericentromeres During meiosis, chromosomes undergo remarkable remodelling for transmission into gametes. Chromosomes are broken and reciprocally exchanged in prophase, specifically cohered at centromeres during meiosis I and permanently separated at meiosis II. Chromosome morphogenesis begins before S phase, when cohesion establishment links sister chromatids coupled to DNA replication. The cohesin complex is a major definer of chromosome structure, establishing intra and inter-sister chromatid linkages and providing the context for spatial control of homolog interactions. Our group revealed how chromosomes are structured for their segregation during mitosis. We established the region around the centromere, C called the pericentromere, as a paradigm for chromosomal domain organisation. We discovered that cohesin is loaded at the centromere and that the borders of pericentromeres are marked by convergent genes that trap cohesin. This folds pericentromeres into a looped structure that is important for accurate chromosome segregation. Our current work is aimed at understanding how pericentromere structure is adapted during meiosis to suppress meiotic recombination and to accommodate the co-segregation of sister chromatids during meiosis I. Specialization of meiotic kinetochores Kinetochores link centromeric nucleosomes to microtubules for chromosome segregation. Our goal is to understand how the kinetochore is adapted to perform its meiosis-specific functions in suppression of meiotic recombination, directing the co-segregation of sister chromatids during meiosis I, and maintaining linkages between sister chromatids until meiosis II. Recently, we defined the proteomic landscape of yeast kinetochores and centromeric chromatin during meiosis, revealing extensive remodelling during prophase and meiosis I. We are now addressing the mechanism of kinetochore remodelling, as well as its functional importance. In many organisms, sister kinetochores are fused in meiosis I, while a lack of fusion in human oocytes may account for susceptibility to segregation errors and fertility problems. Ongoing work in Xenopus, mouse and human oocytes aims to test this hypothesis.

Selected Publications Borek WE, Vincenten N, Duro E, Makrantoni V, Spanos C, Sarangapani KK, de Lima Alves F, Kelly DA, Asbury CL, Rappsilber J and A. Whole genome Hi-C view showing chromosomal contacts of budding yeast arrested in mitotic metaphase. Marston AL. (2021) The proteomic landscape of centromeric chromatin reveals an essential role for the Ctf19CCAN complex in meiotic B. The Mcm21 protein is required for kinetochore remodeling in yeast meiosis. Live cell imaging of kinetochore protein Mtw1- kinetochore assembly. Current Biology 31, 283-296. tdTomato in wild type and mcm21D cells. Paldi F, Alver B, Robertson D, Schalbetter SA, Kerr A, Kelly D, Baxter J, Neale MJ and Marston AL (2020). Convergent genes shape C. Human metaphase II oocyte stained for tubulin (orange), DNA (blue) and shugoshin 2 (green). budding yeast pericentromeres. Nature 582, 119-123. Galander S, Barton RE*, Borek WE*, Spanos C, Kelly DA, Robertson D, Rappsilber J and Marston AL (2019) Reductional meiosis I chromosome segregation is established by coordination of key meiotic kinases. Developmental Cell 49, 526-541. 20 2121 Dónal O'Carroll Regulation of gene expression by non-coding RNA Co-workers: Pedro Moreira, Ansgar Zoch, Yuka Kabayama, Azzurra Codino, Azzurra DePace, and RNA modification Theresa Schöpp, Gabriela Konieczny, Madeleine Heep, Christopher Mapperly, Louie Van De Lagemaat, Hanna Fieler

The O’Carroll laboratory has a longstanding interest in mechanisms that regulate gene and transposon expression. Using the germline and haematopoiesis as model systems, our research explores the importance and molecular mechanisms of regulatory RNA pathways in dynamic developmental and physiological contexts. We currently focus on the PIWI-interacting RNA (piRNA) and RNA modification pathways. The piRNA pathway In mammals, the acquisition of the germline from the soma provides the germline with an essential challenge, the necessity to erase and reset genomic methylation. This is one of the most drastic epigenetic events in mammalian life. De novo genome methylation re-encodes the epigenome, imprinting and transposable element (TE) silencing. In the male germline piRNA-directed DNA methylation silences young active TEs. Antisense TE-derived piRNAs generated from intricate biogenesis pathways act to guide the nuclear PIWI protein MIWI2 to instruct TE DNA methylation. piRNAs are proposed to tether MIWI2 to the young transcriptionally active TE loci that escape the first phase of genome methylation by base pairing to nascent transcripts. The recruitment of MIWI2 sets in motion TE silencing and methylation through the recruitment of unknown effector molecules. Recently we have found the first such effectors of nuclear MIWI2 function. Both SPOCD1 and TEX15 interact with MIWI2 and are essential for piRNA-directed DNA methylation (Nature 2020 and Nature Communications 2020). SPOCD1 interacts with the de novo methylation machinery, whereas the molecular function of TEX15 remains unknown. Our future goal is to fully understand the mechanism by which MIWI2 instructs TE methylation and epigenetic silencing (Figure 1). RNA modification The role of RNA modification in the post-transcriptional regulation of gene expression is only beginning to be understood. The O'Carroll laboratory focuses on understanding the function of N6-methyladenosine (m6A) and 3' terminal uridylation mRNA modifications, both of which can promote RNA degradation. TUT4 and TUT7 mediate 3' uridylation of mRNAs with short poly(A) tails that primes these transcripts for degradation, whereas the binding of YTHDF2 to m6A-modified mRNA promotes transcript decay. We have shown critical functions for these pathways in the metabolism of the maternal transcriptome and oocyte competence. We have also demonstrated that YTHDF2 is overexpressed in multiple sub-types of acute myeloid leukaemia (AML) and its deletion selectively compromises cancer stem cells, without grossly perturbing normal haematopoiesis (Cell Stem Cell 2019). We are currently exploring basic questions regarding the mechanism, regulation and redundancy of the YTH domain family of m6A readers.

Selected Publications Genomic DNA methylation is erased (reprogramming) and reset (de novo methylation) during germ cell development. Our current Zoch, A., Auchynnikava, T., Berrens, R. V., Kabayama, Y., Schöpp, T., Heep, M., Vasiliauskaite, L., Pérez-Rico, Y. A., Cook, A. G., Shkumat- model of MIWI2-piRNA directed TE methylation based on functionally defined interactions is presented. ava, A., Rappsilber, J., Allshire, R. C., & O'Carroll, D. (2020). SPOCD1 is an essential executor of piRNA-directed de novo DNA methyla- tion. Nature, 10.1038/s41586-020-2557-5. https://doi.org/10.1038/s41586-020-2557-5 Schöpp, T., Zoch, A., Berrens, R. V., Auchynnikava, T., Kabayama, Y., Vasiliauskaite, L., Rappsilber, J., Allshire, R. C., & O'Carroll, D. (2020). TEX15 is an essential executor of MIWI2-directed transposon DNA methylation and silencing. Nature Communications, 11(1), 3739. https://doi.org/10.1038/s41467-020-17372-5 Paris J, Morgan M, Campos J, Spencer GJ, Shmakova A, Ivanova I, Mapperley C,Lawson H, Wotherspoon DA, Sepulveda C, Vukovic M, Allen L, Sarapuu A, Tavosanis A, Guitart AV, Villacreces A, Much C, Choe J, Azar A, van de Lagemaat LN,Vernimmen D, Nehme A, Mazurier F, Somervaille TCP, Gregory RI, O'Carroll D*, Kranc KR*. Targeting the RNA m(6)A Reader YTHDF2 Selectively Compromises Cancer Stem Cells in Acute Myeloid Leukemia. Cell Stem Cell. 2019 Apr 24. pii:S1934-5909(19)30120-1. doi: 10.1016/j.stem.2019.03.021. * 22 co-corresponding authors 2323 Hiro Ohkura The meiotic spindle and chromosomes in oocytes Co-workers: Fiona Cullen, Jule Nieken, Charlotte Repton, Emiliya Taskova, Dan Toddie-Moore, Gera Pavlova, Xiang Wan Downloaded from http://rupress.org/jcb/article-pdf/220/2/e202009167/1407234/jcb_202009167.pdf by guest on 31 December 2020 Downloaded from http://rupress.org/jcb/article-pdf/220/2/e202009167/1407234/jcb_202009167.pdf by guest on 31 December 2020 Downloaded from http://rupress.org/jcb/article-pdf/220/2/e202009167/1407234/jcb_202009167.pdf by guest on 31 December 2020 Downloaded from http://rupress.org/jcb/article-pdf/220/2/e202009167/1407234/jcb_202009167.pdf by guest on 31 December 2020

Accurate segregation of chromosomal DNA is essential for life. An error in this process could result in cell death or A B aneuploidy. Furthermore, chromosome segregation in oocytes is error-prone in humans, and mis-segregation is a major A A B Downloaded from http://rupress.org/jcb/article-pdf/220/2/e202009167/1407234/jcb_202009167.pdf by guest on 31 December 2020 B Downloaded from http://rupress.org/jcb/article-pdf/220/2/e202009167/1407234/jcb_202009167.pdf by guest on 31 December 2020 cause of infertility, miscarriages and birth defects. Chromosome segregation in oocytes shares many similarities with those Downloaded from http://rupress.org/jcb/article-pdf/220/2/e202009167/1407234/jcb_202009167.pdf by guest on 31 December 2020 in somatic divisions, but also has notable differences. Distinct features of oocytes potentially hinder accurate chromosome A A B Downloaded from http://rupress.org/jcb/article-pdf/220/2/e202009167/1407234/jcb_202009167.pdf by guest on 31 December 2020 B segregation. They include (1) lack of centrosomes, the major microtubule nucleation centres in mitosis, (2) exceptionally large cell volume, and (3) cell cycle arrests at two stages. Oocytes are likely to have specific molecular mechanisms which mitigate negative impacts of these features, but little is known about how oocytes set up the chromosome segregation machinery. Defining the oocyte-specific mechanisms would be crucial to understand error-prone chromosome segregation in human oocytes. Furthermore, it may provide an insight into whether and how cancer cells might gain resistance to anti- mitotic drugs by activating these pathways. To understand the molecular pathways which set up the chromosome segregation machinery in oocytes, we take Downloaded from http://rupress.org/jcb/article-pdf/220/2/e202009167/1407234/jcb_202009167.pdf by guest on 31 December 2020 Downloaded from http://rupress.org/jcb/article-pdf/220/2/e202009167/1407234/jcb_202009167.pdf by guest on 31 December 2020 advantage of Drosophila oocytes as a "discovery platform" because of their similarity to mammalian oocytes and suitability Downloaded from http://rupress.org/jcb/article-pdf/220/2/e202009167/1407234/jcb_202009167.pdf by guest on 31 December 2020 Downloaded from http://rupress.org/jcb/article-pdf/220/2/e202009167/1407234/jcb_202009167.pdf by guest on 31 December 2020

for a genetics-led mechanistic analysis. In Drosophila oocytes, as in human oocytes, meiotic chromosomes form a Downloaded from http://rupress.org/jcb/article-pdf/220/2/e202009167/1407234/jcb_202009167.pdf by guest on 31 December 2020 Downloaded from http://rupress.org/jcb/article-pdf/220/2/e202009167/1407234/jcb_202009167.pdf by guest on 31 December 2020 Downloaded from http://rupress.org/jcb/article-pdf/220/2/e202009167/1407234/jcb_202009167.pdf by guest on 31 December 2020 compact cluster called the karyosome within the nucleus. Later, meiotic chromosomes assemble a bipolar spindle without Downloaded from http://rupress.org/jcb/article-pdf/220/2/e202009167/1407234/jcb_202009167.pdf by guest on 31 December 2020 centrosomes in the large volume of the cytoplasm, and establish bipolar attachment. We have identified genes/proteins C C D D and regulations specifically important for chromosome organisation and/or spindle formation in oocytes. C C DD The synaptonemal complex assembles during meiotic prophase I and assists faithful exchanges between homologous C D chromosomes, but how its assembly/ disassembly is regulated remains to be understood. We showed how two major post- translational modifications, phosphorylation and ubiquitination, cooperate to promote synaptonemal complex assembly. We found that the ubiquitin ligase complex SCF is important for assembly and maintenance of the synaptonemal complex in female meiosis. This function of SCF is mediated by two substrate-recognizing F-box proteins, Slmb/βTrcp and Fbxo42. SCF-Fbxo42 down-regulates the phosphatase subunit PP2A-B56, which is important for synaptonemal complex assembly FigureFigure 4. Fbxo42 4. Fbxo42 depletion depletion increases increases PP2A-B56 PP2A-B56 (Wrd) (Wrd)levels, levels, which whichdestabilize destabilize the synaptonemal the synaptonemal complex. complex. (A) A volcano (A) A volcano plot of plot proteins of proteins im- im- and maintenance. munoprecipitatedmunoprecipitated with GFP-Fbxo42 with GFP-Fbxo42 or GFP fromor GFP ovaries. from ovaries. For each For protein, each protein, the mean the ratio mean of ratio signal of intensities signal intensities found in found immunoprecipitates in immunoprecipitates of GFP-Fbx of GFP-Fbxo42 ando42 and GFP aloneGFP was alone plotted was plotted against against the statistical the statistical significance significance (the P value (the P given value by givent test) by fromt test) biological from biological triplicates. triplicates. SCF subunits, SCF subunits, PP2A core PP2A subunits core subunits and 14– and3-3 14–3-3 proteinsproteinsFigure were 4. significantly wereFbxo42 significantly depletion enriched enriched in increases GFP-Fbxo42 in GFP-Fbxo42 PP2A-B56 immunoprecipitates. immunoprecipitates. (Wrd) levels,(B) whichLocalization(B) destabilizeLocalization of the the synaptonemalof synaptonemal the synaptonemal complex complex. complex component (A) componentA volcano C(3)G in C(3)G plot meiotic of in meioticproteins cells cells im- Figure 4. Fbxo42 depletion increases PP2A-B56 (Wrd) levels, which destabilize the synaptonemal complex. (A) A volcano plot of proteins im- expressingexpressingmunoprecipitated GFP-Wrd GFP-Wrd (PP2A-B56) with (PP2A-B56) GFP-Fbxo42 and a control. and or a GFP control. Scale from bar Scale ovaries. = 3 bar µm. For = GFP-Wrd, 3each µm. GFP-Wrd,protein,nos-GAL4(MVD1) thenos-GAL4(MVD1) mean ratio Wrd of+/UASp-GFP-Wrd signal Wrd+/UASp-GFP-Wrd intensities Wrd found+; control, Wrd in immunoprecipitates+; control,nos-GAL4(MVD1)nos-GAL4(MVD1)of Wrd GFP-Fbx+/Wrd Wrd+o42.+/Wrd and+. munoprecipitated with GFP-Fbxo42 or GFP from ovaries. For each protein, the mean ratio of signal intensities found in immunoprecipitates of GFP-Fbxo42 and (C) C(3)GGFP(C) localizationC(3)G alone localization was patterns plotted patterns againstin meiotic in the meiotic cells statistical overexpressing cells significance overexpressing GFP-Wrd (the GFP-Wrd P (PP2A-B56)value given (PP2A-B56) andby t controltest) and from control cells biological throughout cells throughout triplicates. meiotic meiotic SCFprogression. subunits, progression. Error PP2A bars coreError repr subunits barsesent repr SEM andesent 14– SEM3-3 GFP alone wasproteins plotted were against significantly the statistical enriched significance in GFP-Fbxo42 (the P value immunoprecipitates. given by t test) from(B) biologicalLocalization triplicates. of the synaptonemal SCF subunits, complex PP2A core component subunits and C(3)G 14– in3-3 meiotic cells proteins wereexpressing significantly GFP-Wrd enriched (PP2A-B56) in GFP-Fbxo42 and a control. immunoprecipitates. Scale bar = 3 µm.(B) GFP-Wrd,Localizationnos-GAL4(MVD1) of the synaptonemal Wrd+/UASp-GFP-Wrd complex component Wrd+; control, C(3)Gnos-GAL4(MVD1) in meiotic cells Wrd+/Wrd+. + + + + expressing GFP-Wrd(C) C(3)G (PP2A-B56) localization andpatterns a control. in meiotic Scale cells bar =overexpressing 3 µm. GFP-Wrd, GFP-Wrdnos-GAL4(MVD1) (PP2A-B56) Wrd and/UASp-GFP-Wrd control cells throughout Wrd ; control, meioticnos-GAL4(MVD1) progression. Error Wrd bars/Wrd repr. esent SEM FigureFigure 4. Fbxo42 4. Fbxo42 depletion depletion increases increases PP2A-B56 PP2A-B56 (Wrd) (Wrd) levels, levels, which which destabilize destabilize the synaptonemal the synaptonemal complex. complex. (A) A volcano (A) A volcano plot of plot proteins of(C) proteins im-C(3)GBarbosa localizationim-Barbosa et al. et patterns al. in meiotic cells overexpressing GFP-Wrd (PP2A-B56) and control cells throughout meiotic progression. Error barsJournal reprJournal ofesent Cell SEM of Biology Cell Biology6of126of12 FigureFigure 3. SCF 3. complexesSCF complexes containing containing Slmb/ Slmb/βTrcpβ orTrcp CG6758/Fbxo42 or CG6758/Fbxo42 mediate mediate regulation regulation of the of synaptonemal the synaptonemal complex. complex. (A) The (A) synaptonemalThe synaptonemal complex complex munoprecipitatedmunoprecipitated with GFP-Fbxo42 with GFP-Fbxo42 or GFP orfrom GFP ovaries. from ovaries. For each For protein, each protein, the mean the ratio mean of ratio signal of intensities signal intensities found in found immunoprecipitates in immunoprecipitates of GFP-Fbx of GFP-Fbxo42 andSCF-Fbxo42o42 andSCF-Fbxo42 promotes promotes the synaptonemal the synaptonemal complex complex https://doi.org/10.1083/jcb.202009167https://doi.org/10.1083/jcb.202009167 GFP aloneGFP was alone plotted was plotted against against the statistical the statistical significance significance (the P value (thecomponent P given valuecomponent by givent test) C(3)G by fromt test) C(3)Gand biological from DNA and biological in triplicates. DNA control, in triplicates. control, SCFSlmb/ subunits, SCFβSlmb/Trcp subunits,, PP2Aβ andTrcp coreFbxo42, PP2A and subunits coreFbxo42RNAi subunits and throughoutRNAi 14– and3-3 throughout 14–3-3 meiotic meiotic progression. progression. Scale barScale = 2 bar µm. = 2(B) µm.C(3)G(B) C(3)G localization localization patterns patterns in control, in control, Figure 4. A volcano plot of proteins im- proteinsproteins were significantly wereFbxo42 significantly depletion enriched enriched in increases GFP-Fbxo42 in GFP-Fbxo42 PP2A-B56 immunoprecipitates. immunoprecipitates. (Wrd) levels,(B) whichLocalization(B) destabilizeLocalization of the the ofsynaptonemal synaptonemal the synaptonemal complex complex. complex component (A) component C(3)G in C(3)G meiotic in meiotic cells cellsBarbosa et al. – – Journal of Cell Biology 6of12 Figure 4. Fbxo42munoprecipitated depletion with increases GFP-Fbxo42 PP2A-B56 or GFP (Wrd) from ovaries. levels, For which eachSlmb/ destabilize protein,βFigureSlmb/Trcp theβRNAi, 3.Trcp themeanSCFRNAi, and synaptonemal ratio complexesFbxo42 andof signalFbxo42RNAi. intensities complex. containingRNAi. Error found (A) barsErrorA Slmb/ in representvolcano bars immunoprecipitatesβ representTrcp plot SEM ofor proteins CG6758/Fbxo42 from SEM of triplicatedGFP-Fbx from im-Barbosa triplicatedo42 experimentsetand mediate al. experiments regulation representing representing of the 20 synaptonemal31 20 germaria/oocytes31 germaria/oocytes complex. for (A) each forThe region/stage. each synaptonemal region/stage. *, P complex < *, P < Journal of Cell Biology 6of12 expressingexpressing GFP-Wrd GFP-Wrd (PP2A-B56) (PP2A-B56) and a control. and a control.SCF-Fbxo42 Scale bar Scale = 3 bar µm.Figure = GFP-Wrd, 3 µm. promotes 3. GFP-Wrd,nos-GAL4(MVD1)SCF complexesnos-GAL4(MVD1) synaptonemal Wrd containing+/UASp-GFP-Wrd Wrd+/UASp-GFP-Wrd Slmb/ Wrd complexβ+Trcp; control, Wrd+ or; control,nos-GAL4(MVD1) CG6758/Fbxo42 assemblynos-GAL4(MVD1) Wrd mediate+/Wrdby Wrd +downregulating.+/Wrd regulation+SCF-Fbxo42. of promotes the synaptonemal thePP2A-B56. synaptonemal complex. complex (A) The synaptonemal complex https://doi.org/10.1083/jcb.202009167 Selected Publications munoprecipitatedGFP alone with was GFP-Fbxo42 plotted against or GFP the from statistical ovaries. significanceFor each protein, (the0.05; P the value mean **,component0.05; given P ratio < **, by 0.01; oft Ptest) signal < C(3)G ***,0.01; from intensities andP biological***, < DNA0.001. P found < in triplicates. 0.001. control, in immunoprecipitates SCFSlmb/ subunits,βTrcp, PP2A of and GFP-Fbx coreFbxo42 subunitso42RNAi andSCF-Fbxo42 and throughout 14–3-3 promotes meiotic the progression.synaptonemal complex Scale bar = 2 µm. (B) C(3)G localization patterns in control, https://doi.org/10.1083/jcb.202009167 GFP(C) aloneC(3)G was(C) localizationC(3)G plotted localization against patterns the patterns statisticalin meiotic in meiotic significancecells overexpressing cells overexpressing(the P value GFP-Wrdcomponent given GFP-Wrd (PP2A-B56)by t test) C(3)G (PP2A-B56) from and and biological control DNAand control cells in triplicates. control, throughout cells throughout SCFSlmb/ meiotic subunits,βTrcp meiotic progression., PP2A and progression. coreFbxo42 Error subunitsRNAi bars Error and reprthroughout bars 14esent–3-3 repr SEMesent meiotic SEM progression. Scale bar = 2 µm. (B) C(3)G localization patterns in control, proteins were significantly enriched inA. GFP-Fbxo42 Two substrate-recognition immunoprecipitates.Slmb/β(B)TrcpLocalizationRNAi, and subunits ofFbxo42 the synaptonemalRNAi. of ErrorSCF, complex bars Slmb/ representcomponent βTrcp SEMC(3)G fromand in meiotic triplicated Fbxo42, cells experiments are required representing for 20synaptonemal–31 germaria/oocytes complex for each region/stage. assembly *, P < Barbosa, P., Zhaunova, L., Debilio, S., Steccanella, V., Kelly, V., Ly, T., and Ohkura H. (2021) SCF-Fbxo42 promotes synaptonemal proteins wereexpressing significantly GFP-Wrd enriched (PP2A-B56) in GFP-Fbxo42 and a control. immunoprecipitates. Scale barSlmb/ = 3 µm.β(B)Trcp GFP-Wrd,LocalizationRNAi,nos-GAL4(MVD1) and ofFbxo42 the synaptonemalRNAi. Wrd+/UASp-GFP-Wrd Error complex bars representcomponent Wrd+; control, SEMC(3)Gnos-GAL4(MVD1) from in meiotic triplicated cells Wrd experiments+/Wrd+. representing 20–31 germaria/oocytes for each region/stage. *, P < 0.05; **, P < 0.01;+ ***, P < 0.001.+ + + complex assembly by downregulating PP2A-B56. J. Cell Biol. 220, e202009167. expressing GFP-Wrd(C) C(3)G (PP2A-B56)localization patternsand a control. in meiotic ScaleB. cells bar Fbxo42 overexpressing= 3 µm. GFP-Wrd,0.05; co-immunoprecipitatesimmunoprecipitated GFP-Wrd **,nos-GAL4(MVD1) Pimmunoprecipitated < (PP2A-B56) 0.01; ***, Wrd and P/UASp-GFP-Wrd control< 0.001. from cells from throughoutdissectedwith Wrd dissected;PP2A control, meiotic ovariesnos-GAL4(MVD1) and progression. ovaries using14-3-3 Error using Wrd an barsin/Wrd anti-GFP anaddition repr. anti-GFPesent SEM Deshaies,to SCFDeshaies, subunits 2005 2005), the ), Fbxo42-interacting the Fbxo42-interacting proteins proteins we identified we identified (C) C(3)G localization patterns in meiotic cells overexpressing GFP-Wrd (PP2A-B56) and control cells throughout meiotic progression. Error bars represent SEM BarbosaBarbosa et al. et al. nanobody.nanobody. Using Using GFP-expressing GFP-expressing ovaries ovaries as aJournal ascontrol, aJournal of control, Cell quanti- of Biology Cell quanti- Biology6of12are6of12 goodare good candidates candidates for SCF-Fbxo42 for SCF-Fbxo42 substrates. substrates. Costa, M. F. A., and Ohkura, H. (2019) The molecular architecture of the meiotic spindle is remodeled during metaphase arrest in oocytes.SCF-Fbxo42 SCF-Fbxo42 promotes promotes the synaptonemal the synaptonemal complexC. complex Fbxo42 downregulates the PP2A-B56 subunithttps://doi.org/10.1083/jcb.202009167https://doi.org/10.1083/jcb.202009167 Wrd tativetativeimmunoprecipitated mass mass spectrometry spectrometry from was performed.dissectedwas performed. ovaries In addition In using addition an to SCFanti-GFP to SCF ToDeshaies, testTo whether test 2005 whether any), the of any Fbxo42-interacting these of these are critical are critical substrates, proteins substrates, we we identifiedgen- we gen- J. Cell Biol. 218, 2854-2864. Barbosa et al. D. A schematicimmunoprecipitated model showing from that dissected SCF-Fbxo42 ovaries using down-regulates anJournal anti-GFP of Cell Biology Deshaies,PP2A-B566of12 2005 to), tip the the Fbxo42-interacting balance of phosphorylation proteins we identified towards Barbosa et al. subunits,subunits,nanobody. 4 and 4 37Using and proteins 37 GFP-expressing proteins were were coimmunoprecipitated coimmunoprecipitated ovariesJournal as of Cella control, Biology specif- quanti- specif-6of12eratederatedare transgenic good transgenic candidates flies fliesexpressing for expressing SCF-Fbxo42 GFP-tagged GFP-tagged substrates. 14–3-3 14– isoforms3-3 isoforms Romé, P., and Ohkura, H. (2018) A novel microtubule nucleation pathway for meiotic spindle assembly in oocytes. J. Cell Biol. 217, 3431- SCF-Fbxo42 promotes the synaptonemal complex nanobody. Using GFP-expressing ovaries ashttps://doi.org/10.1083/jcb.202009167 a control, quanti- are good candidates for SCF-Fbxo42 substrates. SCF-Fbxo42 promotes the synaptonemal complex synaptonemalically complexicallytative with with massFbxo42 assembly. Fbxo42 spectrometry (Fig. ( 4Fig. Barbosa A) 4 andhttps://doi.org/10.1083/jcb.202009167 wasA) Slmb/ and performed. et Slmb/alβTrcp (2021).βTrcp (Fig. In addition ( S3Fig. D), S3 re- toD), SCF re-and PP2AandTo PP2A subunits test subunitswhether (C and any(C A) and of that these A) interactthat are interact critical with with Fbxo42.substrates, Fbxo42. In ad-we In gen- ad- 3445. tative mass spectrometry was performed. In addition to SCF FigureTo 4.Figure testFbxo42 4. whetherFbxo42 depletion depletion increasesany of increases PP2A-B56these PP2A-B56 are (Wrd) critical levels,(Wrd) levels,whichsubstrates, destabilizewhich destabilize we the gen- synaptonemal the synaptonemal complex. complex. (A) A volcano (A) A volcano plot of plot proteins of proteins im- im- subunits, 4 and 37 proteins were coimmunoprecipitated specif- erated transgenic flies expressing GFP-tagged 14–3-3 isoforms subunits,spectively.spectively. 4 and We 37 decided proteinsWe decided to were focus to focuscoimmunoprecipitated on these on these four Fbxo42-interacting four Fbxo42-interacting specif- munoprecipitatederateddition,munoprecipitated transgenicdition, we with alsoGFP-Fbxo42 we with fliesgenerated alsoGFP-Fbxo42 or generatedexpressing GFP fromor GFPtransgenic ovaries. from transgenic GFP-taggedovaries. For each flies For protein, each thatflies protein, the 14– mean canthat3-3 the ratio express meanisoforms can of ratiosignal express of var-intensities signal var-intensities found in found immunoprecipitates in immunoprecipitates of GFP-Fbx of GFP-Fbxo42 ando42 and proteins:proteins:ically the with catalytic the Fbxo42 catalytic (C) (Fig. subunit (C) 4 subunit A and) and the and Slmb/ structural theβ structuralTrcp (A) (Fig. subunit (A) S3 subunit DGFP), alonere-iousGFP was alone Biousand plotted regulatory was PP2A B plottedagainst regulatory subunits againstthe subunits statistical the subunits statistical (C significance of and PP2A, significance of A) (the PP2A, that including P value (the interact including Pgiven value by Tws givent test) with by (B; Tws fromt test) B55), Fbxo42. biological (B; from B55), Wrd biological triplicates. In Wrd ad- triplicates. SCF subunits, SCF subunits, PP2A core PP2A subunits core subunits and 14– and3-3 14–3-3 ically with Fbxo42 (Fig. 4 A) and Slmb/βTrcp (Fig. S3 D), re- proteinsandproteinsFigure PP2A were 4.significantly were subunitsFbxo42 significantly enricheddepletion (C enriched and in increases GFP-Fbxo42 A) in GFP-Fbxo42 that PP2A-B56 immunoprecipitates. interact immunoprecipitates. (Wrd) with levels,(B) Fbxo42.whichLocalization(B) destabilizeLocalization In of ad-the the synaptonemal of synaptonemal the synaptonemal complex complex. complex component (A) componentA volcanoC(3)G in C(3)G plot meiotic of in meioticproteins cells cells im- Figure 4. A volcano plot25 of proteins im- 24 of proteinofspectively. protein phosphatase phosphatase We decided 2A (PP2A) to 2A focus (PP2A) and on these the and two the four 14 two Fbxo42-interacting–3-3 14– isoforms3-3 isoformsexpressing(BFbxo42expressingmunoprecipitated9; GFP-Wrd B56),(Bdition, depletion9; GFP-Wrd B56), (PP2A-B56) and we with increases Wdb (PP2A-B56) and alsoGFP-Fbxo42 and Wdb (B aPP2A-B56generated control.9 and; B56), or (B a GFP control.9 Scale; (Wrd) B56),from as bar transgenic Scale theovaries. levels, =3 as barµm. B the whichFor =subunits GFP-Wrd, 3each µm. B flies destabilize subunits GFP-Wrd,protein,nos-GAL4(MVD1)that confer thenos-GAL4(MVD1) the mean can confer synaptonemal substrate ratioexpress Wrd of+ substrate/UASp-GFP-Wrd signal Wrd+ complex.var-/UASp-GFP-Wrd intensities Wrd (A) found+; control, Wrd in immunoprecipitates+; control,nos-GAL4(MVD1)25nos-GAL4(MVD1) Wrdof GFP-Fbx+/Wrd Wrd+.o42+/Wrd and+. spectively. We decided to focus on these four Fbxo42-interactingmunoprecipitateddition, we with alsoGFP-Fbxo42 generated or GFP from transgenic ovaries. For each flies protein, that the mean can ratio express of signal var-intensities found in immunoprecipitates of GFP-Fbxo42 and (14–3-3(14proteins:–and3-3 and). the catalytic). (C) subunit and the structural (A) subunit(C) C(3)GspecificityGFP(C) localizationC(3)G alonespecificityious localization was B patterns plottedto regulatory the patterns in toagainst meiotic core the in the meioticcells corePP2Asubunits statistical overexpressing cellsPP2A complex significance overexpressing of complex PP2A, GFP-Wrd (C (the +includingGFP-Wrd P (PP2A-B56) valueA;(CChen + given (PP2A-B56) A; and byChen Tws ett controltest) al., and (B; et from2007control cells B55),al., biological throughout2007). cells Wrd throughout triplicates.). meiotic meioticprogression. SCF subunits, progression. Error PP2A bars coreError repr subunits barsesent repr SEM andesent 14– SEM3-3 proteins: theε catalyticεζ ζ (C) subunit and the structural (A) subunitGFP aloneious wasproteins B plotted regulatory were against significantly the subunits statistical enriched significance of in PP2A, GFP-Fbxo42 (the including P value immunoprecipitates. given by Twst test) (B; from(B) B55), biologicalLocalization Wrd triplicates. of the synaptonemal SCF subunits, PP2A complex core component subunits and C(3)G 14–3-3 in meiotic cells Weofshowed proteinWe showed phosphatasethat SCF-Fbxo42that SCF-Fbxo42 2A (PP2A) regulates regulates and synaptonemal the two synaptonemal 14–3-3 com-proteins isoforms com- wereTheseexpressing significantlyThese(B proteins9; GFP-Wrd B56), enriched proteins (PP2A-B56) and were in GFP-Fbxo42 Wdb were individuallyand (B a control.9individually immunoprecipitates.; B56), Scale as overexpressed bar the = 3 overexpressed µm. B(B) subunits GFP-Wrd,Localizationnos-GAL4(MVD1) in ofconfer otherwise the in synaptonemal otherwise substrate Wrd+/UASp-GFP-Wrd complex component Wrd+; control, C(3)Gnos-GAL4(MVD1) in meiotic cells Wrd+/Wrd+. of protein phosphatase 2A (PP2A) and the two 14–3-3 isoformsexpressing(B9; GFP-Wrd B56), (PP2A-B56) and Wdb and (B a control.9; B56), Scale as bar the = 3 µm. B subunits GFP-Wrd, nos-GAL4(MVD1) confer substrate Wrd+/UASp-GFP-Wrd Wrd+; control, nos-GAL4(MVD1) Wrd+/Wrd+. (14–3-3ε and ζ). (C) C(3)Gspecificity localization patterns to the in meiotic core cellsPP2A overexpressing complex GFP-Wrd (C + (PP2A-B56) A; Chen and et control al.,2007 cells throughout). meiotic progression. Error bars represent SEM (14–plex3-3εplex assemblyand assemblyζ). and karyosome and karyosome formation. formation. Because Because SCF isSCF typi-(C) isC(3)GBarbosaspecificity typi- localizationwild-typeBarbosa et al.wild-type patternset to al. ovaries,the in meiotic core ovaries,and cellsPP2A overexpressing the and complex synaptonemal thesynaptonemal GFP-Wrd (C + (PP2A-B56) A; complexChen andcomplex et control al., and2007 cells the and throughout). kar- the kar- meiotic progression. Error barsJournal reprJournalesent of Cell SEM Biologyof Cell Biology6of126of12 We showed that SCF-Fbxo42 regulates synaptonemalSCF-Fbxo42 com-SCF-Fbxo42 promotesThese promotes the proteins synaptonemal the synaptonemal were complex individually complex overexpressed in otherwise https://doi.org/10.1083/jcb.202009167https://doi.org/10.1083/jcb.202009167 Wecally showedcally known known that to ubiquitinate SCF-Fbxo42 to ubiquitinate substrates regulates substrates forsynaptonemal degradation for degradation com- by the byThese theyosome proteinsyosome were were were examined individually examined by immunostaining. by overexpressed immunostaining. in We otherwise found We found that that 26S proteasome26Splex proteasome assembly (Cardozo and (Cardozo karyosome and Pagano, and Pagano, formation. 2004 2004), we Because), hypothesized we hypothesized SCF is typi-overexpressionBarbosaoverexpressionwild-type et al. ovaries, of GFP-tagged of GFP-taggedand the Wrd, synaptonemal Wrd, one of one two of complex alternative two alternative and B56 the kar- B56 Journal of Cell Biology 6of12 plex assembly and karyosome formation. Because SCF is typi-Barbosawild-type et al. ovaries, and the synaptonemal complex and the kar- Journal of Cell Biology 6of12 cally known to ubiquitinate substrates for degradation by theSCF-Fbxo42yosome promotes were the synaptonemal examined complex by immunostaining. We found that https://doi.org/10.1083/jcb.202009167 callythat known onethat or one to more ubiquitinate or more substrates substrates substrates of SCF-Fbxo42 of SCF-Fbxo42 for degradation need need to be to destroyedby be the destroyedSCF-Fbxo42yosomesubunits promotessubunits were the of synaptonemal examinedPP2A, of PP2A, resulted complex by resulted immunostaining. in delayed in delayed assembly assembly We and found premature and that premature https://doi.org/10.1083/jcb.202009167 26S proteasome (Cardozo and Pagano, 2004), we hypothesized overexpression of GFP-tagged Wrd, one of two alternative B56 26Sfor proteasome properfor proper assembly (Cardozo assembly of and the of Pagano, synaptonemal the synaptonemal 2004), complex we hypothesized complex and karyo- and karyo-overexpressiondisassemblydisassembly of theGFP-tagged of synaptonemal the synaptonemal Wrd, one complex, of complex, two as alternative well as aswell abnormal as B56 abnormal that one or more substrates of SCF-Fbxo42 need to be destroyed subunits of PP2A, resulted in delayed assembly and premature thatsome onesome or formation. more formation. substrates We predicted We of predicted SCF-Fbxo42 that overexpression that need overexpression to be destroyed of this of crit- thissubunits crit-karyosomekaryosome of PP2A, morphologies resulted morphologies in (Fig. delayed (4,Fig. B and assembly4, B C and; and C and;Fig. and premature S2Fig. E), S2 as E seen), as seen for proper assembly of the synaptonemal complex and karyo- disassembly of the synaptonemal complex, as well as abnormal forical proper substrateical assembly substrate in wild-type of in the wild-type synaptonemal would would phenocopy phenocopy complex the and synaptonemal the karyo- synaptonemaldisassemblyin Fbxo42in Fbxo42 of RNAi. the RNAi.synaptonemal In contrast, In contrast, overexpression complex, overexpression as well of as the abnormal of other the other pro- pro- some formation. We predicted that overexpression of this crit- karyosome morphologies (Fig. 4, B and C; and Fig. S2 E), as seen somecomplex formation.complex and/or Weand/or karyosome predicted karyosome that defects overexpression defects of Fbxo42 of Fbxo42 depletion. of this depletion. crit- As an Askaryosome anteinsteins did morphologies not did affect not affect the (synaptonemalFig. the synaptonemal4, B and C complex; and complexFig. or S2 the E or), karyosome, asthe seen karyosome, ical substrate in wild-type would phenocopy the synaptonemal in Fbxo42 RNAi. In contrast, overexpression of the other pro- icalF-box substrateF-box protein in protein wild-type is the is substrate-recognition the would substrate-recognition phenocopy subunit the synaptonemal subunit (Petroski (Petroski andin and Fbxo42althoughalthough RNAi. we cannot Inwe contrast, cannot exclude exclude overexpression the possibility the possibility of that the other thatthey they are pro- still are still complex and/or karyosome defects of Fbxo42 depletion. As an teins did not affect the synaptonemal complex or the karyosome, complex and/or karyosome defects of Fbxo42 depletion. As an teins did not affect the synaptonemal complex or the karyosome, F-box protein is the substrate-recognition subunit (Petroski and although we cannot exclude the possibility that they are still F-boxBarbosa proteinBarbosa et al. is et the al. substrate-recognition subunit (Petroski and although we cannot exclude the possibility thatJournal theyJournal of are Cell of still Biology Cell Biology5of125of12 SCF-Fbxo42SCF-Fbxo42 promotes promotes the synaptonemal the synaptonemal complex complex https://doi.org/10.1083/jcb.202009167https://doi.org/10.1083/jcb.202009167 Barbosa et al. Journal of Cell Biology 5of12 Barbosa et al. Journal of Cell Biology 5of12 SCF-Fbxo42 promotes the synaptonemal complex https://doi.org/10.1083/jcb.202009167 SCF-Fbxo42 promotes the synaptonemal complex https://doi.org/10.1083/jcb.202009167 Juri Rappsilber Cellular Tomography Co-workers: Colin Combe, Georg Kustatscher, Christos Spanos, Juan Zou

Structural biology has made amazing advances on providing us models of proteins alone or in complexes by technologies A C such as crystallography, electron microscopy or nuclear magnetic resonance spectroscopy. These methods continue to advance yet fail mostly on requiring proteins to be extracted from their native environments, a process that many proteins do not honour with keeping their native structure. We are one of the pioneers of developing crosslinking mass spectrometry as an alternative approach. This sees proteins crosslinked in their native environment and the sites of crosslinking then being determined by mass spectrometry and data analysis. We are optimising many steps of this process: choice of crosslink reagents, protein-to-reagent ratio, digestion conditions, sample preparation, mass spectrometric acquisition, peak picking, data base construction. We are also further developing our search software xiSEARCH and integrated data visualisation tool xiVIEW. We fully embrace ideas of open sharing our insights and tools by making use of preprint repositories, providing our code via GitHub, submitting our data to public repositories and establishing field standards in close collaboration with key stakeholders such as HUPO PSI and EBI. The continuous progress on our tools has allowed us now to move into (simple) cells. B Structural biology performed inside cells can capture molecular machines in action within their native context. We developed an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae. We combined whole-cell crosslinking mass spectrometry, cellular cryo-electron tomography (in collaboration with Julia Mahamid at the EMBL Heidelberg), and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at sub-nanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpointed NusA at the interface between a NusG- bound elongating RNAP and the ribosome, and propose it can mediate transcription-translation coupling. Translation inhibition dissociated the expressome, whereas transcription inhibition stalled and rearranged it. Thus, the active expressome architecture requires both translation and transcription elongation within the cell. This is in stalk contrast to structures obtained by single particle cryoEM using reconstituted transcription-translation in E. coli, which see NusG and not NusA at the nexus of RNAP and ribosome. Future work will have to clarify whether this difference can be accounted for by working with different species or that working in cells captures a different state then working in a reconstituted system.

In-cell integrative structural biology reveals the structural basis of coupling between transcription and translation in M. pneumoniae. Selected Publications A. 577 distinct PPIs identified at 5% PPI-level FDR (interactions to 8 abundant glycolytic enzymes and chaperones are removed Dau, T., Bartolomucci, G., Rappsilber, J. (2020) Proteomics Using Protease Alternatives to Trypsin Benefits from Sequential Digestion with for clarity). Membrane-associated proteins are shown in grey. Circle diameter indicates relative protein size. Blue: 50S ribosomal Trypsin. Anal Chem.92, 9523-9527. proteins; yellow: 30S ribosomal proteins; green: RNAP; orange: NusA. Each edge represents one or more crosslinks. O'Reilly, F.J., Xue, L., Graziadei, A., Sinn, L., Lenz, S., Tegunov, D., Blötz, C., Singh, N., Hagen, W.J.H., Cramer, P., Stülke, J., Mahamid, J., B. Interactors of RNAP and NusA. NusA NTD, S1, KH domains, and proline rich region (PR) are annotated. Line thickness Rappsilber, J. (2020) In-cell architecture of an actively transcribing-translating expressome. Science.369, 554-557. represents the number of identified crosslinks. Leitner, A., Bonvin, A.M.J.J., Borchers, C.H., Chalkley, R.J., Chamot-Rooke, J., Combe, C.W., Cox, J., Dong, M.Q., Fischer, L., Götze, M., C. Model of a RNAP-ribosome supercomplexe in untreated cells, the expressome compromises an actively elongating RNAP and Gozzo, F.C., Heck, A.J.R., Hoopmann, M.R., Huang, L., Ishihama, Y., Jones, A.R., Kalisman, N., Kohlbacher, O., Mechtler, K., Moritz, R.L., ribosome. Netz, E., Novak, P., Petrotchenko, E., Sali, A., Scheltema, R.A., Schmidt, C., Schriemer, D., Sinz, A., Sobott, F., Stengel, F., Thalassinos, K., Urlaub, H., Viner, R., Vizcaíno, J.A., Wilkins, M.R., Rappsilber, J. (2020) Toward Increased Reliability, Transparency, and Accessibility in Cross-linking Mass Spectrometry. Structure. 28, 1259-1268.

26 2727 Kenneth E. Sawin Cell polarity and cytoskeletal organisation Co-workers: Tanya Dudnakova, Ye Dee Tay, James Le Cornu, Ankita Gupta, Adam Kovac, Ana Rodriguez, Monique Scott

We are interested in two general areas related to cellular organisation: 1) regulation of cell polarity, under both normal and A B

stress conditions, and 2) the molecular mechanisms underlying microtubule nucleation. In both areas we use fission yeast 4 Schizosaccharomyces pombe as a model single-celled eukaryote. We combine classical and molecular genetic analysis Conventional cargo export with live-cell fluorescence microscopy, biochemistry, proteomics/phosphoproteomics, and structural biology methods. 2 Mto1/2 complex docking Cell polarity in fission yeast is regulated by multiple internal cues that cooperate and compete with each other. The Rho-family GTPase Cdc42 and its associated regulators and effectors control the actin cytoskeleton and exocytosis. -4 -2 2 4 Microtubules provide an additional level of control, through the microtubule-associated protein Tea1 and its interactors. We have shown how the Tea1/microtubule system coordinates polarity regulation by a conventional Cdc42 guanine-nucleotide (fold-change), replicate 1 -2 Cytoplasm 2

exchange factor, Scd1, with regulation by an unconventional exchange factor, Gef1. Our work has also led to the discovery Log of new cell-polarity regulators outside of the Cdc42- and Tea1/microtubule-based systems, and a new understanding Sec3-S201 Sec5-S50 -4 Nucleoplasm of how the conserved NDR kinase Orb6 regulates cell polarity. A major current focus is on how the stress-activated RanGTP RanGDP Log (fold-change), replicate 2 kinase Sty1 (homolog of human p38 MAP kinase) regulates cell polarity; we are addressing this through large-scale 2

E G R N H phosphoproteomics and genetics approaches. K H D A K F Q Q I Conventional Mto1/2 E A L H R G P N H R Q S S R exo70∆ H T K Y V S Crm1 export cargo complex Protein Phosphosite Microtubule nucleation depends on the γ-tubulin complex, a large multi-protein complex enriched at microtubule organising -5 -4 -3 -2 -1 0 Osh2 H A R Q E S 695 centres such as the centrosome. Many aspects of γ-tubulin complex regulation remain a mystery. We discovered the Sec72 H K R G G S 1597 fission yeast proteins Mto1 and Mto2, which form an oligomeric "Mto1/2 complex". The Mto1/2 complex targets the -tubulin Osh2 H H R K E S 679 Symmetric FG-Nup γ Sec5 H E R N R S 50 C complex to different sites in the cell and also activates γ-tubulin complex. Mutations in the human homolog of Mto1 Ppk25 H Q R H H S 404 Eis1 H S R N G S 1095 lead to the brain disease microcephaly. Our current work involves understanding the mechanism of γ-tubulin complexl Sec3 H N R N F S 201 Nup146 (Cytoplasmic FG-Nup) Ppk15 H A R H V S 33 sec3-S201A activation by the Mto1/2 complex, using yeast genetics, microscopy, and biochemistry approaches. In recent work we Ppk25 H Q R S I S 38 exo70∆ Ptc1 H R K N A S 34 FG repeats have reconstituted multi-protein complex-dependent microtubule nucleation in vitro using purified proteins, and we have Pep7 H K R Q D S 75 characterised elements of functional nucleation complexes through cross-linking mass spectrometry as well as X-ray Mmb1 H R H A H S 439 Tea3 H R H Y A S 460 crystallography. We have also developed new methods to interrogate protein-protein interactions in complex “solid-phase” Igo1 H T K R P S 115 Ppk1 H K H H L S 59 subcellular structures in vivo, and we have used these to investigate how Mto1/2 complex is localised to nuclear pores. Cek1 H R R K Q S 748 In all of our work we adopt and develop new tools and techniques as necessary to address the biological questions of interest. A. Global phosphoproteomics after inhibition of NDR kinase Orb6 in vivo. Many phosphorylation sites with decreased phosphorylation after Orb6 inhibition match the NDR consensus. Phosphosite mutation of Sec3 (component of exocyst complex) impairs exocytosis and cell separation after cytokinesis. B. Model for docking Mto1/2 microtubule nucleation complex at the nuclear pore. Mto1 mimics a nuclear export cargo but uses Selected Publications this for docking at the pore, not for nuclear export. Ashraf, S., Tay, Y.D., Kelly, D.A., and Sawin, K.E. (2021). Microtubule-independent movement of the fission yeast nucleus. J Cell Sci. Feb C. Negative-stain electron microscopy of reconstituted fission yeast -tubulin ring complex. 23:jcs.253021 PMID: 33602740 γ Leong, S.L., Lynch, E.M., Zou, J., Tay, Y.D., Borek, W.E., Tuijtel, M.W., Rappsilber, J., and Sawin, K.E. (2019). Reconstitution of Microtubule Nucleation In Vitro Reveals Novel Roles for Mzt1. Curr Biol 29, 2199-2207 e2110. PMID: 31287970 Tay, Y.D., Leda, M., Spanos, C., Rappsilber, J., Goryachev, A.B., and Sawin, K.E. (2019). Fission Yeast NDR/LATS Kinase Orb6 Regulates Exocytosis via Phosphorylation of the Exocyst Complex. Cell Rep 26, 1654-1667 e1657. PMID: 30726745

28 2929 David Tollervey RNA Processing and Quality Control Co-workers: Stefan Bresson, Aziz El Hage, Aleksandra Helwak, Michaela Kompauerova, Laura Milligan, Elisabeth Petfalski, Nic Robertson, Emanuela Sani, Vadim Shchepachev, Tomasz Turowski

RNA-binding proteins (RBPs) have important functions at all steps in gene expression, including transcription, RNA processing and mRNA translation. Defects in RBPs underpin many genetic diseases, while responses to environmental stress are frequently mediated by altered RNA-protein interactions. We examined global RBP dynamics in Saccharomyces cerevisiae in response to stress, using our recently developed technique of total RNA-associated protein purification (TRAPP). Stresses induced very rapid remodeling of the RNA-protein interactome, without corresponding changes in RBP abundance (Bresson et al., 2020). A set of “scanning” translation initiation factors (eIF4A, eIF4B, and Ded1), were remarkably rapidly evicted from mRNAs (<30 sec after glucose withdrawal) driving translation shutdown (Fig). Selective mRNA 5'-degradation by the exonuclease Xrn1 was seen following heat shock, particularly for translation-related factors, reinforcing translational inhibition. Notably, these responses are distinct from previously characterized pathways for stress-induced translation inhibition. An outstanding question in nuclear RNA quality control is how “defective” RNAs are identified and targeted for degradation. During surveillance, the RNA exosome functions together with the TRAMP complexes (Delan-Forino et al., 2020). These include the DEAH-box RNA helicase Mtr4 together with an RBP (Air1 or Air2) and a poly(A) polymerase (Trf4 or Trf5). TRAMP acts to make substrates more susceptible to degradation, by addition of a single-stranded “tail”, and also targets them to the exosome. Combining biochemistry, genetics and genomics, we identified three distinct TRAMP complexes formed in yeast, which preferentially assemble on different classes of transcripts. Surprisingly, the poly(A) polymerases Trf4 and Trf5 emerged as crucial in TRAMP targeting and recruitment. Transcription elongation rates are important for many aspects of RNA processing, defining the “window of opportunity” for interaction and folding. However, sequence-specific regulation of elongation rates is poorly understood. Notably, elongation by RNA polymerases is only moderately processive, being based on a "Brownian Ratchet" rather than an energy-driven mechanism. To analyse RNA polymerase I elongation in S.cerevisiae (Turowski et al., 2020) we combined in vivo RNAPI profiling,in vitro biochemical analyses and a quantitative, mechanistic model of transcription elongation. Unexpectedly, these revealed that folding of the nascent pre-rRNA close to the transcribing polymerase has a major effect on the elongation rate, with a modest contribution from the stability of the RNA-DNA duplex in the active site. RNAPI from S.pombe was similarly sensitive to transcript folding, as were S.cerevisiae RNAPII and RNAPIII. For RNAPII, unstructured RNA, which favours slowed elongation, was associated with faster cotranscriptional splicing and proximal splice site usage, indicating regulatory significance for transcript folding.

Selected Publications Bresson, S., Shchepachev, V., Spanos, C., Turowski, T., Rappsilber, J., and Tollervey, D. (2020). Stress-induced translation inhibition Stress induces very rapid translation and selective mRNA degradation. through rapid displacement of scanning initiation factors. Molecular Cell 80, 470-484. Following glucose starvation or heat shock, translation initiation factors rapidly dissociate from the 5'-end of mRNAs, halting Delan-Forino, C., Spanos, C., Rappsilber, J., and Tollervey, D. (2020). Substrate specificity of the TRAMP nuclear surveillance complexes. translation initiation. Already-initiated ribosomes continue translating, leaving unprotected mRNAs. Following heat shock, Xrn1 is Nature Communications 11, 3122-3122. involved in degradation of a subset of these transcripts. Turowski, T.W., Petfalski, E., Goddard, B.D., French, S.L., Helwak, A., and Tollervey, D. (2020). Nascent transcript folding plays a major role in determining RNA polymerase elongation rates. Molecular Cell 79, 488-503.

30 3131 Philipp Voigt Molecular Mechanisms of Epigenetic Gene Regulation Co-workers: Giulia Bartolomucci, Rachael Burrows, Ethan Hills, Amaka Idigo, Simone Lenci, Reshma Nair, Thomas Sheahan, Devisree Valsakumar, Marie Warburton

The overarching goal of research in our lab is to elucidate how histone modifications regulate gene expression. We are A D keen to understand how different histone modifiers and readers interact to establish complex regulatory systems that A unmodified bivalent D control development and cause disease if misregulated. We are taking a multidisciplinary approach to tackle these questions, combining biochemistry with proteomic, genomic, cell-biological, imaging-based, and systems biology-inspired TAF3 TFIID EED recombinant techniques. nucleosomes SAGA PRC2 CBX We aim to clarify how Polycomb and trithorax group proteins regulate expression of developmental genes in embryonic SET1A/B PRC1 stem cells (ESCs). Bivalent domains harbour a distinctive histone modification signature featuring both the active histone + CHD1 H3 lysine 4 trimethylation (H3K4me3) mark and the repressive H3K27me3 mark. They have been suggested to maintain bead-bound pulldowns

nucleosome ESC NE nucleosomes developmental genes in a poised state, allowing for timely expression upon differentiation while maintaining repression H3K4me3 binders H3K27me3 binders unmodified asymmetric in ESCs. Bivalent nucleosomes adopt a previously unknown asymmetric conformation, carrying the active and repressive bivalent novel mark on opposite copies of histone H3. However, it has remained unclear how bivalent domains function to poise genes for binders expression in ESCs and whether they are essential for proper ESC differentiation and embryonic development. LFQ SRCAP KAT6B intensity LC-MS/MS m/z m/z bivalent binders To address these questions mechanistically, we performed pulldown experiments with recombinant asymmetric, bivalent analysis MaxQuant/DEP/R nucleosomes and ESC nuclear extract (Figure 1A). We found that bivalent nucleosomes recruit repressive H3K27me3 binders but not activating H3K4me3 binders, despite the presence of H3K4me3 (Figure 1B), both in vitro and in ESCs. BB C Moreover, we have discovered readers that specifically recognise the bivalent state, including the histone acetyltransferase 12 12 Actr5 Zfp296 complex KAT6B (MORF) and the histone variant H2A.Z chaperone complex SRCAP (Figure 1C). To determine the role Nfrkb 10 10 Tfpt of these novel bivalent binders in regulation of developmental genes, we performed knockout of KAT6B. Loss of KAT6B Actr8 Srcap Znf652 Srsf2 diminishes neuronal differentiation, indicating that bivalency-specific readers are critical for proper ESC differentiation. Mcrs1 Zbtb2 Yeats4 Pcca Zranb2 Yy1 8 8 Acaca Our findings thus suggest a model where bivalent nucleosomes drive poising by setting up a repressed but plastic state at EPOP Pum1 Tfe3 Dmap1 developmental promoters through recruitment of repressive and bivalent binders and exclusion of activators (Figure 1D). Phc1 Vps72 6 Orc2 6 Kat6b (p value) (p value) Zbtb10

10 Ezh2 Ino80 Hmg20a We are now testing this model by examining how repressive and bivalent readers such as PRC2 and KAT6B cooperate 10 Chd1 Cbx7 Mitf Phf13 Meaf6 to maintain a repressed but plastic state in ESCs while allowing for activation of developmentally regulated genes upon −log

Taf3 Bptf1 −log 4 Ezhip 4 Zfp & Zbtb Brpf1 Chd8 Rnf2 proteins Ing5 differentiation. Dpy30 LRWD1 Ezh1 Cxxc1 Eed Suz12 Brpf3 2 2 Phf2 Kat6a C10orf12/Pali1 Spin1 Bap18 Taf subunits 0 Ing2 Phf21a 0 Selected Publications −8 −6 −4 −2 0 2 4 6 8 −8 −6 −4 −2 0 2 4 6 8 log FC(bivalent vs. unmodified) log FC(bivalent vs. unmodified) Bryan, E., Warburton, M., Webb, K. M., McLaughlin, K. A., Spanos, C., Ambrosi, C., Major, V., Baubec, T., Rappsilber, J., & Voigt. 2 2 P. (2021). Nucleosomal Asymmetry Shapes Histone Mark Binding and Promotes Poising at Bivalent Domains. bioRxiv https://doi. org/10.1101/2021.02.08.430127. Sheahan, T. W., Major, V., Webb, K. M., Bryan, E., & Voigt, P. (2020). The TAZ2 domain of CBP/p300 directs acetylation towards H3K27 A.Outline of the nucleosome pulldown approach used to identify binding proteins of asymmetric bivalent nucleosomes. within chromatin. bioRxiv https://doi.org/10.1101/2020.07.21.214338. Villaseñor, R., Pfaendler, R., Ambrosi, C., Butz, S., Giuliani, S., Bryan, E., Sheahan, T. W., Gable, A. L., Schmolka, N., Manzo, M., Wirz, J., B. H3K27me3 but not H3K4me3 readers are enriched with asymmetric bivalent nucleosomes. Additionally, novel, bivalency- Feller, C., von Mering, C., Aebersold, R., Voigt, P., & Baubec, T. (2020). ChromID identifies the protein interactome at chromatin marks. specific binders are recruited (highlighted inC ). Nat. Biotechnol. 38, 728–736. D. Model illustrating how bivalent nucleosomes support establishment of a poised state.

32 3333 Julie Welburn Microtubule motor cooperation in cell division Co-workers: Thomas Attard, Ben Craske, Agata Gluszek, Thibault Legal, Haonan (Leo) Liu, Toni McHugh

Research A B D To maintain their genomic integrity, eukaryotic cells must replicate their DNA faithfully and distribute it equally to the daughter cells. Mitotic defects lead to aneuploidy and cancer. This indicates that the mitotic mechanisms that are in place to allow faithful division have been compromised. The segregation of chromosomes is mediated by polarized and highly dynamic filaments, termed microtubules. Microtubules depend on motor proteins to assemble into a spindle and segregate chromosomes. These motors play key roles in cytoskeletal organization during cell division but also in cell migration, polarity, and axonal and cytoplasmic transport. However, the reductionist approach to studying these motors in isolation is not sufficient to understand their function in the cellular context. It remains unclear how the activities of individual motors and their interacting regulatory networks cooperate to generate physiological cellular function such as chromosome segregation. We aim to define how kinesin motors are modulated by their cargos to provide a specific output, and how the coordinated activities of kinesin motors are greater than the sum of their individual activities in vitro and in human cells. E F Kinetochores and motors CENP-E is a huge motor (730 kD), recruited to unattached kinetochores. CENP-E moves kinetochores along microtubules to facilitate chromosome alignment. How CENP-E associates with the kinetochore, how human CENP-E is activated to walk C on microtubules and how CENP-E motor ensembles are coordinated to move chromosomes is currently not known. Using a non-biased approach, we recently found the C-terminal kinetochore-targeting region of CENP-E interacts with BubR1, amongst other proteins in nocodazole-arrested mitotic cell extracts. We have defined the molecular determinants that specify the interaction between BubR1 and CENP-E. The basic C-terminal helix of BubR1 is necessary but not sufficient for CENP-E interaction, while a minimal key acidic patch on the kinetochore-targeting domain of CENP-E, is also essential. We then demonstrated that BubR1 is required for the recruitment of CENP-E to kinetochores to facilitate chromosome alignment. In collaboration with the Gruneberg lab, University of Oxford, we showed this BubR1-CENP-E axis is critical to align chromosomes that have failed to congress through other pathways and recapitulates the major known function of CENP-E. Overall, our current studies define the molecular basis and the function for CENP-E recruitment to BubR1 at kinetochores

during mammalian mitosis. Our future work now focused on what is the basis and function for the 2 distinct recruitment A. Top, SEC analysis and elution profile for CENP-E2069-2358-GST (green), BubR1705-1050 (yellow) and CENP-E2069-2358-GST/BubR1705-1050 pathways of CENP-E to kinetochores and defining the activation mechanism of human CENP-E to ensure faithful mitosis. (orange). Bottom, Coomassie-stained gels showing elution profiles for the corresponding protein complexes. B. Thermodynamics of BubR1 /CENP-E -GST interaction determined by isothermal titration calorimetry. The y-axis Mitotic motors and microtubule dynamics 705-1050 2091-2358 indicates kcal/mol of injectant. The dissociation constant (Kd) between BubR1705-1050 and CENP-E2091-2358-GST was determined to be Our lab has made new discoveries on the mechanism of mitotic microtubule depolymerases for two families recently 318 ± 90 nM. published: the Kinesin-8 and the Kinesin-13 motors. Our current work now addresses how MCAK and Kinesin-8 motors C. Sequence alignment of the C terminus of human BubR1 with mouse and Xenopus BubR1, and human Bub1. Boxed red and cooperate to control microtubule length in mitosis. blue are the conserved and similar amino acids across all 4 proteins, respectively. Amino acids in red are those with conserved

properties in at least 3 sequences. The sequence necessary for BubR1 binding to CENP-E2055-2608 is highlighted in orange. Selected Publications D. Representative immunofluorescence images of HeLa cells treated with BubR1 siRNA and induced to express GFP-BubR1 WT and GFP-BubR1 , stained with CENP-E, CENP-C and Hoechst after treatment with MG132 for 2.5 hrs. Scale bar: 10 mm. Legal T., Hayward D., Gluszek-Kustusz A., Blackburn EA., Spanos C., Rappsilber J., Gruneberg U., Welburn JPI. The C-terminal helix of 1-1030 E. Scatter plot showing CENP-E intensity relative to GFP-BuBR1 at individual kinetochores plotted as grey circles, with mean and BubR1 is essential for CENP-E-dependent chromosome alignment. JCS (2020). 133:16. PMID: 32665320. McHugh T, Zou J, Volkov V, Bertin A, Rappsilber J, Dogterom M, Welburn JPI. The depolymerase activity of MCAK shows graded standard deviation represented by black lines. response to Aurora B phosphorylation through allosteric regulation. JCS. (2019). PMID: 30578316 F. Graph showing percentage of cells with at least 1 misaligned chromosome for BubR1-depleted cells induced to express GFP-

McHugh T., Gluszek-Kustusz A., Welburn JPI. Kinesin-8 motor Kif18b tethering to microtubule ends is a requirement for spindle BubR1, BubR11-1030 or without induction. Error bars represent standard deviation. **** indicating P < 0.0001. positioning. JCB. (2018). 217:7. 2403-2416. PMID: 29661912 34 3535 Marcus D Wilson Investigating the reading writing and erasing of epigenetic marks Co-workers: Hayden Burdett, Gillian Clifford, Dhananjay Kumar, Maarten Tuijtel, Hannah Wapenaar, James Watson

We are interested in understanding how epigenetic marks are placed, read and interpreted on chromatin. Chromatin A becomes decorated with a variety of chemical tags or epigenetic marks to control the myriad of DNA-related processes in the cell. Epigenetic modifications are initially deposited by writer enzymes. These are then read and interpreted in a co-operative manner by effector proteins. Epigenetic marks can also be removed by eraser proteins resetting the system (Figure 1 A). We look at this process in the test tube by creating modified chromatin using chemical biology and biochemical methods. We then use our defined modified chromatin to study individual nucleosome-chromatin protein complexes using single-particle cryo-electron microscopy (cryo-EM), Biochemical, Biophysical and Cell Biology approaches. We are particularly interested in understanding how DNA damage repair and DNA methylation pathways are orchestrated by epigenetically-modified nucleosomes. 1. How is DNA Methylation guided by chromatin? DNA methylation is a common epigenetic mark that is often associated with turning off genes and compacting DNA. Other epigenetic marks have the power to regulate DNA methylation, controlling when and where DNA methylation is placed on DNA, but we do not understand how this works. We are rebuilding the DNA methylation machinery within chromatin to help us answer this question. B DNA methylation is a highly regulated process, so by looking at the structure of the methylation machinery and the modified nucleosomes we hope to understand how methylation is targeted at specific times and to specific sites on DNA, hopefully helping us to understand how this process can become faulty leading to disease. 2. How do post-translational modifications foster DNA repair? DNA is under constant attack, which can cause unwanted genetic mutations and cancer. Luckily our cells have a host of DNA repair proteins, which help to fix most of the damage. These highly efficient repair proteins are recruited to sites of damage by recognition of DNA damage-specific marks on chromatin. We are hoping to understand how DNA damage is signalled on chromatin and how this leads to correct repair. Our recent work has shown that multiple DNA repair proteins interact multivalently with the nucleosome, commonly interacting with a conserved region called the acidic patch (Figure 1B). Through our in vitro studies we showed that the negatively charged surface of the nucleosome acidic patch is essential for binding of the listed DNA damage proteins. Mapping of the interaction regions and mutation has shown these are mediated by electrostatic interactions, typically A. Schematic of the cycle of epigenetic modifications in the cell. All modifications focus on the nucleosome hub, which can be through a highly conserved arginine anchor. modified on both the histone proteins and wrapped DNA. Example Reader, writer and eraser proteins DNA damage repair and DNA methylation pathways are labelled.

Selected Publications B. Multivalent interactions in the DNA damage response focus on the acidic patch of the nucleosome. Belotserkovskaya, R., Raga Gil, E., Lawrence, N., Butler, R., Clifford, G., Wilson, M.D., and Jackson, S.P. (2020). PALB2 chromatin recruitment restores homologous recombination in BRCA1-deficient cells depleted of 53BP1. Nat Commun11 , 819. Salguero, I., Belotserkovskaya, R., Coates, J., Sczaniecka-Clift, M., Demir, M., Jhujh, S., Wilson, M.D., and Jackson, S.P. (2019). MDC1 PST-repeat region promotes histone H2AX-independent chromatin association and DNA damage tolerance. Nat Commun 10, 5191. Wilson, M.D., Renault, L., Maskell, D.P., Ghoneim, M., Pye, V.E., Nans, A., Rueda, D.S., Cherepanov, P., and Costa, A. (2019). Retroviral integration into nucleosomes through DNA looping and sliding along the histone octamer. Nat Commun 10, 4189.

36 3737 Sarah Keer-Keer Public Engagement Sarah-Jane Judge Maria Fanourgiaki Neil Bratchpiece

There is no such thing as a standard year in Public Engagement, but 2020 will surely stand out as a time of particular 1 3 challenge, but also new opportunities. With the pandemic putting a stop to public festivals and in-person events, it was time to get even more innovative with our approaches. From online to outdoors, post-out packs to comic strips, 2020 has required creativity to engage with our partners, new and old-alike. Having hosted the Edinburgh Care for Carers for a centre visit in February (figure 1), we were delighted to continue collaborating with this group throughout 2020. With the help of clip-on microscopes for phones, we created a post-out version of our popular workshop ‘Life Through Lens’. Whilst the booklet was self-supporting, our researchers enjoyed meeting the group online and viewing the microscopic images they had taken around their homes (figure 2). In 2020 we were also introduced to a Young Carers group in Edinburgh and worked with them to develop a self-led, outdoor session titled ‘The Cell-ebrating nature scavenger hunt’. This involves participants photographing and collecting interesting biological items during walks (leaves, sticks, etc.) which they can then send back to the centre for feedback and 2 microscopic analysis. We hope to roll this out with more youth groups in 2021. Virtual gatherings such as Explorathon and the Midlothian Science Festival allowed our researchers to practice their online engagement skills (figure 3) including an exciting new international session Research‘ Around the World’. School and public audiences met Nepalese research colleagues, along with our own WCB researchers, to discuss how research is a global endeavour but varies from country to country. Social distancing didn’t stop us from encouraging our audiences to get hands on! Community groups got artistic at home with our cell themed colouring books, science crafts and bake-along stained glass cell biscuit Zoom calls (figure 4). The Haddington Girl Guides made a splash with water and household items to learn about the scientific discovery process in our new ‘Researching research’ session, which has now been requested by various other units in East Lothian. In 2020 we were delighted to support the Marston lab in winning a ScotPen Wellcome Engagement Award which enables us to link researchers and IVF patients through the production of glass art works. Public Engagement is becoming more 4 integrated across the whole centre with PE Champions being recruited from each lab and our first whole centre project in the form of our WCB comic book. Artist in residence, Neil Bratchpeice, will be working with each lab to make an engaging comic strip about their research (figure 5). 2021 is set to be a busy year for us with high demand for our new socially distanced offerings but also plan afoot for in- person activities as soon as restrictions are lifted.

5

38 3939 List of Groups

Robin Allshire Adrian Bird Bill Earnshaw Adele Marston Wellcome Principal Research Fellow Wellcome Investigator in Science Wellcome Principal Research Fellow Wellcome Investigator in Science

Tatsiana Auchynnikava Wellcome Research Associate Beatrice Mar Carmena Wellcome Research Associate Weronika Borek Wellcome Postdoc Roberta Carloni MRC Research Associate Alexander-Howden Wellcome Research Assistant Fernanda Cisneros Wellcome Research Associate Chuanli Huang PhD student Tadhg Devlin BBSRC EASTBIO Graduate Student Kashyap Chhatbar ERC Bioinformatician Lorenza Di Pompeo iCM Ph.D. Student Dilara Kocakaplan Darwin Trust PhD student Andreas Fellas iCM Graduate Student John Connelly RSRT Research Associate Moonmoon Deb Royal Society Newton Fellow Lori Koch Wellcome Postdoc Elisabeth Gaberdiel Wellcome iCM Graduate Student Laura Fitzpatrick Wellcome 4yr Graduate student Natalia Kochanova Wellcome Research Associate Melanie Lim Carnegie Trust PhD student Dominik Hoelper EMBO Long Term Fellow Sarah Giuliani ERC Visiting student Emma Peat Part-time Technician Vasso Makrantoni Wellcome Postdoc Marcel Lafos Wellcome Research Associate Jacky Guy Wellcome Research Associate Elisa Pesenti Lab Manager/Wellcome Research Lucia Massari BBSRC Postdoc Nitobe London Wellcome Research Associate Matthew Lyst SIDB Research Associate Associate Bettina Mihalas Wellcome Postdoc Sunil Nahata University of Grenoble CNRS Baisakhi Mondal ERC Research Associate Bram Prevo Sir Henry Wellcome ,Postdoctoral Anuradha Mukherjee Darwin Trust PhD student Visiting Graduate Student Verdiana Steccanella ERC Research Associate/Lab Manager Fellow Meg Peyton-Jones EASTBIO BBSRC PhD student Alison Pidoux Wellcome Research Associate Christine Struthers Personal Assistant Caitlin Reid SBS Ph.D. student/Technician Gerard Pieper Wellcome Postdoc Severina Pociunaite Utrecht University Erasmus+ Katie Paton BBSRC/SBRC Postgraduate Student Lucy Remnant Wellcome Research Associate Ola Pompa Darwin Trust PhD student MSc Student Raphael Pantier ERC Research Associate Kumiko Samejima Wellcome Research Associate Xue (Bessie) Su Wellcome Postdoc Desislava Staneva MRC Research Associate Itaru Samejima Wellcome Research Associate Aparna Vinod Darwin Trust PhD student Manu Shukla Wellcome Research Associate Menglu (Lily) Wang Wellcome Postdoc Sito Torres-Garcia Wellcome Research Associate Sharon White Wellcome Research Associate Dhanya Cheerambathur Weifang Wu Darwin Trust Graduate Student Sir Henry Dale Fellow Patrick Heun Imtiyaz Yaseen Wellcome Research Associate Wellcome Senior Research Fellow Dónal O'Carroll Mattie Green Wellcome Research Assistant Rebecca Yeboah Darwin Trust Graduate Student Wellcome Investigator in Science Cameron Finlayson Wellcome Research Technician Mathilde Fabe Darwin Trust Graduate Student Vasilis Ouzounidis Darwin Trust Ph.D. Student Meghan Frederick Research Assistant Azzurra Codino Wellcome Graduate student Emmanuel Fiagbedzi Wellcome iCM PhD Student Emöke Gerocz Research Assistant A. Jeyaprakash Arulanandam Azzurra DePace Wellcome Graduate student Charlotte de Ceuninck Visiting Masters Student Meena Krishnan Darwin Trust Graduate Student Hanna Fieler Wellcome Masters Student Wellcome Senior Research Fellow Hwei Ling Tan EASTBIO Graduate Student Madeleine Heep Wellcome Graduate Student Alessandro Stirpe Wellcome Research Associate Maria Alba Abad Yuka Kabayama Wellcome Postdoc George Yankson Darwin Trust Graduate Student Fernandaz Wellcome Research Associate Atlanta Cook Gabriela Konieczny Principal's Career Development Fund Asma Al-Murtadha Darwin Trust Graduate Student Wellcome Senior Research Fellow Graduate student Lana Buzuk Research Technician Louie Van De Lagemaat Visiting Scientist Carla Chiodi Wellcome Research Associate Laura Croenen Cunningham Trust Graduate Student Christopher Mapperly Wellcome Trust Tissue Repair PhD Thomas Davies* iCM PhD student James Le Cornu iCM Graduate Student Programme Lorenza Di Pompeo** iCM Graduate Student Atika Al Haisani Graduate Student Pedro Moreira Laboratory Manager Bethan Medina-Pritchard Wellcome Research Assistant Uma Jayachandran Wellcome Research Assistant Paula Sotelo Parrilla Principal’s Career Development Graduate Theresa Schöpp Wellcome Graduate Student Ola Kasprowicz Wellcome Research Associate student Ansgar Zoch DFG Postdoctoral Research Associate Ignacio Jiménez Sánchez Principal’s Career Development Mickey Oliver Wellcome Research Associate Graduate Student Alexander Will Darwin Trust Graduate Student Pragya Srivastava Darwin Trust Graduate Student Reshma Thamkachy SERB Overseas Postdoctoral Fellow * joint iCM PhD student with Prof. Kevin Hardwick 40 ** joint iCM PhD student with Prof. Bill Earnshaw 4141 List of Groups

Hiro Ohkura David Tollervey Marcus D Wilson Technical support Wellcome Investigator in Science Director, Wellcome Principal Research Fellow Sir Henry Dale Fellow Scott Macrae Fiona Cullen Wellcome Research Associate Stefan Bresson Wellcome Trust Research Associate Hayden Burdett MRC Postdoctoral Researcher Jule Nieken Darwin Trust Graduate Student Aziz El Hage Wellcome Trust Research Associate Gillian Clifford Research Assistant Charlotte Repton BBSRC EASTBIO Graduate Student Aleksandra Helwak Wellcome Trust Research Associate Dhananjay Kumar Darwin Trust Graduate Student Wash-up/Media Emiliya Taskova BBSRC Research Technician Michaela Kompauerova EASTBIO PhD student Maarten Tuijtel Cryo-EM Technologist Dan Toddie-Moore Wellcome Research Associate Laura Milligan Wellcome Trust Research Associate Hannah Wapenaar Wellcome Postdoctoral Researcher Denise Affleck Gera Pavlova BBSRC Research Associate Elisabeth Petfalski Wellcome Trust Research Associate James Watson Wellcome iCM PhD student Andrew Kerr Xiang Wan Darwin Trust Graduate Student Nic Robertson ECAT Fellow Margaret Martin Emanuela Sani Wellcome Trust Research Associate Donna Pratt Vadim Shchepachev SNSF Research Fellow Tomasz Turowski Polish Academy Research Fellow Administration/Support Staff Juri Rappsilber Wellcome Senior Research Fellow Greg Anderson Centre Laboratory Manager Elizabeth Blackburn EPPF Research Associate Colin Combe Wellcome Research Associate Philipp Voigt Hywel Dunn-Davies Bioinformatics Developer Georg Kustatscher Wellcome Research Associate Sir Henry Dale Fellow Maria Fanourgiaki Science Communicator Christos Spanos Wellcome Research Associate Carolyn Fleming Centre Administrative Assistant Juan Zou Wellcome Proteomics Data Giulia Bartolomucci ERC Research Technician Sarah-Jane Judge Centre Public Engagement Manager Analysis Manager Rachael Burrows Wellcome Research Assistant (temporary) Ethan Hills Darwin Trust PhD Student Sarah Keer-Keer Centre Public Engagement Manager Amaka Idigo Wellcome Research Assistant David Kelly Centre Optical Instrumentation Simone Lenci ERC Research Technician Laboratory Manager Kenneth E Sawin Reshma Nair ERC Postdoctoral Research Associate Toni McHugh Centre Imaging Facility Assistant Wellcome Investigator in Science Thomas Sheahan ERC Graduate Student Colin McLaren Computing Support Devisree Valsakumar Darwin Trust PhD Student Karen May Centre Manager/Wellcome 4yr PhD Tanya Dudnakova Wellcome Research Associate Marie Warburton ERC Graduate Student Programme Administrator (temporary) Ye Dee Tay Wellcome Research Associate Daniel Robertson Bioinformatics Support Officer James Le Cornu iCM PhD student, joint with Cook Lab Christine Struthers PA to Adrian Bird/Centre Administrator Ankita Gupta Darwin Trust PhD student Karen Traill Centre Manager/Wellcome 4yr PhD Adam Kovac EASTBIO PhD student Julie Welburn Programme Administrator Ana Rodriguez Darwin Trust PhD student Wellcome Senior Research Fellow Maarten Tuijtel Cryo-EM Technologist Monique Scott Darwin Trust PhD student Shaun Webb Centre Bioinformatics Core Facility Thomas Attard MRC Precision Medicine Student Manager Ben Craske BBSRC DTP Graduate Student Agata Gluszek Wellcome Postdoctoral researcher Thibault Legal Wellcome research technician/part-time PhD student Haonan (Leo) Liu Darwin Trust Graduate Student Toni McHugh Wellcome Trust Postdoctoral Researcher

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54 55 Verzini, S., M. Shah, F.X. Theillet, Belsom, J. Bieschke, E.E. Wanker, J. Rappsilber, A.Binolfi and P. Selenko (2020). Emeritus Centre Member “Megadalton-sized dityrosine aggregates of α-synuclein retain high degrees of structural disorder and internal dynamics”. J Mol Biol. 4;432(24):166689. doi: 10.1016/j.jmb.2020.10.023. PMID: 33211011. Vidakovic, A.T., R. Mitter, G. Kelly, M. Neumann, M. Harreman, M. Rodriguez-Martinez, J. Weems, S. Boeing, V. Encheva, L. Gaul, L. Milligan, D. Tollervey, R.C. Conaway, J.W. Conaway, A.P. Snijders, A. Stewart and J.Q. Svejstrup (2020). “A single RNA polymerase II ubiquitylation site coordinates transcription with the DNA damage response”. Cell 180: 1245-1261. PMCID: PMC7103762. Jean Beggs Vidakovic, A.T., M. Harreman, A.B. Dirac-Svejstrup, S. Boeing, A. Roy, V. Encheva, M. Neumann, M. Wilson, A.P. Snijders and Emeritus Professor of Molecular Biology and Emeritus Member of the Centre. J.Q. Svejstrup (2019). "Analysis of RNA polymerase II ubiquitylation and proteasomal degradation." Methods 159-160: 146- Prior to closing in June 2019 when Jean retired, the Beggs lab used biochemistry, cell biology, genetics 156. and quantitative systems biology approaches to investigate RNA processing pathways in budding Villaseñor, R., R. Pfaendler, C. Ambrosi, S. Butz, S. Giuliani, E. Bryan, T.W. Sheahan, A.L. Gable, N. Schmolka, M. Manzo, J. yeast, with a focus on pre-mRNA splicing and links between RNA splicing, transcription and chromatin Wirz, C. Feller, C. von Mering, R. Aebersold, P. Voigt and T. Baubec, T. (2020). “ChromID identifies the protein interactome at modifications. chromatin marks”. Nat. Biotechnol. 38, 728–736. Welburn, J.P.I. and C. Friel (2020). The Kinesin-13 family: specialist destroyers. Book Chapter. Wilson, M.D., L. Renault, D.P. Maskell, M. Ghoneim, V.E. Pye, A. Nans, D.S. Rueda, P. Cherepanov and A. Costa (2019). "Retroviral integration into nucleosomes through DNA looping and sliding along the histone octamer." Nat Commun 10(1): 4189. Wimbish, R.T., K.F. DeLuca, J.E. Mick, J. Himes, I.J. Sanchez, A.A. Jeyaprakash, and J.G. DeLuca (2019). “Coordination International Scientific Advisory Board of NDC80 and Ska complexes at the kinetochore-microtubule interface in human cells”. Mol Biol Cell Doi: https://doi. org/10.1091/mbc.E20-05-0286 Winz, M.L., L. Peil, T.W. Turowski, J. Rappsilber and D. Tollervey (2019). "Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control." Nat Commun 10(1): 563. PMCID: PMC6362110. Zoch, A., Auchynnikava, T., Berrens, R.V., Kabayama, Y., Schöpp, T., Heep, M., Vasiliauskaité, L., Pérez-Rico, Y.A., Cook. A.G., Margaret Fuller Andrea Brand Shkumatava, A., Rappsilber, J., Allshire, R.C. and D.O'Carroll (2020). Nature. 584, 635-639. doi: 10.1038/s41586-020-2557-5. Department of Developmental Biology Wellcome Trust/Cancer Research UK Gurdon Institute, and Department of Genetics Henry Wellcome Building of Cancer and Developmental Stanford University School of Medicine Biology, 291 Campus Drive, Li Ka Shing Building Tennis Court Road, Stanford, CA 94305-5101 Cambridge CB2 1QN, USA UK Iain Mattaj Elena Conti Fondazione Human Technopole Max-Planck-Institut für Biochemie Palazzo Italia Structural Cell Biology Department Via Cristina Belgioioso, 171 Am Klopferspitz 18 20157 Milan, Italy D-82152 Martinsried Germany Michael Rout The Rockefeller University 1230 York Avenue New York, NY 10021 USA

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