Lab methods and techniques

Developing new methods to study lysosomal function

1. Measuring lysosomal ion (e.g. Ca2+/Zn2+/H+) homeostasis

We are perhaps best known for our development of methods for measuring and monitoring lysosomal ions in situ. Dr. Lloyd-Evans was only the second researcher in the world to quantify lysosomal Ca2+ levels in situ within cells and was the first to associate changes in lysosomal Ca2+ to a human disease (Niemann-Pick type C; Lloyd-Evans et al, Nature Medicine 2008). Since then we have identified changes in lysosomal Ca2+ levels in other lysosomal diseases such as CLN3 (the first disease of elevated lysosomal Ca2+, Chandrachud et al, JBC 2015) and changes in lysosomal Ca2+ signalling in familial Alzheimer’s disease (Lee et al, Cell Reports 2015). More recently we have been looking at Ca2+ homeostasis in vivo in in the brains of zebrafish models of neurodegenerative diseases using lightsheet microscopy.

2. Purification of lysosomes using superparamagnetic iron oxide nanoparticles (SPIONs)

Building on the previous work of the Winchester and Krise labs we have utilised superparamagnetic ferrofluid (Liquids Research Ltd) technology to purify lysosomes. These SPIONs are functonalised by coating in high molecular weight dextran which also protects the particle from degradation within the acidic lumen of the lysosome. Cells are cultured in medium containing these SPIONs which are endocytosed to lysosomes that are subsequently purified by high powered magnetic separation. These lysosomes are ultra-pure, highly functional and can be obtained at high yields. Our method is used by multiple labs worldwide and has been published in Methods in Cell Biology and Cell Reports. We are currently working on a manuscript comparig the cellular toxicity of different SPIONs.

3. Purification, cloning and characterisation of lysenin toxin from European Eisenia foetida

We have cloned and purified the lysenin toxin from the European earthworm (Eisenia foetida). This is extremely similar to the commercially available Japanese lysenin toxin (~95% identical) with a slightly wider sphingomyelin binding region (panel A in the figure on the right). Our lysenin toxin has been engineered to include a His tag for fluorescent labelling and it compares immensely favouribly with the commercial toxin when staining sphingomyelin in fixed cells. As can be seen in figure B on the right our toxin picks up the elevated lysosomal sphngomyelin storage in Niemann-Pick C (NPC) disease cells and wild-type (WT) cells treated with the cholesterol transport ihibitor U18666a which induces an NPC disease like phenotype.

4. Thin layer chromatography for separation of all lipids present in NPC and several other lysosomal storage diseases

Based on an earlier published method (Maue et al, HMG 2012) we have adpated this TLC method for a rapid, sensitive and cost effective method for the separation of the majority of lipid species that accumulate in Niemann-Pick C disease (sphingosine, ceramide, sphingomyelin, glucosylceramide and certain gangliosides). In addition, the TLC separates cholesterol, bis(monoacyl)glycerophosphate (LBPA) and certain phospholipids. Utilising this approach we have been able to separate lipids from starting material as low as 0.5mg, allowing the determination of lipid species in patient drived fibroblasts.

5. Generation of zebrafish models (pharmacological and genetic) of lysosomal storage diseases

One of our emerging interests is the use of zebrafish to model human neurodegenerative disease. Zebrafish share ~70-80% genetic identity to humans and due to their rapid development they have an advanced brain within days after fertilisation. Added benefits include, they are cheap to keep, they produce large numbers of fertilised eggs and their optical transparency when young allows for complex microscopic analysis that would be extremely difficult in other organisms such as mice. As model organisms for studying disease they also come into their own for high throughput in vivo drug screening. We are utilising zebrafish to help us better understand the causes of childhood neurological disease and to develop new therapies for these devastating illnesses.