Biology subjects

Herrmann, A.

Publications and source records attributed to Herrmann, A..

7 recordsLinked to original sources

bioRxiv2018

CDK inhibitors reduce cell proliferation and reverse hypoxia-induced metastasis of neuroblastoma tumours in a chick embryo model

Neuroblastoma is a paediatric cancer with a poor prognosis. This is in part due to the widespread metastasis at time of presentation, which is refractory to current treatment modalities. New therapeutic agents that can control not only tumour growth but also metastasis are urgently needed.\n\nOne current therapeutic option used in the clinic is differentiation therapy with retinoic acid, where the terminal differentiation of the neuroblastoma cells reduces tumour growth in the primary tumour as well as at metastatic sites. However, retinoic acid only works in a subset of patients.\n\nWe investigated the potential of CDK inhibitors on neuroblastoma cell differentiation, tumour progression and metastasis by utilising a 3R compliant cost effective preclinical chick embryo model. In both SK-N-AS and BE(2)C cell lines, when engrafted on the chorioallantoic membrane of chick embryos, we observed a reduction of tumour cell proliferation as well as a reduction in hypoxia preconditioning-driven metastasis by 60%. In addition, the expression of a panel of genes with known roles in metastasis, which increased upon hypoxia-preconditioning, was largely reduced by a CDK1 inhibitor. These results provide a promising alternative to currently existing therapies and might aid the development of new treatment protocols for retinoic acid-resistant patients.

cancer biology↗

bioRxiv2018

On the impact of competing intra- and intermolecular triplet-state quenching on photobleaching and photoswitching kinetics of organic fluorophores

While buffer cocktails remain the gold-standard for photostabilization and photoswitching of fluorescent markers, intramolecular triplet-state quenchers emerge as an alternative strategy to impart fluorophores with self-healing or even functional properties such as photoswitching. In this contribution, we evaluated various combinations of both approaches and show that inter- and intramolecular triplet-state quenching processes compete with each other rather than being additive or even synergistic. Often intramolecular processes dominate the photophysical situation for combinations of covalently-linked and solution-based photostabilizers and photoswitching agents. In this context we identified a new function of intramolecular photostabilizers, i.e., protection of fluorophores from reversible off-switching events caused by solution-additives, which were previously misinterpreted as photobleaching. Our studies also provide practical guidance for usage of photostabilizer-dye conjugates for STORM-type super-resolution microscopy permitting the exploitation of their improved photophysics for increased spatio-temporal resolution. Finally, we provide evidence that the biochemical environment, e.g., proximity of aromatic amino-acids such as tryptophan, reduces the photostabilization efficiency of commonly used buffer cocktails. Not only have our results important implications for a deeper mechanistic understanding of self-healing dyes, but they will provide a general framework to select label positions for optimal and reproducible photostability or photoswitching kinetics.

biophysics↗

bioRxiv2018

Structural determinants of the interactions between Influenza A Virus Matrix protein M1 and lipid membranes

Influenza A virus is a pathogen responsible for severe seasonal epidemics threatening human and animal populations every year. One of the ten proteins encoded by the viral genome, the matrix protein M1, is abundantly produced in infected cells and plays a structural role in determining the morphology of the virus. During assembly of new viral particles, M1 is recruited to the host cell membrane where it associates with lipids and other viral proteins. The structure of M1 is only partially known. In particular, structural details of M1 interactions with the cellular plasma membrane as well as M1-protein interactions and multimerization have not been clarified, yet.\n\nIn this work, we employed a set of complementary experimental and theoretical tools to tackle these issues. Using raster image correlation, surface plasmon resonance and circular dichroism spectroscopies, we quantified membrane association and oligomerization of full-length M1 and of different genetically engineered M1 constructs (i.e., N- and C-terminally truncated constructs and a mutant of the polybasic region, residues 95-105). Furthermore, we report novel information on structural changes in M1 occurring upon binding to membranes. Our experimental results are corroborated by an all-atom model of the full-length M1 protein bound to a negatively charged lipid bilayer.

biophysics↗

bioRxiv2018

Quantifying protein oligomerization in living cells: A systematic comparison of fluorescent proteins

Fluorescence fluctuation spectroscopy has become a popular toolbox for non-disruptive studies of molecular interactions and dynamics in living cells. The quantification of e.g. protein oligomerization and absolute concentrations in the native cellular environment is highly relevant for a detailed understanding of complex signaling pathways and biochemical reaction networks. A parameter of particular relevance in this context is the molecular brightness, which serves as a direct measure of oligomerization and can be easily extracted from temporal or spatial fluorescence fluctuations. However, fluorescent proteins (FPs) typically used in such studies suffer from complex photophysical transitions and limited maturation, potentially inducing non-fluorescent states, which strongly affect molecular brightness measurements. Although these processes have been occasionally reported, a comprehensive study addressing this issue is missing.\n\nHere, we investigate the suitability of commonly used FPs (i.e. mEGFP, mEYFP and mCherry), as well as novel red FPs (i.e. mCherry2, mRuby3, mCardinal, mScarlet and mScarlet-I) for the quantification of oligomerization based on the molecular brightness, as obtained by Fluorescence Correlation Spectroscopy (FCS) and Number&Brightness (N&B) measurements in living cells. For all FPs, we measured a lower than expected brightness of FP homo-dimers, allowing us to estimate, for each fluorescent label, the probability of fluorescence emission in a simple two-state model. By analyzing higher FP homo-oligomers and the Influenza A virus Hemagglutinin (HA) protein, we show that the oligomeric state of protein complexes can only be accurately quantified if this probability is taken into account. Further, we provide strong evidence that mCherry2, an mCherry variant, possesses a superior apparent fluorescence probability, presumably due to its fast maturation. We finally conclude that this property leads to an improved quantification in fluorescence cross-correlation spectroscopy measurements and propose to use mEGFP and mCherry2 as the novel standard pair for studying biomolecular hetero-interactions.

biophysics↗

bioRxiv2018

Roles of G1 cyclins in the temporal organization of yeast cell cycle - a transcriptome-wide analysis

Oscillating gene expression is crucial for correct timing and progression through cell cycle. In Saccharomyces cerevisiae, G1 cyclins Cln1-3 are essential drivers of the cell cycle and have an important role for temporal fine-tuning. We measured time-resolved transcriptome-wide gene expression for wild type and cyclin single and double knockouts over cell cycle with and without osmotic stress. Clustering of expression profiles, peak-time detection of oscillating genes, integration with transcription factor network dynamics, and assignment to cell cycle phases allowed us to quantify the effect of genetic or stress perturbations on the duration of cell cycle phases. Cln1 and Cln2 showed functional differences, especially affecting later phases. Deletion of Cln3 led to a delay of START followed by normal progression through later phases. Our data and network analysis suggest mutual effects of cyclins with the transcriptional regulators SBF and MBF.

systems biology↗

bioRxiv2017

Magnetic resonance imaging for characterization of a chick embryo model of cancer cell metastases

BackgroundMetastasis is the most common cause of death for cancer patients, hence its study has rapidly expanded over the past few years. To fully understand all the steps involved in metastatic dissemination, in vivo models are required, of which murine ones are the most common. Therefore pre-clinical imaging methods have mainly been developed for small mammals. However, the potential of preclinical imaging techniques such as magnetic resonance imaging (MRI) to monitor cancer growth and metastasis in non-mammalian in vivo models is not commonly used. We have here used MRI to measure primary neuroblastoma tumour size and presence of metastatic dissemination in a chick embryo model. We compared its sensitivity and accuracy to end-point fluorescence detection.\n\nMethodsHuman neuroblastoma cells were labelled with GFP and micron-sized iron particles (MPIOs) and implanted on the extraembryonic chorioallantoic membrane of the chick embryo at E7. T2 RARE, T2 weighted FLASH as well as time-of-flight MR angiography imaging was applied at E14. Primary tumours as well as metastatic deposits in the chick embryo were dissected post imaging to compare with MRI results.\n\nResultsMPIO labelling of neuroblastoma cells allowed in ovo observation of the primary tumour and tumour volume measurement non-invasively over time. Moreover, T2 weighted and FLASH imaging permitted the detection of very small metastatic deposits in the chick embryo.\n\nConclusionsThe use of contrast agents enabled the detection of metastatic deposits of neuroblastoma cells in a chick embryo model, thereby reinforcing the potential of this cost efficient and convenient, 3R compliant, in vivo model for cancer research.

cancer biology↗

bioRxiv2017

Relevance of host cell surface glycan structure for cell specificity of influenza A virus

Influenza A viruses (IAV) initiate infection via binding of the viral hemagglutinin (HA) to sialylated glycan receptors on host cells. HAs receptor specificity towards sialic acid (SA) is well studied and clearly critical for virus infection, but the contribution of the highly complex cellular plasma membrane to the cellular specificity remains elusive. In addition, some studies indicated that other host cell factors such as the epidermal growth factor receptor might contribute to the initial virus-cell contact and further downstream signaling1.\n\nHere we use two complementary methods, glycan arrays and single-virus force spectroscopy (SVFS) to compare influenza virus receptor specificity with actual host cell binding. Unexpectedly, our study reveals that HAs receptor binding preference does not necessarily reflect virus-cell specificity. We propose SVFS as a tool to elucidate the cell binding preference of IAV thereby including the complex environment of sialylated receptors within the plasma membrane of living cells.

microbiology↗