Single-cell metabolomics at the single-molecule level

Samenvatting

The metabolic state can identify cancerous cells and reveal hidden heterogeneities of seemingly identical cells. Conventional methods obfuscate such cellular heterogeneity by averaging many cells. Recently, there has been a great interest to develop new analytical tools for single-cell measurements. However, single-cell mass-spectrometry and fluorescence-based methods are either destructive or can only detect few metabolites simultaneously.

I propose to combine two technologies, nanopore-based single-molecule detection of metabolites and patch-clamp of mammalian cells, to develop a system to measure intracellular metabolites in living cells at the single-cell level.

I will achieve this objective in three steps:

1) A single cell will be patch-clamped and a Cytolysin A (ClyA) nanopore will be inserted into the cellular membrane. Substrate-binding proteins (SBPs), dwelling inside ClyA, will allow the measurement of cytosolic metabolites which can diffuse from the cell interior into the nanopore. Binding and unbinding of a metabolite to SBPs elicits an ionic current signal that will allow quantification of the metabolite. Because of the single-molecule nature of the system, a wide range of metabolite concentrations can be measured as shown by preliminary data gained from planar lipid bilayer experiments.

2) I will extend the number of SBPs to test a wider range of metabolites. Hundreds of SBPs are reported in the literature with a high structural similarity but high affinities to different molecules.

3) As the system is amenable for parallelization, I will assess intracellular metabolites such as glutathione in myeloma cells whose sensitivity to bortezomib has been suggested to depend on glutathione with a high-throughput (384 cells) system.

The system will allow assessing cell-to-cell heterogeneity and single-cell fluctuations with temporal resolutions of seconds, contributing to our understanding of the inner workings of a cell. It will also have wide reaching applications in intracellular pharmacokinetics as well as ex vivo sensing applications.

Kenmerken

Projectnummer

722.017.010

Hoofdaanvrager

Dr. C. Wloka-Tjalsma

Verbonden aan

Rijksuniversiteit Groningen, Faculty of Science and Engineering (FSE), Groningen Biomolecular Sciences and Biotechnology Institute (GBB)

Looptijd

01/10/2017 tot 30/09/2020