Fusarin C is a mycotoxin that is produced by a variety of Fusarium species and is therefore a possible contaminant in food and feed. For this reason, a reliable high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the determination of fusarin C in food and feed samples was developed based on dispersive solid phase extraction (DSPE). This method has a limit of detection (LOD) of 2 μg/kg, a limit of quantitation (LOQ) of 7 μg/kg, and a recovery rate of 80%. Fifty different corn samples were analyzed, and fusarin C was detected in 40 of them. The fusarin C level varied in kernels of corn ears from not detectable up to 83 mg/kg and in food samples from not detectable up to 28 μg/kg. The co-occurrence of further structural analogues of fusarin C was confirmed by high-performance liquid chromatography Fourier transformation mass spectrometry (HPLC-FTMS). In addition, the stability of fusarin C under storage conditions was evaluated.

The assembly of respiratory complexes into macromolecular supercomplexes is currently a hot topic, especially in the context of newly available structural details. However, most work to date has been done with purified detergent-solubilized material and in situ confirmation is absent. We here set out to enable the recording of respiratory supercomplex formation in living cells. Fluorescent sensor proteins were placed at specific positions at cytochrome c oxidase suspected to either be at the surface of a CI1CIII2CIV1 supercomplex or buried within this supercomplex. In contrast to other loci, sensors at subunits CoxVIIIa and CoxVIIc reported a dense protein environment, as detected by significantly shortened fluorescence lifetimes. According to 3D modelling CoxVIIIa and CoxVIIc are buried in the CI1CIII2CIV1 supercomplex. Suppression of supercomplex scaffold proteins HIGD2A and CoxVIIa2l was accompanied by an increase in the lifetime of the CoxVIIIa-sensor in line with release of CIV from supercomplexes. Strikingly, our data provide strong evidence for defined stable supercomplex configuration in situ.

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The cell is metabolically highly compartmentalized. Especially, mitochondria host many vital reactions in their different microcompartments. However, due to their small size, these microcompartments are not accessible by conventional microscopy. Here, we demonstrate that time-correlated single-photon counting (TCSPC) fluorescence lifetime-imaging microscopy (FLIM) classifies not only mitochondria, but different microcompartments inside mitochondria. Sensor proteins in the matrix had a different lifetime than probes at membrane proteins. Localization in the outer and inner mitochondrial membrane could be distinguished by significant differences in the lifetime. The method was sensitive enough to monitor shifts in protein location within mitochondrial microcompartments. Macromolecular crowding induced by changes in the protein content significantly affected the lifetime, while oxidizing conditions or physiological pH changes had only marginal effects. We suggest that FLIM is a versatile and completive method to monitor spatiotemporal events in mitochondria. The sensitivity in the time domain allows for gaining substantial information about sub-mitochondrial localization overcoming diffraction limitation. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Stress granules are cytosolic, membraneless RNA-protein complexes that form in the cytosol in response to various stressors. Stress granules form through a process termed liquid-liquid phase separation, which increases the local concentration of RNA and protein within the granules, creates dynamic sorting stations for mRNAs and associated proteins, and modulates the availability of mRNA for protein translation. We introduce the concept that neuronal stress granules act as dynamic cytosolic microcompartments in which their components differentially cycle in and out, monitoring the cellular environment. We discuss that neuronal stress granules have distinctive features and contain substructures in which individual components interact transiently. We describe that neuronal stress granules modulate protein expression at multiple levels and affect the proteoform profile of the cytoskeletal protein tau. We argue that a better knowledge of the regulation of stress granule dynamics in neurons and the modulation of their material state is necessary to understand their function during physiological and pathological stress responses. Finally, we delineate approaches to determine the behavior and regulation of critical stress granule organizers and the physical state of stress granules in living neurons.

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