Projects

Coronavirus cell biology

Like many cell biology labs we have wanted to make a contribution to the effort to fight the global pandemic caused but SARS-CoV-2. To achieve this we are using our expertise of intracelluar trafficking to help elucidate the function of viral proteins and develop new ways to detect antibodies to the virus. This work is being performed in collaboration with a number of groups based around the world including the Krogen lab at UCSF, Edgar lab in Cambridge, Collins lab and de Silva labs based in Sheffield. We recently published a paper with the Krogen lab where we have localised every single viral protein encoded by SARS-CoV-1, 2 and MERS. In this study we also identified that Orf9B from the virus is localised to mitochondria where it co-locazies with TOM70. In infected intestinal cells the levels of TOM70 are down regulated sugesting that in these cells this may interefere with the inate immune system. You can watch a talk where I present some of these findings at the Faculty of Science seminar program.

SNAREs and constitutive secretion

Using functional genomics in combination with our novel assays for measuring constitutive secretion we have identified that SNAP29, STX19, VAMP3 and Ykt6 have a role in this process. STX19 is a really interesting SNARE as it does not have a conventional transmembrane domain but has a C-terminal cysteine rich domain which is S-acylated (palmitoylated). This domain targets STX19 to the plasma membrane and Rab8 positive tubular recycling endosomes. To gain an insight into how STX19 is regulated we have used Y2H screens and BioID based approaches to identify novel binding partners for this protein. We are currently exploring the physiological importance of these interactions.

We are also characterising the biology of VAMP4 in post-Golgi trafficking and membrane contact site formation. VAMP4 is an unusual R-SNARE as it has a N-terminal extension which contains a classical dileucine motif and has the ability to directly bind the AP-1 adaptor complex. Depletion of VAMP4 using RNAi leads to a defect in cholesterol esterification at the ER suggesting that VAMP4 may have a role either in the transport of machinery involved in cholesterol trafficking or the formation of membrane contact sites.

Physiological models of constitutive secretion

Plasma cells are the antibody secreting cells of the immune system and are cable of secreting thousands of antibody molecules per second. Using proteogenomics we have identified a large number of novel factors which are deferentially expressed between B-cells and antibody secreting plasma cells. We think that many of these new factors well be involved in helping plasma cell make and transport antibodies. Part of this work has recently been published in colaboration with Marion Espeli in PNAS. Our proteogenomic data is avaliable via this web reource. Parts of this project was originally started while I was still based in Cambridge and a lot of the intial work was perfomed by the PhS student Simon Gilbert.

In addition to this fundamental research we are also applying our knowledge of these process to develop novel tools and approaches to enhance antibody production in CHO cells. This work is currently being performed in collaboration with AZ and GSK.

Technology development

One area of my work which I have particularly enjoyed over the years is the development of new tools and technologies for measuring intracellular trafficking and protein function. In particular, we have developed a wide range of flow cytometry based assays for measuring endocytic trafficking and constitutive secretion. Our pharmacologically regulated secretory cells lines have been adopted by many groups throughout the world and have proven to be very useful for performing functional genomics. We are also developing new tools and assays for measuring the activity of Botulinum and Clostridial toxins with the aim to replace animal testing in the development of Botulinum and tetanus based vaccines and medicines.