Regulated sub-cellular movements are a fundamental aspect of all living cells and they rely on precisely controlled force generation mechanisms. How are forces imparted, monitored and corrected in response to heterogeneous biochemical and physical cues is a fascinating and challenging biological problem. We address these fundamental questions in the context of dividing human cells and apply the molecular knowledge on force generation and cell division to accelerate cytoskeletal drug discovery and drug resistance biomarker research. Defective force generation during cell division can lead to chromosomal instability and errors in the size, content and fate of daughter cells. Our research findings are therefore relevant to the understanding of irregular chromosome numbers (aneuploidy) and tissue disorganisation found in aggressive cancers and several age-related disorders.
We combine single-cell microscopy with molecular and biochemical approaches. We collaboratively develop computational tools to extract single-cell and population metrics. These approaches form the platform for two streams of studies in our group: (i) To investigate how microtubules are correctly anchored and force generation controlled to power the movements of chromosomes and mitotic spindle, we use high-resolution live-cell imaging of human cells. (ii) To translate the basic knowledge on mitotic microtubule regulation into accelerating the discovery of novel microtubule perturbing drugs and drug resistance biomarkers, we perform pharmacogenomics studies. We are currently refining transcriptional signatures that can predict cells sensitive to Paclitaxel. Visit ongoing research projects for more information.
Another wider aim of our research is to better understand how cells successfully position its spindle to ensure proper cell division in order to generate two genetically identical daughter cells from a single mother cell. Mispositioning of the mitotic spindle can lead to incorrect plane of cell division and consequently altered stem cell fate and disorganized epithelia. In knockdown studies, our lab uncovered a novel role for MARK2 in maintaining the spindle at the cell’s geometric center. Following MARK2 depletion, spindles glide along the cell cortex, leading to a failure in identifying the correct division plane. For more information please visit our recent Publication List.
To better understand spindle dynamics, we developed the spinX Software based on Computer Vision combined with Machine Learning techniques to precisely track spindle movement through time. This computational system allows us to monitor any disruption that may cause by exposing cells to different drugs.
Molecular biology and Biochemistry
To probe the molecular basis of force generation mechanisms.
High-resolution live-cell imaging
Tools to track sub-cellular structures and cellular fate.
Computer Vision
Developing tools for automated microscopy image analysis.
Statistical Analysis
High-throughput assessment of dynamic cellular changes.
What are our Goals?
Chromosome-microtubule attachment
Elucidating how chromosomes captured along microtubule-walls are brought to microtubule-ends
End-on Conversion
Uncovering how cells distinguish chromosomes bound to microtubule-walls versus microtubule-ends
Chromosome missegregation
Determining the immediate and long-term impact of chromosome missegregation
Spindle positioning
Quantitative analysis of how cells pull and rotate their mitotic spindles
(1) Modulating Chromosome-microtubule attachment:
Microtubule targetting drugs have been used for decades to treat aggressive cancers. Understanding how microtubule function is regulated during mitosis will help us improve microtubule targetting drugs. Spindle microtubules (red in cartoon) capture chromosomes (blue) and pull them apart into two chromatids. Microtubule capture is facilitated by the Kinetochore (black-circles), a macromolecular structure that assembles specifically on the centromeric region of chromosomes. We would like to know how kinetochores tethered to microtubule-walls become to tethered to microtubule-ends.
(2) Monitoring Chromosome-microtubule attachment:
The kinetochore acts as a 'platform' that recruits checkpoint proteins, selectively, in the presence of erroneous or immature microtubule attachments. During end-on conversion, the cell has to dynamically distinguish an immature lateral attachment from a mature end-on attachment. How is this achieved? We study the various roles of the kinetochore to understand how it biochemically integrates dynamic temporal and mechanical events.
(3) Impact of Chromosome missegregation:
Errors in chromosome segregation can lead to chromosomal instability - a hallmark of cancers. Understanding the precise nature of lesions can help cancer diagnosis and targetted cancer therapies. By inducing different types of chromosome-microtubule attachment defects we ask how cells respond to different types of chromosome missegregation outcomes.
(4) Mechanisms that govern Spindle movements:
Astral microtubules interact with the cell cortex and they are pulled by cortical forces to rotate the mitotic spindle towards a pre-determined axis. In non-polarised tissue culture cells, the long-axis of the interphase cell acts as the predetermined axis. Spindles are also positioned parallel to the cell-adhesion substratum and maintained at the geometrical centre of the rounded up mitotic cell, allowing us to dissect force generation mechanisms that operate at the cell cortex. How do cells that lack polarity cues orient the spindle precisely within the 3-dimensional space of the cell? Do extracellular forces that change cell shape affect spindle position and cell fate?
Funding Bodies
People
Professor Viji M. Draviam
Principal Investigator (Professor of Quantitative Cell and Molecular Biology and Director of Industrial Innovation at QMUL)
Viji M. Draviam is a Professor in Quantitative Cell and Molecular Biology at the School of Biological and Chemical Sciences, Queen Mary University of London. Her research interest is in the area of cell division, with a focus on the molecular basis for pathologies associated with cell division defects. She started her independent research as a Cancer Research UK Career Development Fellow at the University of Cambridge and a Senior Fellow of Wolfson College, Cambridge. Draviam received a Ph.D. from Trinity College, University of Cambridge and an M.Sc. from National Centre for Biological Sciences, Bangalore. Her post-doctoral work was with Peter Sorger at the Department of Systems Biology, Harvard Medical School and MIT, and PhD work was with Jon Pines at the Gurdon Institute, University of Cambridge. She is a Nehru Scholar and fellow of the Cambridge Commonwealth Trust. She is the cofounder of CellCentives, an international clinical initiative to help eradicate Tuberculosis and a co-mentor of ENERGISE campaign that promotes STEM education among women students Explore more .
Dr. Duccio Conti
BBSRC Post-doc
Duccio graduated from the University of Cambridge (UK) - affiliated to Robinson College. He continues his research in Draviam Lab as a BBSRC-funded post-doc Duccio did his MSc in Molecular Genetics at the University of Leicester (UK) and his BSc in Biological Sciences at the Università degli Studi di Firenze (Florence, Italy). Duccio's PhD project is focused on how kinetochores captured along the walls of microtubules become tethered to the ends of microtubules and how this process is regulated and stabilised by kinase-phosphatase counteraction at the outer-kinetochore level. Duccio has a MM in clarinet performance awarded before starting his biological studies. When not working in the lab, Duccio still plays guitar and enjoys street-skating on inline skates.
Awards:
Cambridge Scholarship (2014-2017)
EMBO fellowship (2019)
Madeleine Hart
PhD student
Maddy is a fourth year PhD student at QMUL, whose project looks into the cellular consequences of kinetochore lesions, with a particular focus on DNA damage. She completed her undergraduate degree at Newcastle University in Biomedical sciences in 2014. After which she worked in an NHS diagnostic lab before then starting her PhD. Outside of the lab Maddy enjoys skiing and reading.
Awards:
Moses Prize for best paper (2019)
David Dang
PhD student
David Dang is a final-year PhD student in the BBSRC-funded London Interdisciplinary Doctoral Training Partnership Programme. As part of the scheme he completed his first rotation with Dr Viji Draviam (Queen Mary University) and Dr Nishanth Sastry (King's College London), where he developed the SpinX Software to track spindle movements in epithelial cells.
Currently, he is working on a feature in spinX Software to expand the application field on 3D live-cell image analysis as an enlightening challenge to investigate spindle movements with the aid of sophisticated and intricate web of information embedded in the images through time lapse and dimensions.
Having been trained as a Statistician and Computer Scientist (University of Tuebingen and University of Bremen, Germany) prior to coming to London, now he sees himself as who combines Computer Vision concepts, biological techniques and statistical ideas together to seek advances to address the ultimate question how subcellular changes behave in cells from a quantitative perspective Explore more.
Beside his research David enjoys coding, composing orchestral music, philosophy and playing handball.
Awards:
BBSRC LIDo DTP (2016-2020)
LSI Showcase Award (2017)
Dr. Asifa Islam
PhD student
Asifa is a final-year PhD student at Queen Mary University of London (UK). Before joining Draviam lab, she did an MRes in Oncology from University of Manchester (UK). Asifa is a registered medical practitioner with Pakistan Medical and Dental Council (PMDC), Pakistan and has passed UK medical licensing exams. Her PhD project looks at role of kinetochore bound microtubule associated proteins in developing chromosomal instability in cancer cells. When not working, Asifa likes to travel and read.
Parveen Gul
PhD student
Parveen is a final-year IDB-funded PhD student at Queen Mary University of London (UK) studying the biochemical interactions between an important kinetochore protein Astrin and PP1 in special regard to its role in kinetochore-microtubule interactions. Parveen uses Biochemistry, Molecular Biology, and cell biology tools to understand how the Astrin has a biologically significance role in cellular events like spindle movements, spindle orientation, regulation of kinetochore-microtubule interactions. She has been an important part of the teams who worked towards several important papers published from Draviam lab.
Parveen like horse-riding, cycling, football, reading history of civilisations, drawing and painting, gardening, and paper art.
Xinhong Song
PhD student
Xinhong graduated from the joint undergraduate program launched by Nanchang University (China) and Queen Mary University of London, with clinical medicine and biomedical sciences double bachelor degrees. She is a second year PhD student at Queen Marry. Her project is focused on detecting the molecular mechanism driving aneuploidy in cancers, especially the molecule interactions involved in the kinetochore-microtubule attachment.
Awards:
CSC studentship (2019-2022)
Christoforos Efstathiou
PhD student
Chris is a first-year PhD student within the BBSRC-funded London Interdisciplinary Doctoral Programme (LIDo). Before joining the Draviam lab, Chris obtained an integrated Masters (MSci) in Cell Biology from University College London (UCL).
Being a cell biologist at heart, Chris is now looking to expand his scientific horizons by undertaking an interdisciplinary iCASE PhD project in collaboration with Image Solutions (IMSOL). His PhD project involves the implementation of the highly innovative Electrically Tunable Lens (ETL) technology for the meticulous investigation of spindle dynamics within the millisecond regime.
In addition, Chris will be developing AI tools that would enable the automated analysis of the large volume of data generated. In his free time, Chris enjoys playing volleyball, philosophy, and cooking.
Awards:
BBSRC LIDo iCASE (2020-2024)
Dr. Dijue Sun
Visiting Post-Doc
Dr Dijue Sun is a visiting postdoctoral research scientist at the Draviam lab. She is a cell biologist specialised in chromosome segregation and DNA double-strand breaks in meiosis. During her PhD, she designed a yeast artificial chromosome transfer method to study non-exchange chromosome segregation in meiosis using live-cell imaging. After her PhD, she went on to study how protein affects the adaptive immune system and cell signalling at Williams Harvey Research Institute (QMUL), leading to a first author paper in Cell Reports 2019. After one period of postdoctoral study, Dr Sun took a gap to raise her children. During this period, Dr Sun worked on developing Deep Learning Methods for automated analysis of mitotic spindle movements in the Draviam group. She has been awarded a Janet Thornton return Fellow at Sanger institute, where she will be studying saturated genome editing on DNA Mismatch repair proteins that affects Lynch Syndrome.
Awards:
Janet Thornton return Fellow (2020)
Best presentation in Immunology conferences at Queen Mary University (2017)
Previous lab members:
Daniel Grant
PhD student
Tami Kasichiwin
Msci student
Tami Kasichiwin is currently undertaking an intercalated Masters in Biochemistry at Queen Mary University of London. Her previous work focused on pharmacology and structural biology, specifically the mode of action of COX inhibitors. Having a strong interest in the cell cycle and proteins, her MSci project is to investigate mechanisms involved in microtubule formation and stability. Outside the laboratory, her hobbies include badminton and baking.
Nadia Osumanu
Msci student
Nadia Osumanu is in the process of completing an integrated Masters in Biochemistry. As part of her undergraduate research project she used NMR to investigate the substrate recognition mechanism of the L. pneumophila secretion system. In her free times she loves to read, listen to Ted talks and podcasts and is currently learning Arabic.
Ihsan Zulkilpi
Current position: Lecturer at University Brunei Darussalam (Brunei)
Ihsan Zulkilpi obtained a B.Sc. in Biochemistry at the University of Bristol before moving to Imperial College London to complete an M.Sc. in Human Molecular Genetics. For her PhD Ihsan investigated how MARK2/Par1 kinase controls spindle movements. She also contributed to automated analysis of spindle movements in human epithelial cells. Ihsan is now back in her home country, Brunei Darussalam, as an Assistant Professor of Haematology.
Naoka Tamura
Naoka Tamura completed her Masters by research in Cell biology in Wellcome trust of Cell biology in the University of Edinburgh after obtaining a B.Sc. Biology in the University of Nottingham. As part of her PhD project, Naoka investigated how microtubule ends recruit distinct protein complexes during distinct stages of mitosis. Naoka is now a post-doctoral fellow at the Barts Institute in London.
Roshan Shrestha
Current position: Research Fellow at National Cancer Institute at The National Institutes of Health (US)
Roshan Shrestha is a Research assistant and graduate student studying the molecular mechanisms involved in ensuring proper kinetochore-microtubule attachments. Roshan's work recently showed how chromosomes bound to the walls of microtubules convert their lateral interaction with walls into an end-on interaction with microtubule ends (Shrestha and Draviam, 2013, under review). Roshan works on the anti-microtubule drug discovery project as well. Roshan received his Masters degree (by Research) in Medical Biosciences from Northumbria University, UK. As a part of his MRes thesis he completed a research project based on DNA repair proteins and Topoisomerase II. During this work, Roshan investigated the in vivo protein-protein interaction of human DNA Topoisomerase II with DNA repair proteins, Meiotic Recombinant 11 and Tyrosyl DNA Phosphodiesterase.
Adam Corrigan
Current position: Principal Data Scientist at AstraZeneca (UK)
Adam Corrigan was a postdoctoral research associate investigating the effect of external factors on mitotic spindle orientation. This work is a collaboration with Professor Athene Donald in the sector of Biological and Soft Systems (BSS) at the Department of Physics, and combines high-throughput microscopy with the development of automated image processing tools to measure and model the 3D spindle orientation in non-polarised cells. Adam generated the first version of Spindle3D. Adam is currently working with Dr. Jonathon Chubb's group, UCL.
Arnab Nayak
Arnab Nayak worked on understand the regulation and checkpoint role of TAO1 kinase with the help of high-throughput immunoprecipitation and mass spectrometry tools. Arnab is currently working with Prof. Stefan Muller's group, IBCII.
Judith Simon
Judith Simon worked for her final year master's research project, University of Gottingen. Judith helped us with yeast 2 hybrid studies of interaction between KMN network and microtubule plus-end associated proteins. Judith is currently working in Foijer group at the University of Groningen.
Short-term members:
Maria worked as undergraduate research student and is currently persuing her PhD at King's College London.
Yingjun Liu worked as a research assistant working on Spindle positioning mechanisms in nonpolarsied and improved the spindle 3D software.
Bing Yang worked as a summer intern and improved the spindle 3D software.
Rhys Grant worked for his fourth year undergraduate research project in part III Biochemistry, Natural Sciences, University of Cambridge. He is currently working with Lindon and Glover groups at the department of Genetics as a post-graduate student.
Dan Lu worked for his second year undergraduate Part IB project, Natural Sciences and is a member of St. John's College Cambridge. He had previous research experience working on cell adhesion molecules at Angiogenesis and Metastasis Research Group in Cardiff University. Dan is currently working at the stem cell institute, Cambridge.
Alexia Hapeshi worked for her fourth year undergraduate research project in part III Biochemistry,Natural Sciences. She is currently in the University of Edinburgh as a Postgraduate Student (2013).
Abhishek Dev worked as a summer student and contributed to elucidating TAO1's role in regulating the density of EB1 comets.
Raphael Kelch worked as a summer student and contributed to elucidating TAO1's role in regulating the density of EB1 comets.
Mathieu Vieira worked as an Erasmus summer student and contributed to studies of microtubule stability in MARK2 depleted cells together with Ihsan.
Jess Patel worked for her fourth year undergraduate research project in part III Systems Biology, University of Cambridge.
Current & Past Collaborators:
Dr. Noriko Hiroi, Keio University (Japan)
Prof. Athene Donald, University of Cambridge (United Kingdom)
Prof. Stephen J. Elledge, Harvard Medical School (USA)
Dr. Akira Funahashi, Keio University (Japan)
Dr. Fanni Gergely, Hutchison/MRC Research Centre (United Kingdom)
Prof. Stephen C. Harrison Harvard Medical School (USA)
Dr. Jeff Karp Harvard Medical School (USA)
Dr. Sumeet Mahajan, University of Cambridge (United Kingdom)
Dr. Andrew D. McAinsh, Marie Curie Research Institute (United Kingdom)
Dr. Patrick Meraldi, ETH Zurich (Switzerland)
Dr. Jordan Raff, University of Cambridge (United Kingdom)
Dr. Frank Stegmeier, Novartis, Cambridge (United Kingdom)
Dr. Jason Swedlow, University of Dundee (United Kingdom)
Dr. Vinay Tergaonkar, National University of Singapore (Singapore)
Dr. Jonas Ries, EMBL - Heidelberg (Germany)
Recent publications:
Kinetochores attached to microtubule-ends are stabilised by Astrin bound PP1 to ensure proper chromosome segregation Duccio Conti, Parveen Gul, Asifa Islam, José M Martín-Durán, Richard W Pickersgill & Viji M. Draviam (2019).
Available here: [pdf] Abstract:
Microtubules segregate chromosomes by attaching to macromolecular kinetochores. Only microtubule-end attached kinetochores can be pulled apart; how these end-on attachments are selectively recognised and stabilised is not known. Using the kinetochore and microtubule-associated protein, Astrin, as a molecular probe, we show that end-on attachments are rapidly stabilised by spatially-restricted delivery of PP1 near the C-terminus of Ndc80, a core kinetochore-microtubule linker. PP1 is delivered by the evolutionarily conserved tail of Astrin and this promotes Astrin's own enrichment creating a highly-responsive positive feedback, independent of biorientation. Abrogating Astrin:PP1-delivery disrupts attachment stability, which is not rescued by inhibiting Aurora-B, an attachment destabiliser, but is reversed by artificially tethering PP1 near the C-terminus of Ndc80. Constitutive Astrin:PP1-delivery disrupts chromosome congression and segregation, revealing a dynamic mechanism for stabilising attachments. Thus, Astrin-PP1 mediates a dynamic 'lock' that selectively and rapidly stabilises end-on attachments, independent of biorientation, and ensures proper chromosome segregation.
MARK2/Par1b kinase present at centrosomes and retraction fibres corrects spindle off-centring induced by actin disassembly Madeleine Hart, Ihsan Zulkipli, Roshan Lal Shrestha, David D Dang, Duccio Conti, Parveen Gul, Izabela Kujawiak & Viji M. Draviam (2019).
Available here: [pdf] Abstract:
Tissue maintenance and development requires a directed plane of cell division. While it is clear that the division plane can be determined by retraction fibres that guide spindle movements, the precise molecular components of retraction fibres that control spindle movements remain unclear. We report MARK2/Par1b kinase as a novel component of actin-rich retraction fibres. A kinase-dead mutant of MARK2 reveals MARK2's ability to monitor subcellular actin status during interphase. During mitosis, MARK2's localization at actin-rich retraction fibres, but not the rest of the cortical membrane or centrosome, is dependent on its activity, highlighting a specialized spatial regulation of MARK2. By subtly perturbing the actin cytoskeleton, we reveal MARK2's role in correcting mitotic spindle off-centring induced by actin disassembly. We propose that MARK2 provides a molecular framework to integrate cortical signals and cytoskeletal changes in mitosis and interphase.
Spindle rotation in human cells is reliant on a MARK2-mediated equatorial spindle-centering mechanism Ihsan Zulkipli, Joanna Clark, Madeleine Hart, Roshan L. Shrestha, Parveen Gul, David Dang, Tami Kasichiwin, Izabela Kujawiak, Nishanth Sastry & Viji M. Draviam (2018).
Available here: [pdf] Abstract:
The plane of cell division is defined by the final position of the mitotic spindle. The spindle is pulled and rotated to the correct position by cortical dynein. However, it is unclear how the spindle’s rotational center is maintained and what the consequences of an equatorially off centered spindle are in human cells. We analyzed spindle movements in 100s of cells exposed to protein depletions or drug treatments and uncovered a novel role for MARK2 in maintaining the spindle at the cell’s geometric center. Following MARK2 depletion, spindles glide along the cell cortex, leading to a failure in identifying the correct division plane. Surprisingly, spindle off centering in MARK2-depleted cells is not caused by excessive pull by dynein. We show that MARK2 modulates mitotic microtubule growth and length and that codepleting mitotic centromere-associated protein (MCAK), a microtubule destabilizer, rescues spindle off centering in MARK2-depleted cells. Thus, we provide the first insight into a spindle-centering mechanism needed for proper spindle rotation and, in turn, the correct division plane in human cells.
Aurora-B kinase pathway controls the lateral to end-on conversion of kinetochore-microtubule attachments in human cells Roshan L. Shrestha, Duccio Conti, Naoka Tamura, Dominique Braun, Revathy A. Ramalingam, Konstanty Cieslinski, Jonas Ries & Viji M. Draviam (2017).
Available here: [pdf] Abstract:
Human chromosomes are captured along microtubule walls (lateral attachment) and then tethered to microtubule-ends (end-on attachment) through a multi-step end-on conversion process. Upstream regulators that orchestrate this remarkable change in the plane of
kinetochore-microtubule attachment in human cells are not known. By tracking kinetochore
movements and using kinetochore markers specific to attachment status, we reveal a
spatially defined role for Aurora-B kinase in retarding the end-on conversion process.
To understand how Aurora-B activity is counteracted, we compare the roles of two outer-
kinetochore bound phosphatases and find that BubR1-associated PP2A, unlike KNL1-
associated PP1, plays a significant role in end-on conversion. Finally, we uncover a novel
role for Aurora-B regulated Astrin-SKAP complex in ensuring the correct plane of
kinetochore-microtubule attachment. Thus, we identify Aurora-B as a key upstream regulator
of end-on conversion in human cells and establish a late role for Astrin-SKAP complex in the
end-on conversion process.
Key Publications
Shrestha RL and Draviam VM.Lateral to End-on Conversion of Chromosome-Microtubule Attachment Requires Kinesins CENP-E and MCAK. Current Biology 2013 23(16):1514-26.[pdf] Background: Proper attachment of chromosomes to microtubules is crucial for the accurate segregation of chromosomes. Human chromosomes attach initially to lateral walls of microtubules. Subsequently, attachments to lateral walls disappear and attachments to microtubule ends (end-on attachments) predominate. While it is known in yeasts that lateral to end-on conversion of attachments occurs through a multistep process, equivalent conversion steps in humans remain unknown. Results: By developing a high-resolution imaging assay to visualize intermediary steps of the lateral to end-on conversion process, we show that the mechanisms that bring a laterally bound chromosome and its microtubule end closer to each other are indispensable for proper end-on attachment because laterally attached chromosomes seldom detach. We show that end-on conversion requires (1) the plus-end-directed motor CENP-E to tether the lateral kinetochore onto microtubule walls and (2) the microtubule depolymerizer MCAK to release laterally attached microtubules after a partial end-on attachment is formed. Conclusions: By uncovering a CENP-E mediated wall-tethering event and a MCAK-mediated wall-removing event, we establish that human chromosome-microtubule attachment is achieved through a set of deterministic sequential events rather than stochastic direct capture of microtubule ends.
Corrigan AM*, Shrestha RL*, Ihsan Zulkipli*, Noriko Hiroi, Yingjun Liu, Naoka Tamura, Bing Yang, Jessica Patel, Akira Funahashi, Athene Donald, Draviam VM.Automated tracking of mitotic spindle pole positions shows that LGN is required for spindle rotation but not orientation maintenance. Cell Cycle 2013 12(16):2643-2655.[pdf] Abstract: Spindle orientation defines the plane of cell division and, thereby, the spatial position of all daughter cells. Here, we develop a live cell microscopy-based methodology to extract spindle movements in human epithelial cell lines and study how spindles are brought to a pre-defined orientation. We show that spindles undergo two distinct regimes of movements. Spindles are first actively rotated toward the cell's long-axis and then maintained along this pre-defined axis. By quantifying spindle movements in cells depleted of LGN, we show that the first regime of rotational movements requires LGN that recruits cortical dynein. In contrast, the second regime of movements that maintains spindle orientation does not require LGN, but is sensitive to 2ME2 that suppresses microtubule dynamics. Our study sheds first insight into spatially defined spindle movement regimes in human cells, and supports the presence of LGN and dynein independent cortical anchors for astral microtubules.
Pagliuca C*, Draviam VM*, Marco E, Sorger PK, De Wulf P.Roles for the Conserved Spc105p/Kre28p Complex in Kinetochore-Microtubule Binding and the Spindle Assembly Checkpoint. PLOS one. 2009 4(10): e7640.[pdf] Background: Kinetochores attach sister chromatids to microtubules of the mitotic spindle and orchestrate chromosome disjunction at anaphase. Although S. cerevisiae has the simplest known kinetochores, they nonetheless contain ~70 subunits that assemble on centromeric DNA in a hierarchical manner. Developing an accurate picture of the DNA-binding, linker and microtubule-binding layers of kinetochores, including the functions of individual proteins in these layers, is a key challenge in the field of yeast chromosome segregation. Moreover, comparison of orthologous proteins in yeast and humans promises to extend insight obtained from the study of simple fungal kinetochores to complex animal cell kinetochores.
Principal Findings: We show that S. cerevisiae Spc105p forms a heterotrimeric complex with Kre28p, the likely orthologue of the metazoan kinetochore protein Zwint-1. Through systematic analysis of interdependencies among kinetochore complexes, focused on Spc105p/Kre28p, we develop a comprehensive picture of the assembly hierarchy of budding yeast kinetochores. We find Spc105p/Kre28p to comprise the third linker complex that, along with the Ndc80 and MIND linker complexes, is responsible for bridging between centromeric heterochromatin and kinetochore MAPs and motors. Like the Ndc80 complex, Spc105p/Kre28p is also essential for kinetochore binding by components of the spindle assembly checkpoint. Moreover, these functions are conserved in human cells. Conclusions: Spc105p/Kre28p is the last of the core linker complexes to be analyzed in yeast and we show it to be required for kinetochore binding by a discrete subset of kMAPs (Bim1p, Bik1p, Slk19p) and motors (Cin8p, Kar3p), all of which are nonessential. Strikingly, dissociation of these proteins from kinetochores prevents bipolar attachment, even though the Ndc80 and DASH complexes, the two best-studied kMAPs, are still present. The failure of Spc105 deficient kinetochores to bind correctly to spindle microtubules and to recruit checkpoint proteins in yeast and human cells explains the observed severity of missegregation phenotypes.
Draviam VM*, Stegmeier F*, Nalepa G, Sowa ME, Chen J, Liang A, Hannon GJ, Sorger PK, Harper JW, Elledge SJ.A functional genomic screen identifies a role for TAO1 kinase in spindle-checkpoint signalling. Nature Cell Bio. 2007 9(5):556-64.[pdf] Abstract: Defects in chromosome-microtubule attachment trigger spindle-checkpoint activation and delay mitotic progression. How microtubule attachment is sensed and integrated into the steps of checkpoint-signal amplification is poorly understood. In a functional genomic screen targeting human kinases and phosphatases, we identified a microtubule affinity-regulating kinase kinase, TAO1 (also known as MARKK) as an important regulator of mitotic progression, required for both chromosome congression and checkpoint-induced anaphase delay. TAO1 interacts with the checkpoint kinase BubR1 and promotes enrichment of the checkpoint protein Mad2 at sites of defective attachment, providing evidence for a regulatory step that precedes the proposed Mad2-Mad1 dependent checkpoint-signal amplification step. We propose that the dual functions of TAO1 in regulating microtubule dynamics and checkpoint signalling may help to coordinate the establishment and monitoring of correct congression of chromosomes, thereby protecting genomic stability in human cells.
Stegmeier F*, Rape M*, Draviam VM*, Nalepa G, Sowa ME, Ang XL, McDonald ER 3rd, Li MZ, Hannon GJ, Sorger PK, Kirschner MW, Harper JW, Elledge SJ.Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities. Nature 2007 446(7138):876-81.[pdf] Abstract: The spindle checkpoint prevents chromosome mis-segregation by delaying sister chromatid separation until all chromosomes have achieved bipolar attachment to the mitotic spindle. Its operation is essential for accurate chromosome segregation, whereas its dysregulation can contribute to birth defects and tumorigenesis. The target of the spindle checkpoint is the anaphase-promoting complex (APC), a ubiquitin ligase that promotes sister chromatid separation and progression to anaphase. Using a short hairpin RNA screen targeting components of the ubiquitin-proteasome pathway in human cells, we identified the deubiquitinating enzyme USP44 (ubiquitin-specific protease 44) as a critical regulator of the spindle checkpoint. USP44 is not required for the initial recognition of unattached kinetochores and the subsequent recruitment of checkpoint components. Instead, it prevents the premature activation of the APC by stabilizing the APC-inhibitory Mad2-Cdc20 complex. USP44 deubiquitinates the APC coactivator Cdc20 both in vitro and in vivo, and thereby directly counteracts the APC-driven disassembly of Mad2-Cdc20 complexes (discussed in an accompanying paper). Our findings suggest that a dynamic balance of ubiquitination by the APC and deubiquitination by USP44 contributes to the generation of the switch-like transition controlling anaphase entry, analogous to the way that phosphorylation and dephosphorylation of Cdk1 by Wee1 and Cdc25 controls entry into mitosis.
McAinsh A*, Meraldi P*, Draviam VM*, Toso A and Sorger PK.The human kinetochore proteins Nnf1R and Mcm21R are required for accurate chromosome segregation. EMBO J. 2006 25 (17): 4033-49.[pdf] Abstract: (KTs) assemble on centromeric DNA, bi-orient paired sister chromatids on spindle microtubules (MTs) and control cell-cycle progression via the spindle assembly checkpoint. Genetic and biochemical studies in budding yeast have established that three 'linker' complexes, MIND, COMA and NDC80, play essential but distinct roles in KT assembly and chromosome segregation. To determine whether similar linker activities are present at human KTs, we have compared the functions of Nnf1R and Mcm21R, recently identified MIND and COMA subunits, and Nuf2R, a well-characterized NDC80 subunit. We find that the three proteins bind to KTs independent of each other and with distinct cell-cycle profiles. MT-KT attachment is aberrant in Nnf1R- and Mcm21R-depleted cells, whereas it is lost in the absence of Nuf2R. Defective attachments in Nnf1R-depleted cells prevent chromosome congression, whereas those in Mcm21R-depleted cells interfere with spindle assembly. All three human KT proteins are necessary for correct binding of spindle checkpoint proteins to KTs. The differing functions and KT-binding properties of Nnf1R, Mcm21R and Nuf2R suggest that, like their yeast counterparts, the proteins act independent of each other in KT assembly, but that their combined activities are required for checkpoint signaling.
Draviam VM, Shapiro I, Aldridge B and Sorger PK.Misorientation and reduced stretching of aligned sister kinetochores promote chromosome missegregation in EB1- or APC-depleted cells. EMBO J. 2006 25 (12): 2814-27 [pdf] Abstract: The correct formation of stable but dynamic links between chromosomes and spindle microtubules (MTs) is essential for accurate chromosome segregation. However, the molecular mechanisms by which kinetochores bind MTs and checkpoints monitor this binding remain poorly understood. In this paper, we analyze the functions of six kinetochore-bound MT-associated proteins (kMAPs) using RNAi, live-cell microscopy and quantitative image analysis. We find that RNAi-mediated depletion of two kMAPs, the adenomatous polyposis coli protein (APC) and its binding partner, EB1, are unusual in affecting the movement and orientation of paired sister chromatids at the metaphase plate without perturbing kinetochore-MT attachment per se. Quantitative analysis shows that misorientation phenotypes in metaphase are uniform across chromatid pairs even though chromosomal loss (CIN) during anaphase is sporadic. However, errors in kinetochore function generated by APC or EB1 depletion are detected poorly if at all by the spindle checkpoint, even though they cause chromosome missegregation. We propose that impaired EB1 or APC function generates lesions invisible to the spindle checkpoint and thereby promotes low levels of CIN expected to fuel aneuploidy and possibly tumorigenesis.
DMeraldi P*, Draviam VM*, and Sorger PK.Timing and Checkpoints in the regulation of mitotic progression.Dev Cell. 2004 7:1-20 [pdf] Abstract: Accurate chromosome segregation relies on the precise regulation of mitotic progression. Regulation involves control over the timing of mitosis and a spindle assembly checkpoint that links anaphase onset to the completion of chromosome-microtubule attachment. In this paper, we combine live-cell imaging of HeLa cells and protein depletion by RNA interference to examine the functions of the Mad, Bub, and kinetochore proteins in mitotic timing and checkpoint control. We show that the depletion of any one of these proteins abolishes the mitotic arrest provoked by depolymerizing microtubules or blocking chromosome-microtubule attachment with RNAi. However, the normal progress of mitosis is accelerated only when Mad2 or BubR1, but not other Mad and Bub proteins, are inactivated. Moreover, whereas checkpoint control requires kinetochores, the regulation of mitotic timing by Mad2 and BubR1 is kinetochore-independent in fashion. We propose that cytosolic Mad2-BubR1 is essential to restrain anaphase onset early in mitosis when kinetochores are still assembling.
We are happy to announce that we are have two exciting PhD projects available in our lab.
(i) Combining high-throughput live-cell imaging and artificial intelligence tools for Cancer studies
A fully funded PhD studentship is available for working with cutting-edge technologies in the field of (Optics) Microscopy, Machine Learning (Computer Vision) and Cell Biology.
Investigation of highly dynamic biological processes like cell division requires high-speed imaging techniques. Conventional microscope systems for live-imaging in 3D are restricted in their spatial-temporal resolution. Therefore, the Draviam group at QMUL will use a highly innovative Electrically Tunable Lens technology that allows precise high-throughput image acquisition in the millisecond regime in 3D. The large volume of live-cell movie data generated through such microscopes offers a unique opportunity for the student to use and develop Machine Learning tools for analysing the images of cancer cells. He/she will work closely with optical engineers, Computer Scientists, Statisticians and Biologists within a highly interdisciplinary and collaborative research environment. As a LIDo-interdisciplinary and iCASE PhD student, you will be assigned to SysMIC courses at UCL and be part of the LIDo networking consortium.
For more information click HERE or write an Email to: Dr Viji M Draviam
(ii) How do human cells ensure the accurate segregation of chromosomes?
Aneuploidy (incorrect number of chromosomes) is a hallmark of aggressive cancers, but the underlying molecular cause is unclear. The PhD student will aim to discover molecular mechanisms that prevent aneuploidy in human cells.
This multi-disciplinary project is ideal for students interested in working at the interface of cancer and basic biology research.
For more information click HERE or write an Email to: Dr Viji M Draviam
Lab Location
School of Biological and Chemical Sciences Queen Mary University of London Mile End Road E1 4NS London
Contact
Dr Viji M Draviam Telephone: +44 (0)20 7882 5020 Email: v.draviam@qmul.ac.uk
