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

10 25 50
 
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 09/2014 
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 09/2013 
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 04/2014 
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 04/2012 
End 03/2017
 
Description MRC Confidence in Concept
Amount £22,506 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 08/2017 
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 04/2012 
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 10/2013 
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 10/2017 
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