Seminars

Daniel Jarosz, Stanford University School of Medicine, Stanford, California

Wellcome iCM PhD student seminar - Protein-based Inheritance: Epigenetics Beyond the Chromosome

Survival in changing environments requires phenotypic diversification. Yet mechanisms safeguarding genome integrity often limit sources of biological novelty. My lab studies how organisms balance competing needs for robustness and evolvability. I will discuss a pervasive form of transgenerational epigenetic inheritance driven by self-assembly of intrinsically disordered proteins, which alters the apparent native conformation of many proteins depending on life history. Despite its ubiquity, this form of epigenetics ‘beyond the chromosome’ has been hidden in plain sight from most omics technologies, leaving us blind to its importance.

Host: Andreas Fellas and Liz Gaberdiel

Michal Wieczorek, Rockefeller University, New York

Wellcome iCM PhD student seminar - Deciphering the nature of the gamma-tubulin ring complex using cryo-EM and biochemical reconstitutions

The formation of diverse microtubule cytoskeleton networks depends on the γ-tubulin ring complex (γ-TuRC). Discovered over 25 years ago, this essential multi-subunit protein complex is thought to facilitate microtubule assembly by mimicking a microtubule nucleation intermediate. However, a molecular description of how γ-TuRC constituents come together to facilitate microtubule assembly has been lacking, severely limiting biochemical dissections of proposed functional models.

In this talk, I will describe our recent work on generating a near-complete molecular model of the endogenous human γ-TuRC using cryo-EM. We find that the γ-TuRC adopts a cone-shaped structure ~30 nm in diameter, in which a unique arrangement of the γ-tubulin ring complex proteins (GCPs) 2-6 collectively position 14 copies of γ-tubulin into a quasi-helical “ring”. An unexpected structural feature also spans the lumen of the γ-TuRC and contains monomeric actin in complex with the microprotein MZT1. Using the structure of the endogenous complex as a guide, we also successfully reconstitute the γ-TuRC from ten recombinant human proteins. Remarkably, the γ-tubulin “rings” in both endogenous and reconstituted human γ-TuRCs do not match the arrangement of α/β-tubulin in the microtubule lattice, raising important questions about how the γ-TuRC regulates microtubule assembly. Our near-complete molecular picture of the γ-TuRC answers long-standing questions in the cytoskeleton field and paves the way towards functional studies of this enigmatic complex using bottom-up approaches.

Host: James Le Cornu and Lorenza Di Pompeo

Andrea Musacchio - Max-Planck Institute of Molecular Physiology, Dortmund

Wellcome iCM PhD student seminar - The kinetochore, an intrinsically divisive molecular machine

Chromosome bi-orientation is the pre-condition for successful cell division, but how it is achieved on the molecular level in settings as diverse as mitosis and meiosis remains poorly understood. Kinetochores are crucial for chromosome bi-orientation and impart fidelity to the chromosome segregation process. In addition to binding microtubules, they recognize and correct improper microtubule attachments, and act as control centers to make the timing of cell division contingent on completion of bi-orientation through the spindle assembly checkpoint. How are these different activities regulated and integrated within the kinetochore’s structure? To answer this question, our laboratory took up the long-term goal of reconstituting kinetochores and their functions in vitro, focusing on human kinetochores as a model system. The reconstitution is challenging, because kinetochores consist collectively of ~35 core subunits, and several additional regulatory subunits, for a total of ~100 different polypeptides. The challenge is compounded by the embedding of kinetochores in the complex and incompletely understood environment of the centromere, a specialized chromatin domain whose organization promotes epigenetic propagation of the kinetochore assembly site through cell generations. I will illustrate what organizational principles have emerged from this work, and how they are inspiring our current attempts to build the entire kinetochore and its functions in vitro. The ultimate challenge for future in vitro work on the kinetochore, and a more general challenge for any in vitro reconstitution, is to ignite regulatory energy-dissipating reactions. We would like to build particles that, like their cellular counterparts, sense bi-orientation (or lack thereof) and turn the checkpoint on or off depending on context. This will require the addition of enzymes, most notably mitotic kinases and phosphatases, whose opposing regulation determines, at any given time, appropriate context-dependent signaling outcomes.

Host: Thomas Davies and James Watson

Julie Canman - Columbia University, New York

Wellcome iCM PhD student semianr - "FLIRTing with cell type-differences in cell division"

Canman Lab Research Program Summary:

There are two main arms of the Canman lab 1) understanding cytokinesis, the physical division of one cell into two, and 2) developing new microscopy-based thermo-genetic technology to further studies of cytokinesis and other cellular events.  Failure in cytokinesis, resulting in a bi-nucleated cell with two (or more) times the normal chromosome number, is emerging as an important contributor to many human diseases including blood diseases, neurological disorders, and cancer.  Using the worm model system Caenorhabditis elegans, the Canman lab studies the molecular, temporal, spatial, and cell type-specific regulation of cytokinesis—a process that requires rapid and precise coordination of multiple structural and regulatory pathways to ensure accuracy.  The lab also explores how the mechanisms of cytokinesis are modified in specific cell types (or cell lineages) to make cell division more robust and prevent cytokinesis failure in that cell type and/or in other neighboring cells.  In parallel to conducting direct experiments on cytokinesis, the Canman lab is equally committed to the development of new microscopy-based technology to better enable us to probe cytokinesis both temporally and spatially with thermo-genetics—that is, the use of temperature to control protein function via genetically encoded, fast-acting (<20 seconds) temperature-sensitive mutations. 

Selected Canman Lab Publications:

1)      S.M. Hirsch^#, S. Sundaramoorthy^#, T. Davies, Y. Zhuravlev, J. Waters, M. Shirasu-Hiza, J. Dumont, and J.C. Canman. 2018. FLIRT: Fast, Local, Infrared Thermogenetics. Nature Methods. 15:921-923.  ^#Authors contributed equally.

2)      T. Davies, N. Romano Spica, B. Lesea-Pringle, J. Dumont, M. Shirasu-Hiza, and J.C. Canman. 2018. Cell-intrinsic and extrinsic control of cytokinetic diversity. eLife. 7:e36204.

3)      Y. Zhuravlev, S. Hirsch, S.N. Jordan, M. Shirasu-Hiza, J. Dumont, and J.C. Canman. 2017. CYK-4 regulates Rac, but not Rho, during cytokinesis. Molecular Biology of the Cell. 28(9):1258-1271.

4)      S. Sundaramoorthy, S.M. Hirsch, A.G. Badaracco, J.H. Park, T. Davies, J. Dumont, M. Shirasu-Hiza, A.C. Kummel, and J.C. Canman. 2017.  Low efficiency upconversion nanoparticles for high-resolution co-alignment of near-infrared and visible light paths on a light microscope. ACS Applied Materials and Interfaces. 9(9):7929-7940.

5)      S.N. Jordan, T. Davies, Y. Zhuravlev, J. Dumont, M. Shirasu-Hiza, and J.C. Canman.  2016. Cortical PAR proteins promote robust cytokinesis during asymmetric cell division. The Journal of Cell Biology. 221:39-49.

6)      G. Maton, F. Edwards, B. Lacroix, M. Stefanutti, K. Laband, T. Lieury, T. Kim, J. Espeut, J.C. Canman, and J. Dumont. 2015. Kinetochore components are required for central spindle assembly. Nature Cell Biology. 17:697-705.

Host: Tamina Lebek and Sofia Esteban Serna

Tom Deegan, University of Dundee

WCB Adhoc Seminar - Molecular Mechanism and Regulation of DNA replication termination

Eukaryotic DNA replication is carried out by a large and complex macromolecular machine called the replisome, as the core of which lies the CMG replicative helicase. The final step of DNA replication is the disassembly of the CMG-replisome, which is driven by the Cdc48 / p97 AAA+ ATPase, and is triggered by ubiquitylation of CMG by a cullin-RING type E3 ubiquitin ligase. CMG ubiquitylation must be strictly regulated, such that replisome disassembly always happens upon replication termination, but never occurs prematurely at active replication forks. Until now, the molecular mechanisms that underpin this exquisite regulation have remained unknown. Here, I will describe the biochemical reconstitution of eukaryotic replisome ubiquitylation and disassembly with purified proteins, which has identified a novel role for the DNA structure of a replication fork in suppressing premature replisome ubiquitylation before termination. I will also share unpublished data, based on several cryo-EM structures of replisome-E3 ligase assemblies, which reveals the molecular basis for this regulation.

Host: Adele Marston

Kikue Tachibana, Institute of Molecular Biotechnology, Vienna

WCB Renowned Seminar Series - Genome architecture of totipotent mouse embryos

Eukaryotic genomes are compacted into loops and topologically associating domains (TADs), which contribute to transcription, recombination and genomic stability. Cohesin extrudes DNA into loops that are thought to lengthen until it encounters a barrier. The only known barrier to loop extrusion in vertebrates is the CTCF zinc finger transcription factor. Little is known whether loop extrusion is impeded by macromolecular machines. We demonstrate that the replicative helicase MCM2-7 complex is a barrier that restricts loops and TADs in G1 phase. Single-nucleus Hi-C (snHi-C) of mouse one-cell embryos revealed that MCM loading reduces CTCF-anchored loops and increases TAD boundary insulation, suggesting loop extrusion is impeded before reaching CTCF sites. Single-molecule imaging provides evidence that MCMs are physical barriers that constrain cohesin translocation in vitro. Simulations are consistent with MCMs as abundant, randomly positioned barriers. We conclude that distinct loop extrusion barriers contribute to shaping 3D genomes.

Bart Dequeker 1), Hugo B. Brandão 2,3), Matthias J. Scherr 4), Johanna Gassler 1), Sean Powell 1), Imre Gaspar 1,7), Ilya M. Flyamer 5), Ian F. Davidson 6), Jan-Michael Peters 6), Karl Duderstadt 4), Leonid Mirny 3), Kikue Tachibana 1,7)

1) Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna BioCenter, Vienna, Austria 2) Harvard Program in Biophysics, Harvard University, Cambridge, Massachusetts, USA 3) Department of Physics, Massachusetts Institute of Technology, Cambridge, USA 4) Department Structure and Dynamics of Molecular Machines, Max Planck Institute of Biochemistry, Martinsried, Germany 5) MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, UK 6) Research Institute of Molecular Pathology, Vienna BioCenter, Vienna, Austria 7) Department of Totipotency, Max Planck Institute of Biochemistry, Martinsried, Germany

Host: Adele Marston