Monitoring of intracellular and extracellular toxin/bioactive peptide production under physiological stress conditions through labeling by chemical modification.
Start Date
24-5-2022 2:30 PM
End Date
24-5-2022 2:45 PM
Abstract
CyanoHAB forming cyanobacteria Microcystis and Planktothrix produce high intracellular amounts of cyanotoxins and other bioactive peptides such as microcystins (MCs) and anabaenopeptins (APs). Often the production of cyanopeptides has been related to physiological stress conditions, for example induced by low light or nutrient limitation. Both MC and AP peptides are produced via Non-Ribosomal Peptide Synthesis (NRPS). Natural mutations in adenylation domains can lead to an unspecific amino acid incorporation which can be exploited to incorporate non-natural amino acids (AA) carrying azide or alkyne moieties producing chemically modified MC/AP molecules. Modified MC/AP molecules are subsequently labeled via copper-catalyzed azide-alkyne cycloaddition (CuACC) using various fluorophores. This labeling technique has been established previously, showing a rather specific increase in fluorophore signal. High resolution imaging is subsequently used to localize and track modified MC/AP-molecules. The CuAAC based labeling which is considered as a rather specific chemical reaction is applied in parallel with conventional staining techniques of intracellular structures or other organelles. Thus by analyzing the spatial distribution of MC/AP in relation to known intracellular structures such as lipids or other organelles we hope to better understand the production, storage and release of MC/AP under the above mentioned physiological stress conditions.
Monitoring of intracellular and extracellular toxin/bioactive peptide production under physiological stress conditions through labeling by chemical modification.
CyanoHAB forming cyanobacteria Microcystis and Planktothrix produce high intracellular amounts of cyanotoxins and other bioactive peptides such as microcystins (MCs) and anabaenopeptins (APs). Often the production of cyanopeptides has been related to physiological stress conditions, for example induced by low light or nutrient limitation. Both MC and AP peptides are produced via Non-Ribosomal Peptide Synthesis (NRPS). Natural mutations in adenylation domains can lead to an unspecific amino acid incorporation which can be exploited to incorporate non-natural amino acids (AA) carrying azide or alkyne moieties producing chemically modified MC/AP molecules. Modified MC/AP molecules are subsequently labeled via copper-catalyzed azide-alkyne cycloaddition (CuACC) using various fluorophores. This labeling technique has been established previously, showing a rather specific increase in fluorophore signal. High resolution imaging is subsequently used to localize and track modified MC/AP-molecules. The CuAAC based labeling which is considered as a rather specific chemical reaction is applied in parallel with conventional staining techniques of intracellular structures or other organelles. Thus by analyzing the spatial distribution of MC/AP in relation to known intracellular structures such as lipids or other organelles we hope to better understand the production, storage and release of MC/AP under the above mentioned physiological stress conditions.