Research group of Professor Peter Cullen

Research group of Professor Peter Cullen

From top left to right :- Dr Colin Traer - post doc, Dr Jacqueline Oakley - post doc, Chris Danson - Phd student, Prof. Pete Cullen, Dr Sam Yarwood - post doc, an Van Weering - post doc, Ian Mcgough - PhD student

Professor Peter Cullen is a Principal Investigator in the Department of Biochemistry at the University of Bristol, UK. Research in the Cullen Lab is focused on endocytosis, and in particular, a family of proteins called the sorting nexins (SNX’s).

The endosomal network of cells is organised into an array of intracellular, membranous compartments, distinguished by subtle variations in lipid composition, associated protein complexes and functionality. The fate of endocytic cargo is reliant on interactions with a number of molecularly distinct sorting complexes which tightly control the relationship between sorting of their respective cargo and the physical morphology of the endosomal network. In the Cullen Laboratory, researchers hope to further the understanding of how molecular mechanisms function to facilitate cargo sorting, how membrane geometry relates to cargo sorting, how the spatial organisation of the early endocytic network is achieved and the links this system has with disease.

Peter and his colleagues are interested in the family of sorting nexins (SNXs), which are characterised by the presence of a Phox Homology (PX) domain. This domain acts as a binding motif for specific phospho-inositides, membrane lipids which are constituents of all endocytic membranes and therefore localize SNXs to endosomal structures. SNXs have diverse roles and interact with accessory protein(s), such as cargo selective components and motor proteins, to contribute towards the highly selective and dynamic nature of endocytic membrane trafficking.

Chris Danson, a Research Assistant in the Cullen Laboratory, uses Volocity to measure the dynamics of the endosomal network. Chris says;

"Volocity provides a multitude of intuitive image analysis tools that enable us to extract statistically analysable data directly from images and movies, much of which will be included in our subsequent publications. Of particular interest is how SNXs relate to the dynamic nature of the endocytic network; below are movies, generated on a PerkinElmer spinning disk system, of cells expressing a fluorescently tagged SNX construct. This SNX localizes to a highly dynamic tubular – vesicular compartment.  By employing Volocity, we are able to generate data concerning the speed, directionality and spatial organisation of such transport vesicles using the tracking and distance measurement tools that are inherent to the software".

Movie A: SNXs associate with a highly dynamic tubular-vesicular endosomal compartment. Hela cells were transiently transfected with a GFP tagged SNX and imaged live following a 24 h incubation period. Frames were captured at half second intervals for 5 min. Cropped regions of the cell (movie B) demonstrate that vesicles positive for this SNX display a high level of motility indicative of transport steps. Movie C: The dynamic tubular structures emanating from vesicles are envisaged as cargo enrichment structures and exit sites from the early endosome.

Volocity Colocalization

Chris also uses Volocity to investigate colocalization. Determining the cellular localization of SNXs is a key preliminary step towards determining SNX function; we employ the co-localization features of Volocity to quantify the degree of co-localization between SNXs and markers of the endocytic system.  The software has the capacity to generate co-localization data via several different algorithms, each of which has its own particular benefits, for example the Pearson’s correlation has the ability to determine whether two proteins are in complex with one another (at least at the resolution of light microscopy).  The figure below displays images of a GFP tagged SNX co-stained with either an early endosomal marker or a late endosomal marker; using Volocity's co-localization software we have been able to confirm this SNX predominantly co-localizes with early endosomes.  We have been working closely with Volocity software engineers to develop co-localization software to further enhance its usability with respect to answering these types of scientific questions”.

low-level expression, GFP–SNX

Figure: At low-level expression, GFP–SNX is predominately associated with elements of the early endosome. HeLa cells were transiently transfected with GFP–SNX and fixed following a 24 h incubation period.  Fixed cells were co-immunostained for endogenous proteins representative of various trafficking compartments and the nuclear stain DAPi.  (A) GFP–SNX displays a high degree of co-localization with early endosome markers, in comparison to that of the late endosomal markers (B).  The scale bars represent 20 ?m. (C) The percentage of GFP–SNX enriched vesicles (n>300) co-localising with Alexa-568 labelled endosomal markers was digitally quantified from confocal images using Volocity.

To find out more visit the Cullen Lab.