Abstract
Stable isotope labelling experiments are used routinely in metabolic flux analysis (MFA) to determine the metabolic phenotype of cells and tissues. A complication arises in multicellular systems because single cell measurements of transcriptomes, proteomes and metabolomes in multicellular organisms suggest that the metabolic phenotype will differ between cell types. In silico analysis of simulated metabolite isotopomer datasets shows that cellular heterogeneity confounds conventional MFA because labelling data averaged over multiple cell types does not necessarily yield averaged flux values. A potential solution to this problem—the use of cell-type specific reporter proteins as a source of cell-type specific labelling data—is proposed and the practicality of implementing this strategy in the roots of Arabidopsis thaliana seedlings is explored. A protocol for the immunopurification of ectopically expressed green fluorescent protein (GFP) from Arabidopsis thaliana seedlings using a GFP-binding nanobody is developed, and through GC-MS analysis of protein hydrolysates it is established that constitutively expressed GFP reports accurately on the labelling of total protein in root tissues. It is also demonstrated that the constitutive expression of GFP does not perturb metabolism. The principal obstacle to the implementation of the method in tissues with cell-type specific GFP expression is the sensitivity of the GC-MS system.
Highlights
The metabolic networks that underpin the functions of living cells support an array of intracellular metabolic fluxes that define a metabolic phenotype [1]
Not all of the amino acids could be detected reliably in both fractions, but the derived from immunopurified green fluorescent protein (GFP), the measured mass isotopomer distributions (MIDs) will only be useful for csMFA if (i) they for report amino acids for which reliable data could be obtained from the same fragments it was apparent accurately on the isotopomer distributions in the amino acid pools that are used for the synthesis of fromcellular the linear regression that the does reporter protein primary were indistinguishable total protein; and (ii) thethe useMIDs of thederived reporterfrom protein not perturb metabolism in from those derived from the total protein fraction
Cellular differentiation arises from changes in gene expression that confer specific functions on particular cell types
Summary
The metabolic networks that underpin the functions of living cells support an array of intracellular metabolic fluxes that define a metabolic phenotype [1]. A common feature in all these applications is that the labelling information required for MFA is determined after extraction of the sample, masking potential differences in metabolic phenotype between cell types. The reporter protein could be endogenously expressed, or it could be the result of targeting a potentially inducible transgene to the tissue of interest In support of the former suggestion, subcellular information on heterotrophic metabolism in plastids is routinely obtained from the starch in whole cell extracts [5], and differences in the labelling of the two subunits of rubisco have been interpreted in terms of the cytosolic and plastidic locations for their synthesis [33]. This procedure is used to establish that constitutively expressed GFP reports accurately on the labelling of total protein in root tissues and that constitutive expression of GFP does not perturb metabolism, thereby laying the foundations for the use of this reporter protein for csMFA in plants
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