Molecular reconstitution of cellular machinery essential for neuronal development
Lead Research Organisation:
Birkbeck, University of London
Department Name: Biological Sciences
Abstract
When you were a child, did you ever get lost amidst the towering shelves of a supermarket? That sense of something very small in a huge and complex environment is reminiscent of the challenge faced by cells of the developing brain. The adult human brain is built from billions of specialised cells called neurons. During embryonic growth, immature neurons undertake an amazing journey that involves finding their way from the centre of the developing brain, pushing past many other neurons to get to specific locations in the brain's complex structure. Successful migration enables the formation of the network of mature neurons that are essential for memory and thought. Severe human diseases including epilepsy and mental retardation, and early death, can be caused if the neurons get lost on their journey. Work in my lab is beginning to provide understanding about components of the machinery that enable neurons to navigate the complex maze of the developing brain.
In the same way as our body has a skeleton, neurons have a skeleton - called the cytoskeleton - which also provides support and strength. The cytoskeleton is involved in many important aspects of the life of the cell, including cell shape and movement and it is essential for brain development. Studying the cytoskeleton is important so we can understand both how healthy cells work and malfunctions of the cytoskeleton in disease. My research team studies the three-dimensional structure of the cytoskeleton, because knowing what the cytoskeleton looks like contributes to our understanding of how it works within the cell. We use a very powerful microscope to take pictures of individual cytoskeleton molecules and then use computers to combine these pictures and calculate their three-dimensional shape. Our current research focuses on a part of the cytoskeleton called microtubules, long cylindrical structures that act as scaffolds to help neurons on the move.
In this project, we will be studying a family of proteins that provide extra stability for microtubules in neurons. These proteins are called the doublecortin family of microtubule associated proteins, and they are essential for human brain development. We have had some exciting recent results from our microscope studies and we want to know more about how these proteins interact with microtubules to provide strength for migrating neurons. We also want to know how microtubule stabilisation by the doublecortin family affects the neuron's transporter motors that use microtubules as tracks to carry cargo around the cell.
With this information, we hope to provide insight into how diseases of brain development occur. Our research should also provide essential clues about how immature brain cells might be used to treat brain damage later in life, for example in stroke patients or in sufferers of neurodegenerative diseases like Alzheimer's disease.
In the same way as our body has a skeleton, neurons have a skeleton - called the cytoskeleton - which also provides support and strength. The cytoskeleton is involved in many important aspects of the life of the cell, including cell shape and movement and it is essential for brain development. Studying the cytoskeleton is important so we can understand both how healthy cells work and malfunctions of the cytoskeleton in disease. My research team studies the three-dimensional structure of the cytoskeleton, because knowing what the cytoskeleton looks like contributes to our understanding of how it works within the cell. We use a very powerful microscope to take pictures of individual cytoskeleton molecules and then use computers to combine these pictures and calculate their three-dimensional shape. Our current research focuses on a part of the cytoskeleton called microtubules, long cylindrical structures that act as scaffolds to help neurons on the move.
In this project, we will be studying a family of proteins that provide extra stability for microtubules in neurons. These proteins are called the doublecortin family of microtubule associated proteins, and they are essential for human brain development. We have had some exciting recent results from our microscope studies and we want to know more about how these proteins interact with microtubules to provide strength for migrating neurons. We also want to know how microtubule stabilisation by the doublecortin family affects the neuron's transporter motors that use microtubules as tracks to carry cargo around the cell.
With this information, we hope to provide insight into how diseases of brain development occur. Our research should also provide essential clues about how immature brain cells might be used to treat brain damage later in life, for example in stroke patients or in sufferers of neurodegenerative diseases like Alzheimer's disease.
Technical Summary
Human brain development involves the coordinated movement of billions of neurons. Microtubules (MTs) are crucial for this, acting as both a dynamic framework for motility and forming tracks for intracellular trafficking by molecular motors. Disruption of these functions causes aberrant migration, resulting in developmental neurological defects and brain malformations. Understanding neuronal migration is also critical in the context of brain repair therapies for adult diseases, for example in the treatment of stroke and neurodegeneration. MT-associated proteins (MAPs) are essential for regulating and organising MTs. However, our mechanistic understanding of MAP function is limited by a lack of integrated structural and functional information. The aim of this research programme is to shed light on the doublecortin (DCX) family of essential neuronal MAPs (DCX-MAPs). We will elucidate the molecular mechanisms by which they regulate MTs and control cargo trafficking through interactions with molecular motors. Using state-of-the-art cryo-electron microscopy and image processing, we will solve sub-nanometre resolution structures of DCX-MAP-MT complexes to precisely reveal their mechanisms of MT regulation. We will also reconstitute complexes of motor-bound DCX-MAP-MT complexes and determine these structures by cryo-electron microscopy, revealing the molecular details of motor-track cross-talk. Biochemical and biophysical assays will provide a functional context for our structural discoveries and allow us to test our hypotheses using protein engineering. To place our molecular discoveries in a cellular context, we will study the function of DCX-MAPs in neurons using cryo-electron tomography. We will visualise the organisation of MTs in neurons and elucidate the impact of depletion of DCX-MAPs on neuronal MTs. Through this integrated programme of research, we will discover regulatory mechanisms of MT dynamics and trafficking, with important implications for human health.
Planned Impact
Who will benefit from this research?
- Patients with genetic disorders of neuronal development
- Patients with brain damage due to mechanical injury or neurodegeneration
- UK economy
- The wider public
- Women in science
How will they benefit from this research?
The work described will lead to a greater understanding of essential mechanisms involved in human brain development. DCX-MAPs are regulators of MTs and disruption of their function causes human diseases including epilepsy, intellectual disability and blindness. The molecular insight that will arise from our studies will, first, contribute to improved diagnosis of particular syndromes associated with these diseases. There is emerging evidence that groups of symptoms cluster with particular types of mutations and our studies will provide greater clarity as to the molecular basis of these phenotypes. Once the molecular basis of these diseases are fully understood, novel treatments for these often devastating conditions can be contemplated. Our work will contribute to this, particularly in bridging the gap between molecular function of DCX-MAPs and their regulation of MTs within neurons. In the future, it is also hoped that the understanding that our work will bring concerning general mechanisms of neuronal development can be brought to bear on treatment of extremely common neurological phenomena such as brain injury and neurodegenerative diseases. Science and technology will lie at the heart of global economic recovery, and we will liase with Birkbeck College Business Relations Department to maximise the impact of our discoveries. This will ultimately have benefits for the economic competitiveness of the United Kingdom.
It is essential to retain talented young researchers in the UK, and the proposed research programme will provide an attractive research opportunity for excellent young scientists looking for multi-disciplinary areas of discovery. In addition, transferable skills - such as time- and project-management, presentation and collaboration - that can be applied in all employment sectors will be acquired, particularly through transferable skills training within the Institute of Structural and Molecular Biology.
We will aim to make the discoveries of our research available not only to the academic community, but also to the general public. I have a proven track-record of public communication of science. The appointed PDRAs and I will undertake to design web-pages for my lab which are accessible for the general public and will seek to participate in other public understanding of science activities, for example by inviting sixth-form students to visit our lab and experience the day-to-day life of scientists. During the project period, I will arrange to visit my former school to inspire future scientists and will seek to become involved in advancing gender equality in science, engineering and technology through involvement with The UKRC and WISE campaign.
- Patients with genetic disorders of neuronal development
- Patients with brain damage due to mechanical injury or neurodegeneration
- UK economy
- The wider public
- Women in science
How will they benefit from this research?
The work described will lead to a greater understanding of essential mechanisms involved in human brain development. DCX-MAPs are regulators of MTs and disruption of their function causes human diseases including epilepsy, intellectual disability and blindness. The molecular insight that will arise from our studies will, first, contribute to improved diagnosis of particular syndromes associated with these diseases. There is emerging evidence that groups of symptoms cluster with particular types of mutations and our studies will provide greater clarity as to the molecular basis of these phenotypes. Once the molecular basis of these diseases are fully understood, novel treatments for these often devastating conditions can be contemplated. Our work will contribute to this, particularly in bridging the gap between molecular function of DCX-MAPs and their regulation of MTs within neurons. In the future, it is also hoped that the understanding that our work will bring concerning general mechanisms of neuronal development can be brought to bear on treatment of extremely common neurological phenomena such as brain injury and neurodegenerative diseases. Science and technology will lie at the heart of global economic recovery, and we will liase with Birkbeck College Business Relations Department to maximise the impact of our discoveries. This will ultimately have benefits for the economic competitiveness of the United Kingdom.
It is essential to retain talented young researchers in the UK, and the proposed research programme will provide an attractive research opportunity for excellent young scientists looking for multi-disciplinary areas of discovery. In addition, transferable skills - such as time- and project-management, presentation and collaboration - that can be applied in all employment sectors will be acquired, particularly through transferable skills training within the Institute of Structural and Molecular Biology.
We will aim to make the discoveries of our research available not only to the academic community, but also to the general public. I have a proven track-record of public communication of science. The appointed PDRAs and I will undertake to design web-pages for my lab which are accessible for the general public and will seek to participate in other public understanding of science activities, for example by inviting sixth-form students to visit our lab and experience the day-to-day life of scientists. During the project period, I will arrange to visit my former school to inspire future scientists and will seek to become involved in advancing gender equality in science, engineering and technology through involvement with The UKRC and WISE campaign.
Organisations
- Birkbeck, University of London (Lead Research Organisation)
- National Institute of Health and Medical Research (INSERM) (Collaboration)
- Paul Scherrer Institute (Collaboration)
- UNIVERSITY OF LEEDS (Collaboration)
- National Institutes of Health (NIH) (Collaboration)
- GlaxoSmithKline (GSK) (Collaboration)
- Utrecht University (Collaboration)
People |
ORCID iD |
Carolyn Moores (Principal Investigator) |
Publications
Atherton J
(2017)
A structural model for microtubule minus-end recognition and protection by CAMSAP proteins.
in Nature structural & molecular biology
Maurer SP
(2012)
EBs recognize a nucleotide-dependent structural cap at growing microtubule ends.
in Cell
Atherton J
(2013)
MAPping out distribution routes for kinesin couriers.
in Biology of the cell
Scarabelli G
(2016)
Mapping the Processivity Determinants of the Kinesin-3 Motor Domain
in Biophysical Journal
Scarabelli G
(2015)
Mapping the Processivity Determinants of the Kinesin-3 Motor Domain.
in Biophysical journal
Atherton J
(2018)
Microtubule architecture in vitro and in cells revealed by cryo-electron tomography
in Acta Crystallographica Section D Structural Biology
Atherton J
(2018)
Microtubule architecture in vitro and in cells revealed by cryo-electron tomography.
in Acta crystallographica. Section D, Structural biology
Manka SW
(2018)
Microtubule structure by cryo-EM: snapshots of dynamic instability.
in Essays in biochemistry
Liu JS
(2012)
Molecular basis for specific regulation of neuronal kinesin-3 motors by doublecortin family proteins.
in Molecular cell
Vemu A
(2016)
Structure and Dynamics of Single-isoform Recombinant Neuronal Human Tubulin.
in The Journal of biological chemistry
Maurer S
(2012)
Systems Biochemistry and Structural Biology of Microtubule End Tracking
in Biophysical Journal
Atherton J
(2017)
The divergent mitotic kinesin MKLP2 exhibits atypical structure and mechanochemistry.
in eLife
Manka SW
(2018)
The role of tubulin-tubulin lattice contacts in the mechanism of microtubule dynamic instability.
in Nature structural & molecular biology
Vemu A
(2017)
Tubulin isoform composition tunes microtubule dynamics.
in Molecular biology of the cell
Description | Building blocks of molecular complexity: the neuronal cytoskeleton in health and disease |
Amount | £1,709,028 (GBP) |
Funding ID | MR/R000352/1 |
Organisation | Medical Research Council (MRC) |
Sector | Public |
Country | United Kingdom |
Start | 12/2017 |
End | 12/2022 |
Title | A microtubule RELION-based pipeline for cryo-EM image processing (MiRP) |
Description | We created a pipeline designed for image processing and high-resolution reconstruction of cryo-electron microscopy microtubule datasets, based in the popular and user-friendly RELION image-processing package, Microtubule RELION-based Pipeline (MiRP). The pipeline uses a combination of supervised classification and prior knowledge about geometric lattice constraints in microtubules to accurately determine microtubule architecture and seam location. The presented method is fast and semi-automated, producing nearatomic resolution reconstructions with test datasets that contain a range of microtubule architectures and binding proteins. |
Type Of Material | Model of mechanisms or symptoms - in vitro |
Year Produced | 2019 |
Provided To Others? | Yes |
Impact | Our image reconstruction procedures have been speeded up and are more robust. We know that others in the community have also tested our pipeline. |
URL | https://github.com/moores-lab/MiRP |
Title | EMDB-Atherton |
Description | EM Database of cryo-EM derived structures and associated models: publically available |
Type Of Material | Database/Collection of data |
Year Produced | 2014 |
Provided To Others? | Yes |
Impact | Other researchers now able to download and use models derived from our published cryo-EM reconstructions (7 in total), all of which are deposited. |
URL | http://emsearch.rutgers.edu/atlas/2765_summary.html |
Title | EMDB-Atherton2 |
Description | The cryo-EM structure and resulting model were submitted to EMDB/PDB |
Type Of Material | Database/Collection of data |
Year Produced | 2016 |
Provided To Others? | Yes |
Impact | None yet |
URL | http://www.ebi.ac.uk/pdbe/entry/emdb/EMD-8150 |
Title | Structure and Dynamics of Single-isoform Recombinant Neuronal Human Tubulin cryo-EM dataset |
Description | EMPIAR, the Electron Microscopy Public Image Archive, is a public resource for raw, 2D electron microscopy images. |
Type Of Material | Database/Collection of data |
Year Produced | 2016 |
Provided To Others? | Yes |
Impact | Others in the field have been able to use this data to improve image processing tools |
URL | https://www.ebi.ac.uk/pdbe/emdb/empiar/entry/10071 |
Description | Akmanova_CAMSAP |
Organisation | Utrecht University |
Country | Netherlands |
Sector | Academic/University |
PI Contribution | Structural analysis using cryo-EM of the CAMSAP family of neuronal microtubule regulators |
Collaborator Contribution | Biochemical and cell biological analysis of the CAMSAP family of neuronal microtubule regulators |
Impact | Multi-disciplinary project ongoing using structural, biochemical and cell biological techniques |
Start Year | 2013 |
Description | EML proteins and their MT regulatory activities |
Organisation | University of Leeds |
Department | Astbury Centre for Structural Molecular Biology |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Cryo-EM sample preparation, imaging and structure determination of a range of EML-tubulin co-complexes |
Collaborator Contribution | Provision of EML samples and EML expertise |
Impact | No outputs yet - ongoing |
Start Year | 2017 |
Description | GSK |
Organisation | GlaxoSmithKline (GSK) |
Country | Global |
Sector | Private |
PI Contribution | Consultation and possibility of research supervision to generate material for assay development |
Collaborator Contribution | Initiation of collaboration and research context, subject to agreement on Contract/Confidentiality agreement |
Impact | Collaboration was terminate due to change of direction at GSK. |
Start Year | 2014 |
Description | Kinesin binding protein by cryo-EM |
Organisation | Paul Scherrer Institute |
Country | Switzerland |
Sector | Academic/University |
PI Contribution | We are undertaking cryo-EM studies of kinesin-kinesin-binding-protein complex formation. The collaboration partner has attempted to crystalise this complex for some time. |
Collaborator Contribution | The collaboration partner will be sharing protein reagents with us. |
Impact | Work ongoing |
Start Year | 2016 |
Description | Recombinant tubulin |
Organisation | National Institutes of Health (NIH) |
Department | National Institute of Neurological Disorders and Stroke (NINDS) |
Country | United States |
Sector | Public |
PI Contribution | Cryo-EM reconstructions of microtubules polymerised from purified tubulin |
Collaborator Contribution | Purification and biophysical characterisation of purified tubulin |
Impact | Manuscripts in preparation |
Start Year | 2015 |
Description | Using cryo-electron tomography to study the neuronal cytoskeleton |
Organisation | National Institute of Health and Medical Research (INSERM) |
Department | UMR-S 839 Institut du Fer à Moulin |
Country | France |
Sector | Public |
PI Contribution | We will be using cryo-electron tomography to study the ultrastructural of developing neurons, specifically track the effect of genetic mutations in doublecortin on neuronal microtubules |
Collaborator Contribution | Our partners provide us with cultured neurons |
Impact | Ongoing work |
Start Year | 2014 |
Description | Careers lunch round table (The EMBO Meeting) |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | Yes |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Young scientists attending the EMBO Meeting were invited to join established research scientists to discuss mentoring and career planning hard to monitor |
Year(s) Of Engagement Activity | 2013 |
URL | http://2013.the-embo-meeting.org/ |
Description | Hosted a 6th form work shadow student for 1 week |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | A 6th form student visited our lab for one week and had the chance to shadow various members of the group and to participate in meetings etc. She had lots of questions about our research and about careers in science. She has subsequently been accepted to read Biochemistry at Oxford University and let me know that she had been able to discuss her experience with us in her interview. |
Year(s) Of Engagement Activity | 2018 |
Description | Hosting 6th form work shadow student |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | A sixth form biology student from my local area work-shadowed me for a week. She had experience in the lab and did some online research related to this. This visit will help her in her university applications this year. |
Year(s) Of Engagement Activity | 2013 |
Description | Hosting two 6th form work shadow students |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | Two 6th form students from an all-girls school visited the lab during the work placement visit to experience the day to day life of research scientists. They shadowed members of the research group and discussed a number of different career options. They reported that they would be more likely to consider studying science at university as a result of this visit. |
Year(s) Of Engagement Activity | 2016 |
Description | How to become a Group Leader/PI EMBO Meeting Youtube discussion |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | Yes |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Informal discussion with other PIs filmed by EMBO team and posted on Youtube hard to measure |
Year(s) Of Engagement Activity | 2013 |
URL | http://www.youtube.com/watch?v=7zSNTlzbkqc&feature=youtu.be |
Description | Invited lunchtime speaker at the St Olave's School Natural Sciences Club |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | I was invited to attend the St Olave's School Natural Sciences Club lunchtime meeting to talk about my research. This sparked lots of questions and discussions afterwards which the school reported excited further discussion about science research among the attendees. |
Year(s) Of Engagement Activity | 2016 |
Description | Soapbox Science |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | "Soapbox Science is a novel public outreach platform for promoting women scientists and the science they do. Our events transform public areas into an arena for public learning and scientific debate; they follow the format of London Hyde Park's Speaker's Corner, which is historically an arena for public debate. With Soapbox Science, we want to make sure that everyone has the opportunity to enjoy, learn from, heckle, question, probe, interact with and be inspired by some of our leading scientists. " |
Year(s) Of Engagement Activity | 2017 |
URL | http://soapboxscience.org |