Elucidating the on/off switch for an essential mitotic motor

Lead Research Organisation: Birkbeck College
Department Name: Biological Sciences

Abstract

The purpose of this research is to discover how cell multiplication is controlled and how it might be turned on and off. In the same way as our bodies have a skeleton that provides us with support and strength, the cells of our bodies have a skeleton - called the cytoskeleton - which also provides support and strength. The cytoskeleton is involved in many important aspects of the life of the cell, including cell movement, architecture and multiplication. Studying the cytoskeleton is important both so we can understand how healthy cells work, but also to understand malfunctions of the cytoskeleton in disease. My research team studies the three-dimensional structure of the cytoskeleton, because knowing what the cytoskeleton looks like contributes to our understanding of how it works in the cell. We use a very powerful microscope to take pictures of individual cytoskeleton molecules and then use computers to combine these pictures and calculate their three-dimensional shape. Our current research focuses on a part of the cytoskeleton called microtubules. These are long cylindrical structures that act like tracks around the cell and along which the cell's transporter motors travel; these motors are called kinesins and there are lots of different types. In this project, we will be studying a kinesin type-5, which is very important for accurate cell multiplication. We want to know how kinesin-5s use cellular fuel to move along microtubules and how this activity is controlled. We have had some exciting recent results from our microscopy studies and we want to know more about how this tiny, essential motor works. Previous experiments, including our own research, have suggested that kinesin-5 motors have an on-off switch that tells them when to move, but we don't understand very well how this works. The aim of our new project is to understand this switch and what controls it. This is particularly important because we know that cancer results from out-of-control cell multiplication, so we also hope that by understanding how kinesin-5 switches itself on and off, we might be able to provide more insight into how cancer can be treated.

Technical Summary

The microtubule-based spindle orchestrates accurate chromosome segregation, facilitated by many microtubule-associated proteins. Kinesin-5 proteins - members of the ATP- and MT-dependent kinesin nanomotor superfamily - are essential for cell division in most eukaryotes. Kinesin-5s are involved in generation and maintenance of spindle bipolarity by microtubule cross-linking, and drugs that allosterically inhibit motor ATPase also block cell division. However, the structural mechanism of allosteric communication between the ATP-, MT- and inhibitor binding sites are not understood. Kinesin-5s have been identified as targets for novel cancer therapies, but elucidation of the kinesin-5 molecular mechanism is essential to the ongoing development of clinically effective anti-mitotic drugs. The goal of the proposed work is to elucidate the kinesin-5 ATP-dependent, force-generating mechanism and to understand how this can be switched on and off. Structures of the motor-MT interaction <10Å resolution provide essential information about motor mechanisms and we will use cryo-electron microscopy (EM) and image processing to reveal the kinesin-5-MT interaction and identify secondary structural elements in the kinesin motor engine. We will track the movement of the energy-transducing neck-linker element of the kinesin-5 motor domain by covalently attaching a gold label to visualise its nucleotide-dependent movements in cryo-EM reconstructions. We will investigate the importance of conformational flexibility within the kinesin-5 motor domain by artificially cross-linking the kinesin-5-specific, drug-binding loop to restrict its movement. We will also determine the role of the kinesin-5 tail domain in kinesin-5 regulation, investigate how it binds to microtubules and whether it interacts directly with the motor domain to regulate enzyme activity. Recombinantly expressed kinesin-5 domains will thus be characterised using biochemical, biophysical and structural techniques.

Planned Impact

There will be multiple beneficiaries from the proposed research outside the immediate academic community. The work described will lead to a greater understanding of essential mechanisms involved in cell division. Kinesin-5 motors have been identified as potential targets of chemotherapeutics and our research will aid the development of more effective drugs. Beneficiaries from this aspect of the research will include the biotechnology industry, as greater understanding from academic studies such as ours leads to more effective generation of improved drugs and, therefore, improved sales. We would aim to engage these beneficiaries immediately. In the longer term, improving drugs for cancer treatment will also benefit the millions of patients who battle this disease every year and improve their quality of life. Science and technology will lie at the heart of global economic recovery, and we will liase with Birkbeck College Business Relations Department to maximise the impact of our discoveries. We will aim to make the discoveries of our research available not only to the academic community, but also to the general public. I have a proven track-record of public communication of science, was the 2006 winner of the prestigious DeMontfort medal for science communication (SET for Britain) and have also attended the BBSRC Media Training Day. The appointed PDRA and I will undertake to design web-pages for my lab which are accessible for the general public and will seek to participate in other public understanding of science activities, for example by inviting sixth-form students to visit our lab and experience the day-to-day life of scientists. During the project period, I will arrange to visit my former school to inspire future scientists and will apply to become an 'Inspirational Woman' for Women Into Science, Engineering and construction (http://www.wisecampaign.org.uk/inspirational_women.cfm).

Publications

10 25 50
publication icon
Behnke-Parks WM (2011) Loop L5 acts as a conformational latch in the mitotic kinesin Eg5. in The Journal of biological chemistry

publication icon
Britto M (2016) Schizosaccharomyces pombe kinesin-5 switches direction using a steric blocking mechanism. in Proceedings of the National Academy of Sciences of the United States of America

publication icon
Britto M (2014) Cut7-Driven Microtubule Sliding Reverses Direction Depending on Motor Density in Biophysical Society Annual Meeting

publication icon
Goulet A (2013) New insights into the mechanism of force generation by kinesin-5 molecular motors. in International review of cell and molecular biology

publication icon
Goulet A (2012) The structural basis of force generation by the mitotic motor kinesin-5. in The Journal of biological chemistry

publication icon
Goulet A (2014) Comprehensive structural model of the mechanochemical cycle of a mitotic motor highlights molecular adaptations in the kinesin family. in Proceedings of the National Academy of Sciences of the United States of America

 
Description We have used cryo-electron microscopy (cryo-EM) to determine the microtubule-bound structures of human kinesin-5 motor domain at successive steps in its ATPase cycle, before and after ATP binding. In collaboration with Prof. Steven Rosenfeld (Cleveland Clinic, Ohio, USA), our sub-nanometre resolution reconstructions enable visualisation of coupled nucleotide-dependent conformational transitions of kinesin-5-specific structural elements. This reveals the structural basis of kinesin-5 force generation and the mechanism for their inhibition by small molecules.
Exploitation Route The purpose of this research was to uncover aspects of how cell multiplication is controlled and how it might be turned on and off. In the same way as our bodies have a skeleton that provides us with support and strength, the cells of our bodies have a skeleton - called the cytoskeleton - which also provides support and strength. The cytoskeleton is involved in many important aspects of the life of the cell, including cell movement, architecture and multiplication. Studying the cytoskeleton is important both so we can understand how healthy cells work, but also to understand malfunctions of the cytoskeleton in disease. My research team studies the three-dimensional structure of the cytoskeleton, because knowing what the cytoskeleton looks like contributes to our understanding of how it works in the cell.



In particular, this project has focused on the part of the cytoskeleton called microtubules. These are long cylindrical structures that act like tracks along which molecular transport motors carry cellular cargo. The motors that we have studied are called kinesins and there are many different types. In this project, we have been focusing on a kinesin type-5, which is very important for accurate cell replication. We wanted to know how kinesin-5s use cellular fuel to move along microtubules during cell replication and how this activity is controlled. Previous experiments, including our own research, have suggested that kinesin-5 motors have an on-off switch that tells them when to move, but we don't understand very well how this works. Therefore, the aim of our project was to understand this switch and what controls it. This is particularly important because

we know that cancer results from out-of-control cell multiplication, so we also hope that by understanding how kinesin-5 switches itself on and off, we might be able to provide more insight into how cancer can be treated. Our structural insights could be used in the design of more specific and effective kinesin-5 inhibitors for use in cancer chemotherapy.
Sectors Pharmaceuticals and Medical Biotechnology

URL http://www.bbk.ac.uk/biology/our-staff/academic/carolyn-moores/current-projects/kinesin5
 
Description Our findings can be used by other researchers working on the fundamental mechanisms of kinesin molecular motors. These findings can also be put to use by researchers investigating small molecules that specifically block mitotic kinesins as candidates for novel anti-mitotics.
First Year Of Impact 2010
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description BBSRC Project Grant
Amount £351,254 (GBP)
Funding ID BB/L00190X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2014 
End 02/2017
 
Description Structural studies of mitotic kinesins 
Organisation Mayo Clinic
Country United States 
Sector Charity/Non Profit 
PI Contribution Structural studies of mitotic kinesins using cryo-EM
Collaborator Contribution Sample preparation and expert knowledge
Impact Several publications together with our collaborator Prof Steve Rosenfeld
Start Year 2011
 
Description How to become a Group Leader/PI EMBO Meeting Youtube discussion 
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 Informal discussion with other PIs filmed by EMBO team and posted on Youtube

hard to measure
Year(s) Of Engagement Activity 2013
URL http://www.youtube.com/watch?v=7zSNTlzbkqc&feature=youtu.be
 
Description Under the Microscope: Kinesin Motors and Cancer (Birkbeck Science Week) 
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 Presentation as part of Birkbeck Science Week, 2012 - ~30 audience members with plenty of insightful questions after

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
URL http://blogs.bbk.ac.uk/events/2012/04/11/under-the-microscope-kinesin-motors-and-cancer/