Extracellular signals need to be processed in a correct manner for a cell to function properly. In many cases the signals are internalised via endocytosis and then processed in a specific manner, e.g. by sorting it to a specific destination. My lab not only tries to understand these basic sorting mechanisms but also tries to exploit our understanding of these routes to deliver cargo to specific destinations in the cell.

Our research thus focuses on the visualisation and perturbation of endocytic events both at the light and electron microscopical level by Correlative Light Electron Microscopy (CLEM) and 3-dimensional electron tomography (see Figure and Brown et al., 2012).

Figure: Internalisation of Epidermal Growth Factor (EGF) coupled to a green fluorophore and a gold particle and Transferrin (Tf) coupled to a red fluorophore and 10 nm gold. Using live light microscopy we can follow the fluorophores moving live inside the cell (A, time in seconds). At timepoint 0 both marker segregate and when we trace back the same cell in the electron microscope (B), we can visualise this segregation event in more detail at higher resolution (C, red arrows point to Tf particles and red arrows towards EGF.

In order to be able to perform such experiments we have to develop software and hardware tools suited for the job. As such, our research is of an interdisciplinary nature and we collaborate a lot with chemists and engineers to develop such tools.

Below are some of the topics that we are currently working on:

1. To study the very early steps of endocytosis we are using Total Internal Reflection Fluorescence (TIRF) and aim to combine those with our CLEM approach.
2. The formation of tubular extensions from endosomes and lysosomes (Brown et al., 2012).
3. Development of CLEM probes that are both fluorescent and electron dense for use in intracellular transport studies (Hodgson and Verkade, 2014). In collaboration with Prof. Dek Woolfson, Chemistry, UoB.
4. Targeting and delivery of molecules to specific destinations in cells, either through tagging molecules (Benito et al., 2014) or encapsulating them (Fletcher et al., 2013). This work is done in collaboration with Carmen Galan and Dek Woolfson in Chemistry.
5. Retracing and overlay in light and electron microscopy images. Work done in collaboration with Alin Achim in Engineering (Nam et al., 2014).

References:

- D. Nam, J. Mantell, D. Bull, P. Verkade^*, and A. Achim^*. (2014). A Novel Framework for Segmentation of Secretory Granules in Electron Micrographs. Med Image Anal. 18:411-424.

- Hodgson L, J. Tavaré, and P. Verkade. (2014). Development of a quantitative Correlative Light Electron Microscopy technique to study GLUT4 trafficking. Protoplasma. DOI 10.1007/s00709-013-0597-5.

- Benito-Alifonso D, S. Tremel, B. Hou, H. Lockyear, J. Mantell, D.J. Fermin, P. Verkade, M. Berry, and M.C. Galan. (2014). Lactose as a "trojan horse" for quantum dot cell transport.Angew Chem Int Ed Engl. 53:810-814.

- Fletcher, J., R. Harniman, F. Barnes, A. Boyle, A. Collins, J. Mantell, T. Sharp, M. Antognozzi, P. Booth, N. Linden, M. Miles, R. Sessions, P. Verkade and D. Woolfson(2013). Self-assembling cages from coiled-coil peptide modules. Science, 340: 595-599.

- Brown, E., J., Van Weering, T. Sharp, J. Mantell, and P. Verkade (2012). Capturing endocytic segregation events with HPF-CLEM. Methods in Cell Biology, Volume 111: Correlative Light and Electron Microscopy, 175-201.