Research groups
Paul Verkade
Senior Lecturer
+44 (0)117 33 12179 (tel)
+44 (0)117 33 12168 (fax)
p.verkade@bristol.ac.uk
Research
Studying Fusion and Segregation Events during Intracellular Transport
Our research focuses on understanding and visualising intracellular transport processes, both biosynthetic as well as endocytic. Cells have to make new proteins and transport these to the correct places for proper functioning of the cell. Likewise they have to do this for signals (proteins) from the outside environment. Cells have to interpret these signals and then route them correctly. What carriers are being used to convey these signals? Where and how do they meet or segregate?, and How do they concentrate cargo? These are the questions we are trying to solve.
We mainly do this by combining light microscopy with electron microscopy. In these so-called Correlative Light Electron Microscopy (CLEM) experiments (Figure A) we make use of the advantages of doing live cell imaging at the light microscopy level (i.e. time resolution, the history of a structure) and using the high spatial resolution of the electron microscope (Brown et al., 2009). In this way we can capture specific segregation and fusion events taking place inside the cell (Verkade, 2008). In order to visualise such events we are using 3-dimensional EM Tomography (Figure B). Once we have established the “normal” situation we can start to introduce mutant proteins that we think are involved in the process.
Examples from a CLEM Experiment: A. Overlay of a fluorescence image onto an EM image. Epidermal Growth Factor (EGF) is labeled with a fluorescent (green) and electron dense label and is internalised into the cell. We can follow the internalisation into the cell using live cell imaging. Once we have captured a particular event we can analyse that at high resolution in 3 dimensions using electron tomography and draw a model (B, not from the same sample as A!).
The major project that is currently ongoing is to study the segregation
of recycling and degradative cargo in the endocytic pathway. It is known
that in the early endosome cargo destined for degradation (for instance
Epidermal Growth Factor, EGF) is segregated from cargo that is recycled
back to the plasma membrane (Transferrin, TF). We have marked both EGF and
TF with markers that are both visible in the light microscope (fluorescent)
and the electron microscope (gold). We first follow the cargo live in the
light microscope. When we see an interesting event happening we rapidly
transfer the sample into a high-pressure freezer (Verkade, 2008) and process
it for electron microscopy. We trace back that same structure and can study
that structure at high resolution in 3 dimensions. In this way we can clearly
state if a structure is connected or not and we know its origin (fusing
or segregating).
Group
Edward Brown, Judith Mantell.
Recent publications
Brown, E., J. Mantell, D.A. Carter, G. Tilly, and P. Verkade (2009). Studying intracellular transport using High-Pressure Freezing and Correlative Light Electron Microscopy. Seminars in Cell and developmental Biology. doi: 10.1016/j.semcdb.2009.07.006.
Hughes, H., A. Budnik, K. Schmidt, K. Palmer, J. Mantell, C. Noakes, A. Johnson, D.A. Carter, P. Verkade, P. Watson, and D.J. Stephens (2009). Organisation of human ER-exit sites: requirements for the localisation of Sec16 to transitional ER. Journal of Cell Science, 122: 2924-2934.
Wassmer, T., N. Attar, M. Harterink, J.R.T. van Weering, C.J. Traer, J. Oakley, B. Goud, D.J. Stephens, P. Verkade, H.C. Korswagen, and P.J. Cullen (2009). The retromer coat complex coordinates endosomal sorting and dynein-mediated transport, with carrier recognition by the trans-Golgi network. Developmental Cell, 17: 110-122.
P. Verkade (2008). Moving EM: The Rapid Transfer System as a New Tool for Correlative Light and Electron Microscopy and High Throughput for High-Pressure Freezing. Journal of Microscopy. 230: 317-328.