3D structure of dynein motor domain and sub-domain mapping by cryo-electron microscopy
Lead Research Organisation:
University of Leeds
Department Name: Inst of Molecular & Cellular Biology
Abstract
Dynein is a molecular motor that moves along molecular tracks called microtubules inside cells. Its function is to deliver cargos to their correct locations within the cell and to help control the pulling apart of chromosomes during cell division. A set of highly-related molecular motors are also found in long, whip-like structures that protrude from many cell types, including those lining the airways and the Fallopian tubes. Sperm cells carry one of these structures in their tails. Here, coordinated dynein motor activity causes repeated bending of these structures which the cells use either to propel fluids over their surfaces, for example, to remove debris from the lungs or to move the unfertilized egg towards the uterus, or to propel a cell, for example, a sperm cell, through its environment. Dyneins from both sources are thought to move along microtubules in a similar way, but probably very differently to other types of molecular motors in cells. Dynein is ten times bigger than an unrelated molecular motor called kinesin. This is surprising since kinesin moves along the same microtubule track as dynein. The structure of dynein is not known in detail. Consequently, its mechanism of force generation is not known. Some clues have come from electron microscope images of individual molecules showing a large change in shape of the motor, before and after it has bound and split ATP, the fuel it uses to move. One of these shapes shows the motor before it exerts force, the other after force has been exerted, providing clues about the moving parts. However, images have only shown the shape-change in two-dimensions. In the proposed research, we will obtain the three-dimensional structure of the dynein motor. Differences between the two conformations will show the moving parts and reveal dynein's mechanism. We will also obtain structures of dynein modified to carry probes, visible by electron microscopy, inserted at specific sites within the protein sequence of the molecule. These will provide information on how the protein folds up to form the motor and how the positions of these probes move during force production.
Technical Summary
Cytoplasmic dynein is a minus-end directed microtubule motor essential in many cellular trafficking events. The considerable size of its motor domain (380 kDa) and its origin within the AAA+ superfamily, distinguish it from the two other classes of linear motor proteins myosin and kinesin, suggesting its mechanism of force generation is novel. However, no atomic resolution structures have been obtained of dynein's motor domain, nor of any of its sub-fragments, so its mechanism remains mysterious. Here we propose to determine the structure and mechanism of a recombinant cytoplasmic dynein motor domain. Using cryo-electron microscopy and single-particle image processing we will determine both pre- and post-power stroke conformations of the motor. Using a series of fusion proteins labeled with green and blue fluorescent proteins, we will map the positions of several of its AAA+ domains within the motor domain, as well as the position of the N- and C-termini.
Organisations
People |
ORCID iD |
Stanley Alan Burgess (Principal Investigator) |
Publications
Roberts AJ
(2009)
AAA+ Ring and linker swing mechanism in the dynein motor.
in Cell
Roberts AJ
(2009)
Electron microscopic imaging and analysis of isolated dynein particles.
in Methods in cell biology
Roberts AJ
(2013)
Functions and mechanics of dynein motor proteins.
in Nature reviews. Molecular cell biology
Kon T
(2009)
Helix sliding in the stalk coiled coil of dynein couples ATPase and microtubule binding.
in Nature structural & molecular biology
Roberts AJ
(2012)
ATP-driven remodeling of the linker domain in the dynein motor.
in Structure (London, England : 1993)
Description | Structure and mechanism of cytoplasmic dynein |
Amount | £800,000 (GBP) |
Funding ID | RGP0009/2008-C |
Organisation | Human Frontier Science Program (HFSP) |
Sector | Charity/Non Profit |
Country | France |
Start | 09/2008 |
End | 09/2011 |
Description | Transport to the centre of the cell: discovering dynein's stepping mechanism by cryo-electron microscopy |
Amount | £497,727 (GBP) |
Funding ID | BB/K000705/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2012 |
End | 09/2015 |
Description | Discovery Zone |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | Yes |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | unaware of any none to report |
Year(s) Of Engagement Activity | 2008 |
Description | Maya workshop |
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 | learned to use Maya- for molecular animation taught school teacher skills in molecular Maya for his education of students acquired skills in molecular animation for my own lab |
Year(s) Of Engagement Activity | 2010 |
URL | http://www.molecularmovies.com/toolkit/ |
Description | Scientific Advisor |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | Yes |
Geographic Reach | International |
Primary Audience | Media (as a channel to the public) |
Results and Impact | led to TV film on molecular biology "Journey to the Inside" broadcast in the US and Eurpoe none |
Year(s) Of Engagement Activity | 2009 |
Description | Seminar Scripps |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | Yes |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | talk sparked questions and discussion no direct impacts to be reported |
Year(s) Of Engagement Activity | 2009 |
Description | seminar Edinburgh |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | Yes |
Geographic Reach | Regional |
Primary Audience | Professional Practitioners |
Results and Impact | sparked discussion none to report |
Year(s) Of Engagement Activity | 2009 |
Description | seminar London |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | Yes |
Geographic Reach | Regional |
Primary Audience | Professional Practitioners |
Results and Impact | talk sparked questions and discussion afterwards none to report |
Year(s) Of Engagement Activity | 2009 |
Description | seminar Warwick |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | Yes |
Geographic Reach | Regional |
Primary Audience | Professional Practitioners |
Results and Impact | talk sparked questions and discussion none |
Year(s) Of Engagement Activity | 2010 |