Single-molecule studies of kinesin biophysics using DNA-kinesin chimeras
Lead Research Organisation:
University of Oxford
Department Name: Oxford Physics
Abstract
Kinesins are a family of motor proteins that have a wide range of functions including transport of molecular cargoes within cells, assembly and disassembly of networks of rigid microtubules that are used to organize cell contents, and the separation of chromosomes when cells divide. Conventional kinesin has two identical heads that can each bind to a microtubule. It 'walks' hand-over-hand, which requires coordination - it is important that at least one head remains bound at all times, otherwise the motor is likely to lose contact with its track. The mechanisms by which the motor coordinates its heads and generates force are still not clear. It is important to understand them, because defective kinesins can cause disease and because kinesin and microtubules are drug targets in some cancer therapies. We are studying the mechanism of kinesin by creating artificial motors incorporating elements of kinesin attached to frameworks assembled from short strands of DNA. This allows us to alter the number of kinesin heads that are working together, or the length or the elasticity of the link between them, in ways that are not possible when working with the natural protein alone. Our aims are to improve the precision of measurements of single kinesin molecules walking, to study how kinesin's heads are coordinated, and to investigate how many kinesins work together to create larger forces.
Technical Summary
We will address key problems in the kinesin mechanism by using DNA self-assembly to produce assemblies of motors containing a defined number of kinesin units (single heads or dimers) arranged with a defined attitude, spacing and mechanical coupling. No other system that provides this degree of architectural control currently exists. We have developed a system for attaching kinesin molecular motors to double stranded DNA using Zn-finger DNA recognition domains fused to the C-terminus of kinesin. We can programme the number, spacing and relative orientation of binding sites into the DNA template. We have assembled teams of kinesin dimers and single kinesin heads and have used gliding assays, in which motor teams are anchored to a cover slip and fluorescently labelled microtubules move over them, to measure gliding velocity for teams of different compositions over a wide range of temperatures. We have also demonstrated the inverted assay in which motor teams labelled with single fluorescent quantum dots are observed to move along immobilized microtubules. We will use an optical trap to measure force-velocity and force-displacement curves for single motors and motor teams. We will use self-assembled DNA templates to achieve precise control of the number of motors attached to the trapped bead and the geometry with which they interact with a microtubule. These structures will be designed to improve the mechanical properties of the linkage between the trapped bead and motor, increasing the temporal and spatial resolution of the trap. Velocity and run length of fluorescently labelled motor teams on immobilized microtubules, and microtubule gliding velocities on surface-bound teams, will also be measured. We will thus be able to investigate the basic mechanisms by which kinesin motors generate force, how individual kinesin heads are coordinated and how motors cooperate - including the structural requirements and limits for effective function within multimotor teams.
Publications
Wollman AJ
(2014)
Transport and self-organization across different length scales powered by motor proteins and programmed by DNA.
in Nature nanotechnology
Description | Kinesins are a family of motor proteins that have a wide range of functions including transport of molecular cargoes within cells, assembly and disassembly of networks of rigid microtubules that are used to organize cell contents, and the separation of chromosomes when cells divide. Conventional kinesin has two identical heads that can each bind to a microtubule. It 'walks' hand-over-hand, which requires coordination - it is important that at least one head remains bound at all times, otherwise the motor is likely to lose contact with its track. The mechanisms by which the motor coordinates its heads and generates force are still not clear. It is important to understand them, because defective kinesins can cause disease and because kinesin and microtubules are drug targets in some cancer therapies. We are studying the mechanism of kinesin by creating artificial motors incorporating elements of kinesin attached to frameworks assembled from short strands of DNA. This allows us to alter the number of kinesin heads that are working together, or the length or the elasticity of the link between them, in ways that are not possible when working with the natural protein alone. Our aims are to improve the precision of measurements of single kinesin molecules walking, to study how kinesin's heads are coordinated, and to investigate how many kinesins work together to create larger forces. We have measured the properties of synthetic teams of kinesin motor proteins and are using the results to develop a model for how these teams generate force (work on this aspect of the grant is still in progress). We have also used controllable kinesin teams to create synthetic molecular transport systems, inspired by biological structures, in which motor proteins can be programmed by molecular signals to create ordered arrays of microtubule tracks (and to destroy them), and to use these arrays to scavenge, concentrate and release cargo molecules. |
Exploitation Route | Discoveries made by using novel DNA-kinesin hybrids are of interest to the academic community working on kinesin and related molecular motors and on their roles within cells. In the longer term, significant discoveries may influence the development of drugs and therapies for use in the treatment of diseases related to motor malfunction and for cancer. Biomimetic systems powered by fast and efficient kinesin motors may form the basis of future self-organized molecular factories and theranostic devices. |
Sectors | Healthcare Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology |
Description | Our work on the development of dynamic DNA devices and molecular machinery has contributed to a huge growth in the international research community that studies DNA self-assembly. Our demonstration that kinesin can assemble micrometre-scale structures which can be used to manipulate molecular cargoes, under the control of molecular programming instructions, is proof of principle that synthetic self-organized systems can operate across multiple length scales. Practical applications of this revolutionary new technology are still just around the corner, but promising directions include molecular manufacture and molecular electronics, drug discovery, drug delivery and structural biology. |
Sector | Other |
Description | 14-ERASynBio BioOrigami |
Amount | £415,854 (GBP) |
Funding ID | BB/M005739/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 08/2014 |
End | 08/2017 |
Description | Artificial synthesis of the bacterial flagellar motor with DNA nanostructures |
Amount | $1,200,000 (USD) |
Funding ID | RGP0030/2013 |
Organisation | Human Frontier Science Program (HFSP) |
Sector | Charity/Non Profit |
Country | France |
Start | 08/2013 |
End | 08/2016 |
Description | Bio-Inspired Quantum Technologies |
Amount | £1,500,000 (GBP) |
Organisation | University of Oxford |
Department | Oxford Martin School |
Sector | Academic/University |
Country | United Kingdom |
Start | 03/2013 |
Description | EPSRC & BBSRC Centre for Doctoral Training in Synthetic Biology |
Amount | £8,261,498 (GBP) |
Funding ID | EP/L016494/1 |
Organisation | Engineering and Physical Sciences Research Council (EPSRC) |
Sector | Public |
Country | United Kingdom |
Start | 03/2014 |
End | 09/2022 |
Description | EScoDNA Marie Curie Initial Training Network |
Amount | € 4,070,204 (EUR) |
Funding ID | 317110 |
Organisation | Marie Sklodowska-Curie Actions |
Sector | Charity/Non Profit |
Country | Global |
Start | 02/2013 |
End | 01/2017 |
Description | Extending the Boundaries of Nucleic Acid Chemistry |
Amount | £1,659,227 (GBP) |
Funding ID | BB/J00054X/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 03/2012 |
End | 03/2017 |
Description | MRC Confidence in Concept |
Amount | £22,506 (GBP) |
Organisation | Medical Research Council (MRC) |
Sector | Public |
Country | United Kingdom |
Start | 07/2017 |
End | 04/2018 |
Description | Marie Sklodowska Curie Innovative Training Network |
Amount | € 3,979,633 (EUR) |
Funding ID | 765703 |
Organisation | European Commission H2020 |
Sector | Public |
Country | Belgium |
Start | 01/2018 |
End | 12/2021 |
Description | Royal Society Wolfson Research Merit Award |
Amount | £100,000 (GBP) |
Funding ID | WM110130 |
Organisation | The Royal Society |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 03/2012 |
End | 03/2017 |
Description | SynbiCITE - an Imperial College led Innovation and Knowledge Centre (IKC) in Synthetic Biology |
Amount | £5,074,190 (GBP) |
Funding ID | EP/L011573/1 |
Organisation | Engineering and Physical Sciences Research Council (EPSRC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2013 |
End | 09/2018 |
Description | University of Oxford John Fell Fund |
Amount | £72,096 (GBP) |
Organisation | University of Oxford |
Sector | Academic/University |
Country | United Kingdom |
Start | 09/2017 |
End | 09/2018 |
Description | RC |
Organisation | Marie Curie |
Department | Marie Curie Research Institute |
Country | United Kingdom |
Sector | Charity/Non Profit |
PI Contribution | Joint research on kinesin biophysics and kinesin-DNA hybrid devices |
Collaborator Contribution | Joint research on kinesin biophysics and kinesin-DNA hybrid devices |
Impact | See outcomes of grants BBG0191181 and EP/G037930/1 Multidisciplinary: physics, molecular and cellular biology |
Start Year | 2009 |
Description | RC |
Organisation | University of Warwick |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Joint research on kinesin biophysics and kinesin-DNA hybrid devices |
Collaborator Contribution | Joint research on kinesin biophysics and kinesin-DNA hybrid devices |
Impact | See outcomes of grants BBG0191181 and EP/G037930/1 Multidisciplinary: physics, molecular and cellular biology |
Start Year | 2009 |
Description | Cherwell 2014 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | Discussion with sixth formers - particularly interested in cross-disciplinary aspect of research described School has departmental contact to arrange future speakers |
Year(s) Of Engagement Activity | 2014 |
Description | Marston 2014 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | Questions about principles of self-assembly Talk to scout troop - enabled engagement with school-age children, many of whom would not normally come to a talk on science |
Year(s) Of Engagement Activity | 2014 |
Description | Oxfordshire Science Festival |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | Yes |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | Engagement and discussion with a very wide age range. Engagment with members of public with a very wide range of backgrounds and interests. |
Year(s) Of Engagement Activity | 2014 |
Description | Royal Society Summer Exhibition |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | Yes |
Geographic Reach | National |
Primary Audience | Schools |
Results and Impact | Lively interest from school children with a wide range of ages N/A |
Year(s) Of Engagement Activity | 2012 |
Description | WowHow |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | Yes |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | Lively engagement with children of both primary and secondary age Schools asked for teaching materials |
Year(s) Of Engagement Activity | 2012,2014 |