The lab is interested in cell biology at the molecular level. We work on two processes that are fundamental to all eukaryotic cells: mitosis and endocytosis. Understanding these processes will give us insight into human diseases and conditions such as cancer, viral infection and birth defects. Use the tabs above to find out more about our research.
Mitotic spindle stability
How are kinetochore fibres stabilised?
Kinetochore fibres transduce large forces to move chromosomes in the cell. Cells have evolved mechanisms to prevent the fibres from buckling and breaking.
Kinetochore fibres (K-fibres) of the mitotic spindle connect the spindle pole to the kinetochore and allow chromosome movement. These fibres are bundles of microtubules that are tethered together by inter-microtubule bridges. We are investigating non-motor proteins of the mitotic spindle and the contribution they make to kinetochore fibre stability by forming bridges between adjacent microtubules.
In collaboration with Ian Prior we have developed a correlated light-electron microscopy (CLEM) approach to examine bridges within kinetochore fibres. We have found that a subset of these bridges are composed of TACC3/ch-TOG/clathrin (Booth et al., 2011). Using 3D EM we found that the bridges are actually part of a "mesh", a large network of microtubule connectors (Nixon et al., 2015). We are investigating which proteins make up the other connectors.
TACC3 ch-TOG and clathrin
Clathrin is a protein involved in membrane trafficking in interphase cells. When the cell enters mitosis, membrane trafficking is shut-down and clathrin localises to the mitotic spindle.
We discovered a novel role for clathrin in mitotic spindle function (Royle et al., 2005). Clathrin, together with at least two other spindle proteins (TACC3 and ch-TOG), stabilises kinetochore fibres (K-fibres). We think this works by the complex acting as an inter-microtubule bridge (Booth, et al., 2011). Our lab have developed methods to rapidly remove the complex to understand the role of these bridges in kinetochore fibre function (Cheeseman et al., 2013).
Regulation of endocytosis during mitosis
We are trying to unravel the mechanism by which endocytosis is switched off by cells during mitosis. We are interested in this process as many trafficking proteins have alternative functions during mitosis. A process called Moonlighting.
We have known for half a century that cells suppress endocytosis during mitosis. The reason why cells do this and the mechanisms they use to do it are obscure. Endocytosis is switched off at the level of invagination of the coated pit. We are using proteomics to understand which clathrin-coated vesicle proteins are involved in the shutdown of endocytosis.
We are also interested in the changes in the clathrin-coated vesicle proteome during mitosis as these proteins may have other roles during mitosis, a process called moonlighting (Royle, 2011).
Synaptic vesicle endocytosis
The molecular mechanisms of clathrin-mediated endocytosis are under investigation in the lab. Particularly, we are interested in clathrin-adaptor interactions. We do this work in non-neuronal cells, but also by measuring endocytosis at neuronal synapses
Clathrin cannot bind to membrane and cargo without using adaptor proteins. We are working on how clathrin binds the adaptors and which adaptors are used at synapses of hippocampal neurons.
We showed that in non-neuronal cells the clathrin AP-2 interaction occurs via four interaction sites in the N-terminal domain of clathrin (Willox & Royle, 2012a). We also carried out a screen of adaptor protein candidates in neurons and found that stonin 2 is a major adaptor for synaptic vesicle retrieval (Willox & Royle, 2012b).
We are housed in the Mechanochemical Cell Biology Building. This is a new purpose-built facility which is home to a number of other labs interested in related questions and using similar methodology. In addition to excellent imaging facilities in the building some equipment is specific to our lab.
Nikon widefield microscope
This Ti-U microscope has a range of objectives (100x, 60x, 40x, 20x, 40x ELWD) and fluorescence cubes for imaging fluorescent proteins. Images are captured using NIS Elements AR. It also has excellent DIC.
The picture is out-of-date as this scope has been upgraded to give z-motorisation and an environmental chamber.
Olympus widefield microscope for pHluorin imaging
This IX71 microscope is set up for imaging pHluorins (pH-sensitive GFPs). It has 60x and 40x oil objectives and fluorescence cubes for specific for GFP and mCherry. It has an Hamamatsu ORCA ER camera.
Stimulation of neurons is via a Grass electrical stimulator. The computer syncs image acquisition and stimulation. Fast perfusion via a Warner solution switcher is also installed.
Light sheet microscope (diSPIM)
This microscope from ASI/3i uses two orthogonal light sheets for imaging of thick specimens. This microscope was bought with an award from BBSRC ALERT14 round.
The HPM100 is our high-pressure freezer for electron microscopy. This system has the advantage of freezing relatively large samples. It also has the capability to do CLEM.
Following freezing we typically do freeze substitution in our ASF2.
This is our Leica UC7 microtome for cutting ultra-thin sections from resin blocks for EM. Typical sections are between 60-120 nm thick.
We also have a benchtop sputter coater, knife maker and other apparatus for EM sample prep.
We are working on two things that cells do: how they divide and how they 'eat' things from the outside. Dividing and eating are as important for cells as for people! So any problems with these jobs can lead to disease. This is why we are trying to understand these jobs, so that we can think of new ways to treat human diseases.
Our work on mitosis
When a cell divides, each of the two new cells must get one copy of the genome each. The cell makes a machine called the mitotic spindle to share the chromosomes (genome) equally between the two cells. This is a very important task: cells that have too few or too many chromosomes can become cancerous. Our lab is trying to figure out how the mitotic spindle does this so efficiently. There are certain fibres within the spindle that pull chromosomes around the cell to share them out. These fibres are made up of many smaller tubules that are held together by "bridges". We are using powerful microscopes to study these bridges and find out what they are made of.
So far, we have found that some of these bridges are made by at least three different proteins. Two of these proteins are overexpressed in cancer. This means that in patients with these tumours, the cancer cells are making too much of these two proteins. We are figuring out how this might cause the tumour, or how it might make the cancer get worse.
Our work on membrane trafficking
Cells can be thought of as islands – they are closed to the outside world because they have a plasma membrane that doesn't let anything through. However, cells have evolved tiny portals for things to enter in a highly controlled way. These portals are working all the time, but they are shut down when the cell needs to divide. It is important that no mistakes are made at this time. Otherwise the cell could die or it could start to grow in an unusual way leading to disease e.g. cancer. For many years nobody knew how this shutdown worked, but our lab have found out how it happens.
The same portals are used by brain cells for recycling! Nerve cells in the brain talk to each other by releasing small packets of chemicals. Once the chemical has been released, the packet needs to be retrieved so it can be refilled and released again. This is just like recycling of milk bottles. Our lab is trying to understand how cells do the retrieval part of the recycling programme.
We have a blog called quantixed. Part of this blog aims to describe the papers that we publish in simple terms that can be understood by non-specialists. For example this post. These posts are tagged with outreach. Feel free to take a look.
We are very grateful for the generous support from the following sources.
Cancer Research UK | Steve is a Senior Research Fellow for Cancer Research UK (2013-2019). Before this the lab was funded by a Career Establishment Award from Cancer Research UK (2007-2012).
Biotechnology and Biological Sciences Research Council | Funded a project grant in the lab to find new moonlighting proteins and understand the regulation of endocytosis by the cell cycle. Also, funded an ALERT14 equipment grant to purchase equipment for correlative light-electron microscopy experiments.
North West Cancer Research | NWCR fund a project grant to analyse the material connecting kinetochore fibre microtubules in the spindle. This project is in collaboration with Ian Prior in Liverpool.
The Wellcome Trust | The Wellcome Trust have supported the lab in the past with a project grant to look at synaptic vesicle recycling and with PhD studentships and summer studentships.
If you'd like to request any of our published reagents, please check the lab addgene page
in the first instance.
If the plasmid you're looking for is not there, feel free to get in touch