Award details

Transporter, Creator, Destroyer: How is the kinesin motor domain tuned to specific functions?

Reference	BB/K006398/1
Principal Investigator / Supervisor	Dr Claire Therese Friel
Co-Investigators / Co-Supervisors
Institution	University of Nottingham
Department	Sch of Biomedical Sciences
Funding type	Research
Value (£)	448,560
Status	Completed
Type	Research Grant
Start date	17/06/2013
End date	16/06/2017
Duration	48 months

Abstract

Kinesins are crucial engines of eukaryotic self-organisation. Members of the kinesin superfamily interact with the microtubule cytoskeleton and play a vital role in transport of cellular cargo and in cell division. The aim of the proposed work is to understand structure-function relationships across the kinesin superfamily. The proposed experiments are designed to test the hypothesis that kinesin motor domains are built from 'mini-modules' that confer and tune properties required for emergent function, such as translocase, polymerase or depolymerase activity. Such modules may include those that provide properties such as microtubule lattice recognition and motor domain coordination, in the case of a translocase; or severing of inter- or intra-microtubule protofilament interactions and microtubule end recognition, for a depolymerase. The model for the study is the kinesin-13 family of microtubule depolymerases, specifically full-length human mitotic centromere-associated kinesin, MCAK. Variants designed based on available structural information plus sequence comparisons both within the kinesin-13 subfamily and between the larger kinesin superfamily, will be evaluated in terms of nucleotide turnover, using transient kinetic fluorescence spectroscopy, and in terms of microtubule binding and depolymerization kinetics, using conventional fluorescence and single-molecule TIRF microscopy. The goal is to discover a set of modules that specify kinesin motor domain function, such that behaviour can be switched between family members by rationally designed mutation. I expect to engineer new tools for cell biology and propose a collaboration to allow use of the MCAK variants produced in investigating the role of microtubule depolymerisation on kinetochore-driven force generation in mitosis. The output of this work is expected to have considerable impact on our understanding of the molecular mechanisms of motorized self-organization in eukaryotic systems.

Summary

A network of proteinacous filaments, called the cytoskeleton, is found within cells. The cytoskeleton, like the bone skeleton of the human body, provides cells with a scaffold allowing them to maintain the shape required for their function. Also, like our bone skeleton, the cytoskeleton provides a platform against which to generate forces within the cell. This works in a similar way to the way in which our muscles generate forces by pulling against our bones. Unlike our bone skeleton, the cytoskeleton is highly dynamic. The filaments that make up the cytoskeleton can be formed, broken down and reformed within seconds. The cytoskeleton comprises three different types of filament, one of which is the microtubule cytoskeleton. Microtubules, as the name suggests, are long tubes with a diameter of less than one ten-thousandth of a millimetre. Microtubules are constructed from a building block called 'tubulin'. The protein tubulin possesses the fascinating ability to spontaneously assemble, disassemble, and reassemble many times over. By adding a fluorescent dye label to the tubulin building blocks and using powerful microscopes, we can see the microtubules formed from tubulin and watch their assembly and disassembly. In this way, we can observe a process that is happening right now in our own cells, but in isolation so that we can observe it without any other parts of the cell around. This provides us with a wonderful opportunity to study and understand the dynamics of microtubule assembly and disassembly. Both the microtubules themselves and their ability to assemble and disassemble perform vital functions in our cells. Microtubules act as rails upon which proteins, called kinesins (from the word kinetic, indicating their ability to move) walk, allowing them to carry cargo from where it is made to where it is needed in the cell. The ability of microtubules to assemble and disassemble allows them to form structures required temporarily by cells, such as the apparatus required to separate the duplicate DNA when a cell divides. To create this apparatus, known as the mitotic spindle, the assembly and disassembly of microtubules must be carefully controlled. Therefore, the cell contains an array of proteins responsible for coordinating the building and destruction of microtubules. Amazingly, one protein that disassembles microtubules is a kinesin and has the same structure as kinesins that walk along microtubule rails. How structurally homologous proteins can carry out such different jobs is a fascinating question. The components of kinesins that allow them to carry out different functions are not fully understood. To answer this question, we will remove parts of the kinesin motor domain and also swap parts between the motor domains of different kinesins and watch the effect on their behaviour. This will allow us to identify the important pieces, how they work and how we can control them. Microtubules and kinesins are a vital part of the mechanisms that keep our cells, and therefore our bodies, in order and able to grow and function correctly. Perhaps their most important role is in controlling cell division. Failure of the mechanisms controlling cell division results in multiple human diseases, including cancer and developmental disorders. The more we can find out about the proteins involved in the control of cell division the more power we have to prevent and repair disruption of this process.

Impact Summary

Scientific Impact: I expect the work to provide an unprecedented ability to control and alter the function of kinesins and through this control their effect on the microtubule cytoskeleton and microtubule dynamics. The kinesin-microtubule system is a prime target for use in nanotechnological applications, for example in the construction of nanotransport systems. The findings of the proposed work could, therefore, be of great importance to researchers developing new bioscience based technologies. The proposed work is therefore of direct relevance to the BBSRC priority area of 'systems biology and bionanotechnology'. The kinesin superfamily of motor proteins is crucial to the healthy functioning of cells, primarily regarding intracellular transport and cell division. Microtubule depolymerising kinesins are involved in correction of inappropriate kinetochore-chromosome attachments during mitosis. The proposed work will provide new knowledge and a deeper understanding of the molecular mechanism of kinesins in general and microtubule depolymerising kinesins in particular. Furthermore, the cell biological tools created through this work will be used to deliver information on the precise role of microtubule depolymerising kinesins at the kinetochore during mitosis. This will provide a great opportunity to identify novel therapeutic targets to combat uncontrolled cell division and errors in chromosome segregation and as such has a direct relationship to the BBSRC priority areas of 'basic biosciences underpinning health' and 'Aging research: lifelong health and well-being'. Delivering highly skilled people: The proposed project will train a post-doctoral research assistant (PDRA) in cutting edge biophysical techniques. Therefore, the opportunity to work on this project will provide the PDRA with the broad set of skills required to succeed in the increasingly multidisciplinary environment of modern science. The training of the PDRA will be of benefit to the economy and general population by increasing the pool of highly skilled laboratory workers available for employment in the pharmaceutical and biotechnology industries. In addition, gaining transferable skills in presentation, supervision, training and analytical thinking will equip the researcher for a career in any sector of the economy. Pharmaceutical and Biotechnology industries: These industries are a vital part of the economy of the UK and currently employ approximately 150,000 people. In 2009, the UK pharmaceutical industry produced a £9 billion trade surplus. The process of drug discovery relies heavily on a strong, high quality foundation of basic-science research to provide insights into potential drug targets, as well as the development of new assays and technologies. The microtubule cytoskeleton and associated proteins play a vital role in cell division. In particular, microtubule depolymerising kinesins have a role in force generation during mitosis and also have an error correction function in repairing inappropriate kinetochore-microtubule attachments during chromosome segregation. Mis-segregation events due to incorrect kinetochore attachment during mitotic cell divisions leads to aneuploidy, a condition associated with cancer and developmental abnormalities. Indeed, the model depolymerising kinesin for the proposed work (MCAK) has been found to be overexpressed in gastric, breast and colorectal cancer cells. By increasing our knowledge of the individual molecules involved in the mitotic system, in particular those involved in error correction mechanisms, we may pave the way for new therapeutics.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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