How are mono-oriented chromosome-microtubule attachments protected to prevent errors in mitosis and associated cellular ageing?
Lead Research Organisation:
Queen Mary University of London
Department Name: Sch of Biological & Behavioural Sciences
Abstract
When a mother cell divides into two, its chromosomes are pulled apart into two equal sets by rope-like microtubules. Errors in chromosome-microtubule attachment can result in the loss or gain of chromosomes, leading to irregular chromosome numbers in cells - a hallmark of animal and human infertility and several premature ageing syndromes. To precisely pinpoint why chromosome numbers are incorrect in some diseases, a clear molecular understanding of how microtubules capture and pull chromosomes apart is essential.
Chromosome-microtubule attachment is mediated by a macromolecular structure - the kinetochore - made of nearly 100 proteins. The Draviam group reported a protein complex Astrin-SKAP that is recruited to kinetochores soon after the formation of correct chromosome-microtubule attachments, and the complex is required for maintaining correct attachments. How Astrin senses attachment status and how it stabilises them are not known. These will be addressed to explain how Astrin ensures the accurate segregation of chromosomes.
By combining methods in structural biology (Pickersgill lab) and evolutionary biology (Martin-Duran lab), the Draviam group showed that Astrin interacts with an outer-kinetochore protein, HEC1, and Astrin delivers an enzyme PP1-phosphatase. Astrin-mediated delivery of PP1 is carefully scheduled to selectively stabilise correct attachments. In other words, Astrin works like a 'messenger' arriving selectively at correctly attached kinetochores to deliver a 'tool' that is needed to stabilise the attachments. This means that determining how Astrin-HEC1 and Astrin-PP1 interactions are controlled will unravel how cells ensure proper chromosome-microtubule attachments and prevent chromosome missegregation.
This project is timely as it takes advantage of a Super-resolution microscope (funded by BBSRC) to track dynamic changes at the outer kinetochore, at the highest spatial resolution possible. First, Astrin's arrival at kinetochores will be correlated with nanoscale structural changes at the outer-kinetochore to learn about changes specific to correct attachments. Second, the regions of Astrin essential for HEC1 or PP1 interaction will be determined, and mutants of Astrin that cannot bind to HEC1 or PP1 will be expressed in cells to study how Astrin senses attachments and how cells schedule Astrin-PP1 interaction to ensure the accurate segregation of chromosomes.
To further strengthen the research program, two collaborations have been planned:
(i) Pull-downs using Owenia embryo lysates to take clues from evolutionarily conserved Astrin interactions.
(ii) Computational modelling of protein structure to take clues from HEC1 and PP1 crystal structures for designing Astrin interaction mutants.
Astrin mutants that disrupt chromosome-microtubule attachment and chromosome segregation accuracy or timing will be studied for the extent to which they promote cellular ageing, either immediately (within hours) or in the long-term (in days), by tracking markers for stress, DNA damage and repair.
This will be the first nano-scale study of dynamic changes at the outer-kinetochore which protect correct attachments and prevent chromosome missegregation. Simultaneous single-cell tracking of attachment defects, segregation inaccuracy and premature ageing make this project unique and invaluable for isolating mitotic errors that cause ageing. This knowledge can help build biomarkers to predict and track premature ageing in animals and humans.
Fundamental discoveries made here about microtubule-mediated pulling or pushing of chromosomes will be widely useful for other microtubule-mediated processes in our body. For instance, neuronal growth, spindle rotation, immune signalling and cell migration are all reliant on regulatory switches to sense and stabilise microtubules in different parts of the cell. Thus the study will be broadly useful to understand force generation mechanisms within cells.
Chromosome-microtubule attachment is mediated by a macromolecular structure - the kinetochore - made of nearly 100 proteins. The Draviam group reported a protein complex Astrin-SKAP that is recruited to kinetochores soon after the formation of correct chromosome-microtubule attachments, and the complex is required for maintaining correct attachments. How Astrin senses attachment status and how it stabilises them are not known. These will be addressed to explain how Astrin ensures the accurate segregation of chromosomes.
By combining methods in structural biology (Pickersgill lab) and evolutionary biology (Martin-Duran lab), the Draviam group showed that Astrin interacts with an outer-kinetochore protein, HEC1, and Astrin delivers an enzyme PP1-phosphatase. Astrin-mediated delivery of PP1 is carefully scheduled to selectively stabilise correct attachments. In other words, Astrin works like a 'messenger' arriving selectively at correctly attached kinetochores to deliver a 'tool' that is needed to stabilise the attachments. This means that determining how Astrin-HEC1 and Astrin-PP1 interactions are controlled will unravel how cells ensure proper chromosome-microtubule attachments and prevent chromosome missegregation.
This project is timely as it takes advantage of a Super-resolution microscope (funded by BBSRC) to track dynamic changes at the outer kinetochore, at the highest spatial resolution possible. First, Astrin's arrival at kinetochores will be correlated with nanoscale structural changes at the outer-kinetochore to learn about changes specific to correct attachments. Second, the regions of Astrin essential for HEC1 or PP1 interaction will be determined, and mutants of Astrin that cannot bind to HEC1 or PP1 will be expressed in cells to study how Astrin senses attachments and how cells schedule Astrin-PP1 interaction to ensure the accurate segregation of chromosomes.
To further strengthen the research program, two collaborations have been planned:
(i) Pull-downs using Owenia embryo lysates to take clues from evolutionarily conserved Astrin interactions.
(ii) Computational modelling of protein structure to take clues from HEC1 and PP1 crystal structures for designing Astrin interaction mutants.
Astrin mutants that disrupt chromosome-microtubule attachment and chromosome segregation accuracy or timing will be studied for the extent to which they promote cellular ageing, either immediately (within hours) or in the long-term (in days), by tracking markers for stress, DNA damage and repair.
This will be the first nano-scale study of dynamic changes at the outer-kinetochore which protect correct attachments and prevent chromosome missegregation. Simultaneous single-cell tracking of attachment defects, segregation inaccuracy and premature ageing make this project unique and invaluable for isolating mitotic errors that cause ageing. This knowledge can help build biomarkers to predict and track premature ageing in animals and humans.
Fundamental discoveries made here about microtubule-mediated pulling or pushing of chromosomes will be widely useful for other microtubule-mediated processes in our body. For instance, neuronal growth, spindle rotation, immune signalling and cell migration are all reliant on regulatory switches to sense and stabilise microtubules in different parts of the cell. Thus the study will be broadly useful to understand force generation mechanisms within cells.
Technical Summary
Chromosomal instability (CIN) accelerates cellular ageing and tissue degeneration. CIN can arise from defects in chromosome-microtubule attachments. A clear molecular understanding of how kinetochores establish chromosome-microtubule attachments will help explain the precise molecular reason for CIN-induced ageing in animals and humans.
This project builds on the Draviam group's recent success in establishing a new paradigm for how cells ensure proper chromosome-microtubule attachment. The group showed that mono-oriented end-on kinetochores recruit Astrin (a microtubule-end associated protein) while losing classical checkpoint proteins. Astrin's kinetochore recruitment is crucial for maintaining attachments, favouring its further enrichment. Astrin's localisation and function require its interaction with two evolutionarily conserved kinetochore proteins: PP1 and HEC1. Revealing precisely how these two protein interactions are regulated will provide deep insight into previously unrecognised signalling mechanisms that protect mono-oriented attachments to ensure proper chromosome segregation, and in turn prevent CIN.
This project takes advantage of a state-of-the-art Super-Resolution microscope (OMX-Flex, co-funded by BBSRC) to track kinetochore changes at the highest spatial resolution possible. By blending cell, structural and evolutionary biology tools, the study will show (a) nano-scale structural changes specific to mono-oriented end-on kinetochores (b) biochemical regulation of Astrin's interaction with PP1 and HEC1 to ensure proper end-on attachment and (c) the impact of disrupting mono-oriented attachments on segregation accuracy and cellular ageing, explaining how and why cells protect mono-oriented kinetochores.
BBSRC PRIORITY: This 'data-driven biology' study will create world-leading image data and analysis tools, to unravel how cells prevent mitotic errors that cause cellular ageing, relevant to 'Healthy ageing across the life course'.
This project builds on the Draviam group's recent success in establishing a new paradigm for how cells ensure proper chromosome-microtubule attachment. The group showed that mono-oriented end-on kinetochores recruit Astrin (a microtubule-end associated protein) while losing classical checkpoint proteins. Astrin's kinetochore recruitment is crucial for maintaining attachments, favouring its further enrichment. Astrin's localisation and function require its interaction with two evolutionarily conserved kinetochore proteins: PP1 and HEC1. Revealing precisely how these two protein interactions are regulated will provide deep insight into previously unrecognised signalling mechanisms that protect mono-oriented attachments to ensure proper chromosome segregation, and in turn prevent CIN.
This project takes advantage of a state-of-the-art Super-Resolution microscope (OMX-Flex, co-funded by BBSRC) to track kinetochore changes at the highest spatial resolution possible. By blending cell, structural and evolutionary biology tools, the study will show (a) nano-scale structural changes specific to mono-oriented end-on kinetochores (b) biochemical regulation of Astrin's interaction with PP1 and HEC1 to ensure proper end-on attachment and (c) the impact of disrupting mono-oriented attachments on segregation accuracy and cellular ageing, explaining how and why cells protect mono-oriented kinetochores.
BBSRC PRIORITY: This 'data-driven biology' study will create world-leading image data and analysis tools, to unravel how cells prevent mitotic errors that cause cellular ageing, relevant to 'Healthy ageing across the life course'.
