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.

Publications

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Roberts AJ (2013) Functions and mechanics of dynein motor proteins. in Nature reviews. Molecular cell biology

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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 10/2008 
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 10/2012 
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 dynein homepage 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Hosting of the "Dynein Homepage" a resource for all those in the field and the general public for updates on the state of the art of dynein research

none to report
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011,2012,2013,2014,2015
URL http://www.dynein.org/
 
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