Project 1: The role of tubulin modifications in specifying MT functional properties during mitosis

This project will be carried out by the ESR1 under the supervision of Dr. I. Vernos and co-supervision of Dr. C. Janke.

The objective of the project is to determine the role of tubulin isotypes and modifications in establishing different functional MT populations in the spindle. In collaboration with the group of C. Janke (P2a) ESR1 will identify tubulin isotypes and PTMs of the different spindle MT subclasses and study their role in the successful outcome of cell division. ERS1 will use biochemistry, mass spectrometry and functional experiments in the Xenopus Egg extract system. Using commercial or homemade antibodies, the ESR will characterize the distribution of tubulin PTMs in spindles assembled around nuclei or DNA-coated beads by confocal microscopy. ESR1 will identify tubulin-modifying enzymes present in the egg extract using RT-PCR, and will generate specific antibodies to these enzymes to characterize their expression pattern and localization in Xenopus and/or human cells. ERS1 will elucidate the role of these enzymes in the nucleation, dynamic and stability of mitotic MTs through functional experiments in the Xenopus egg extract system and will test and validate candidate drugs identified by ESR8. ERS1 will also study the substrate specificity, the regulation and the interacting partners of the modifying enzymes.

Project 2: Molecular mechanisms of the assembly/disassembly dynamics at the MT-minus end during mitosis

This project will be carried out by the ESR2 under the supervision of Dr. I. Vernos and co-supervision of Dr. T. Müller-Reichert.

The main objective is to elucidate the mechanism controlling the specific dynamic properties of MT minus-ends tissue culture cells, Xenopus egg extracts and in vitro with purified components ESR2 will investigate how the MCRS1 complex identified by P1regulates MT minus-end dynamics. ESR2 in will work together with ESR11 to develop/adapt the Cherry biotech temperature control device to analyse MT dynamics in vitro and in egg extracts. ESR2 will study the MT minus ends structure using EM in collaboration with P7 and the mechanism by which the MCRS1 complex regulates K-fiber minus-end dynamics in cells. Together with P4 and P9, ESR2 will study MT minus end dynamics through in vitro experiments with reconstituted MCRS1 complexes in the presence of the antagonizing MT depolymerases. Together with ESR10, ESR2 will examine the role of the rate of MT minus-end depolymerisation on spindle assembly and stability using computer simulations.

Project 3: Functions of tubulin polyglutamylation in the mammalian mitotic spindle

This project will be carried out by the recruited ESR3 under the supervision of Dr. C. Janke and co-supervision of Dr. T. Müller-Reichert.

The project’s aim is to understand how polyglutamylation of MTs is distributed within the mitotic spindle, and how this modification affects spindle architecture and function. In the first part of the project, ESR3 will use high-resolution microscopy and a panel of modification-specific antibodies to identify MT sub-species in the mammalian mitotic spindle in collaboration with IMBA (see DivIDE’s Associated Partners) who will provide the necessary microscopy expertise. She/he will use synchronized mammalian cells to investigate the tubulin-modifying enzymes by quantitative PCR and biochemical measurements. In the second part, ESR3 will use video microscopy to identify mitotic or/and abscission pheno-types related to changes in tubulin PTMs, and also study the dynamic localization of tubulin-modifying enzymes throughout the cell cycle in established BAC cell lines. In the third part, ESR3 will analyse candidate enzymes that regulate specific MTs in the mitotic spindle by siRNA or knockout approaches (CRISPR), and study the impact of the modification on MT interactions.

Project 4: Fission yeast as a model system to study tubulin modification and its effect on mitosis

This project will be carried out by the recruited ESR4 under the supervision of Dr. P. Tran and co-supervision of Dr. T. Surrey.

Core mechanisms of spindle dynamics and chromosome segregation, and the motors and MAPs involved, are conserved from fission yeast to human. Fission yeast contains no tubulin-modifying enzymes, suggesting that tubulin PTMs have evolved in higher eukaryotes as a fine-tuning mechanism ensuring optimal efficiency of motors and MAPs interacting with MTs to organize the mitotic spindle. ESR4 will genetically engineer new fission yeast strains as a model system to study how tubulin PTMs affect mitosis. She/he will replace the essential - and -tubulin genes of fission yeast with the mammalian tubulin genes, or with chimeras of fission yeast genes carrying the functionally important tails of mammalian tubulin. ESR4 will systematically introduce different mammalian polyglutamylases enzymes into these novel strains, and observe their effects on MT polyglutamylation, as well as related phenotypes. ESR4 will use mutagenesis to identify precise tubulin modification sites of each polyglutamylase in the recombinant system, which until today has remained impossible in other systems. ESR4 will study how specific polyglutamylases affect spindle organization and dynamics by a combination of biochemistry and live-cell imaging in together with A3. ESR4 will validate discoveries from the yeast model by targeted testing of specific tubulin-modifying enzyme, its effect of specific motors and MAPs, and spindle phenotype in collaboration with ESR3 in mammalian cells. ESR4 will complement the in vivo cell studies with in vitro reconstitution studies using purified proteins. In collaboration with P4, she/he will use TIRF microscopy assays to test the mechanisms by which fission yeast and mammalian motors and MAPs can interact differentially with unmodified and modified tubulin. Finally, ESR4 will also work with CherryBiotech to apply microfabrication technologies to control cellular microenvironments such as temperature, cell geometries, and chemical gradients, to further understand how cells and modified tubulins respond.

Project 5: Tubulin biochemical dynamics in human oocyte maturation

This project will be carried out by the recruited ESR5 under the supervision of Dr. R. Vassena and co-supervision of Dr. I. Vernos.

The research project of ESR5 will be focused on a comprehensive study of cytoskeletal changes involved in the early events of human development. Specific to this project is the identification of prospective markers of embryonic developmental competence, achieved by linking early molecular events in oocyte maturation with patient’s data of clinical relevance for reproductive fitness (e.g. age, ovarian reserve, previous parity, and cohort reproductive outcomes). Specifically, ESR5 will investigate the spectrum and frequency of tubulin post translational modifications (PTMs) during human oocyte maturation. This goal will be achieved by adapting the model of in vitro maturation (IVM) of human meiotically competent oocytes used in Clinica EUGIN to oocytes that are arrested in the first meiotic prophase and are commonly discarded, and combining it with high resolution confocal microscopy with Immunohistochemistry.

Project 6: Regulation of dynein function

This project will be carried out by the recruited ESR6 under the supervision of Dr. T. Surrey and co-supervision of Dr. A. Musacchio.

The ESR6 will study the molecular mechanism of the regulation of human dynein/dynactin activity using in vitro approaches and mutational analysis in human cells.

Goal 1 of ESR6 will be to understand the effects of known dynein regulators on the single molecule activity of recombinant human dynein using in vitro assays. Single molecule fluorescence microscopy and force spectroscopy will be performed for various combinations of purified regulators and purified human dynein complex. The regulators to be purified are: dynactin complex (from cultured human cells), LIS1, NudE, NuMA, BicD. Their effects on the single molecule and collective ensemble activity of dynein will be studied.

Goal 2 of ESR6 will be to understand the effects of misregulation of dynein on spindle formation and function in cultured human cells. Based on the understanding of the molecular mechanisms achieved in vitro, experiments with cultured cells will be designed to monitor the effects of misregulation of dynein on cell division, focusing here especially on spindle pole focusing, spindle stability and spindle positioning.

Project 7: A synthetic biology approach to spindle checkpoint silencing by the Dynein-Dynactin complex

This project will be carried out by the recruited ESR7 under the supervision of Dr. A. Musacchio and co-supervision of Dr. T. Surrey.

Kinetochores (KTs) are the largest MT-binding molecular machines in the cell. They coordinate MT binding with the spindle assembly checkpoint (SAC), a crucial cell cycle control that halts mitotic progression until chromosome bi-orientation. How the SAC is silenced to allow mitotic exit on bi-orientation is conjectural. The Dynein-Dynactin complex is recruited to KTs prior to MT attachment and is crucially required for SAC silencing by “accompanying” the SAC proteins away from KTs upon MTs attachment.

The aim of ESR7 will be to reconstitute KT-MT attachment and SAC silencing at the single-molecule level. Following components are available in the lab of A. Musacchio (P5) and T. Surray (P4) and will be shared with ESR7:

Reconstituted KTs (27 subunits), SAC constituents, including the RZZ complex (~12 subunits), and the Spindly protein are already available to P5 in mg amounts and in fluorescent form. P4 has also developed expression of recombinant Dynein. ESR7 will deposit recombinant KTs on appropriate nano-patterned surfaces in microfluidic chambers engineered by P9 for full temperature control and dynamic exchange of components. Molecular interactions of SAC subunits, MTs, and Dynein-Dynactin with artificial KTs will be monitored by fluorescence microscopy and force spectroscopy and analysed by cross-linking and mass spectrometry to identify interaction interfaces. With P8, ESR7 will try to formalize and simulate this process. Reconstitution in egg extracts of Xenopus laevis (with P1) will also be tried in order to isolate new factors influencing KT-MT attachment. Mutants will validate potentially relevant interactions. Additionally, ESR7 will perform an electron microscopy approach, which will reveal structural information of the KT-MT interaction. Effects of tubulin modifiers on KT-MT attachment will be studied in vitro and in cells in a 3-way collaboration (P2, P4 and P5) by the above described approaches.

Project 8: Selective inhibition of tubulin polyglutamylation in the mitotic spindle

This project will be carried out by the recruited ESR8 under the supervision of Dr. B. Klebl and co-supervision of Dr. C. Janke.

The overall goal of this subproject is to identify new selective drug precursors for cell cycle and/or neurodegenerative research with polyglutamylases from the TTLL protein family as a drug targets. ESR8 will generate a chemical tool that selectively inhibits these enzymes, and subsequently chemically optimize it.

ESR8 will express and purify the glutamylase TTLL4 and will learn how to perform radiometric glutamylation assays with P2a. She/he will develop a fluorescence-based assay adaptable to robotics-based industrial scale for high-throughput screening of a large small-molecule library. ESR8 will use the LDC/COMAS library (~440.000 compounds) to identify inhibitors of polyglutamylases with P5. ESR8 will optimize protein production with P2a and P5 (protein facility). Primary hits of the first screen will be identified with the support of computational clustering, and ESR8 will confirm and verify the direct binding of the inhibitor to their target TTLL4 by orthogonal and secondary assays. ESR8 will further establish selectivity assays using related TTLLs enzymes together with P2a. Validated hits from preferred compound clusters will be chemically optimized and tested for their pharmacological properties in LDC’s eADMET panel. Optimized hit candidates will be used as chemical probes to study the functions of polyglutamylation in cell division in close collaboration with ESR3. The best candidates will be further tested in assays with mammalian cells, Xenopus egg extracts and C. elegans by partners P1, P2a, P7, and A2. ESR8 will be trained in project management at the interface of academic and pharmaceutical research, in patent searches and protection of intellectual property, also in collaboration with P2.

Project 9: Functional role of polyglutamylation in C. elegans spindle assembly and mammalian abscission

This project will be carried out by the recruited ESR9 under the supervision of Dr. T. Müller-Reichert and co-supervision of Dr. A. Musacchio.

The aim of the project is to understand the functional role of polyglutamylation in spindle assembly and abscission. ESR9 will apply established protocols to analyze MT dynamics and ultrastructure in mitotic C. elegans embryos and in post-mitotic mammalian tissue culture cells. He/she will characterize the function of specific polyglutamylation enzymes using RNAi together with live-cell imaging. Specific phenotypes will be quantitatively analysed and compared to wild-type structure by using a GFP-β-tubulin strain. Moreover, the impact of MT polyglutamylation on MT dynamics of kinetochore/interpolar and astral MTs in mutant embryos will be determined in an EB1-marker strain. ESR9 will also use staged mammalian cells to investigate the role of tubulin-modifying enzymes in cytokinesis with correlative light and electron microscopy (CLEM) to quantitatively analyse intermediate stages of abscission in 3D. In addition, ESR9 will characterize polyglutamylation phenotypes by electron tomographic analyses to determine whether spastin-mediated MT depolymerisation is influenced by MT polyglutamylation during abscission.

Project 10: Mathematical modelling of mitotic spindle structures

This project will be carried out by the recruited ESR10 under the supervision of Dr. F. Nédélec and co-supervision of Dr. I. Vernos.

The aim of ESR10 will be to model the assembly and dynamics of a metazoan mitotic spindle during metaphase. Using a mathematical model, ESR10 will study two aspects of spindle assembly that will be investigated by experimental partners of the consortium: The formation of the poles (with P4), and the respective role of the different pathways of nucleation (with P1). The methodology will be to simulate the MTs and the associated proteins with computers, to study how the properties of the elements allow them to organize into a functional spindle structure. The simulation will incorporate dynamic instability of MTs and kinesin-5 induced poleward-flux, as done previously by P8. ESR10 will focus on two aspects that were not studied so far: (i) the focusing of MTs into a fusiform shape, and (ii) the nucleation of MTs near the spindle poles. The two processes imply the motor dynein, because it may focus MTs directly by acting as a sliding crosslink between two MTs, and indirectly by transporting enzymes to the spindle-poles. The later activity can lead to spindle pole formation, in particular if these enzymes are able to nucleate or sever MTs. With these additional parameters in the model, ESR10 will study how spindle poles form, and how the length of the spindle is determined.

Project 11: Microfluidic device for medium exchange in a biological sample under highly controlled flow and temperature

This project will be carried out by the recruited ESR11 under the supervision of Dr. T. Guerinier and Dr. J. Cramer and co-supervision of Dr. T. Phong.

ESR11 in collaboration with ESR2, 6, and 7 will work on developing new tools for synthetic biology and microscopy. The goal is to dynamically control at the same time various environmental factors (e.g. temperature, chemical environment) of samples under the microscope. ESR11 will first adapt the current temperature control device to fit partner’s samples. ESR11 will visit P1 and P4 and 5 to discuss with ESR2, 5 and 6 and gather all the needs and limitations associated to their experiments (e.g. size of sample, microscope capability, gas/solution composition, optical, biological, chemical compatibility). The aim is to develop the world fastest control system for the analysis of biological systems, which can be easily adapted to different biological systems. A system of High temporal resolution is important, to be able to control and analyse biochemical mechanisms which happen in a very short time frame. ESR11 will develop a new device for changing the medium of biological samples under highly controlled flow and temperature. To do so, the existing temperature control device will be enriched by a system to exchange liquid with a high temporal resolution. This device will allow adding or removing a chemical compound to/from cells medium in a time frame of less than 10 seconds. This will be an unique tool for studying dynamic systems, and for finely controlling the time of adding and removing of chemical compounds / inhibitors directly on the microscope stage, while filming the cells or the in vitro reconstituted systems. The new products developed by ESR11 will not only address the needs of the partners but will be commercialised. A market study validates the need for such an instrument for the whole scientific community.