Computer aided interactive gene network simulations including stochastic molecular kinetics and noise
Johannes Hettich and Christof M. Gebhardt
Recent next generation sequencing and single molecule methodologies provide functional insights into gene regulatory networks beyond Boolean interactions, including the number, binding modes and kinetic rates of transcription factors and the kinetics of gene bursting. We report CaiNet, a fast computer aided interactive network simulation environment to set up and simulate arbitrary gene regulatory networks. CaiNet automatically compiles a network laid out in a graphical user interface into a fast hybrid stochastic-deterministic simulation framework without further mathematical knowledge or input by the user. Stochastic noise can optionally be omitted for simplified deterministic solutions. We validate CaiNet by comparison with Gillespie simulations using an auto feedback motive. We apply CaiNet to the circadian clock and find that temporally modulated external input signals allow regulating the periodicity of oscillations in a nested network topology. We further use CaiNet to simulate the temporal behavior of the pluripotency network, using published kinetic parameters where possible, as it transits from the naive state to germ layer lineages upon changes in signal inputs.
Artifact Formation in Single Molecule Localization Microscopy
Jochen M. Reichel, Thomas Vomhof, Jens Michaelis
We investigate the influence of different accuracy-detection rate trade-offs on image reconstruction in single molecule localization microscopy. Our main focus is the investigation of image artifacts experienced when using low localization accuracy, especially in the presence of sample drift and inhomogeneous background. In this context we present a newly developed SMLM software termed FIRESTORM which is optimized for high accuracy reconstruction. For our analysis we used in silico SMLM data and compared the reconstructed images to the ground truth data. We observe two discriminable reconstruction populations of which only one shows the desired localization behavior.
bioRxiv preprint first posted online Jul. 12, 2019
Inferring quantity and qualities of superimposed reaction rates in single molecule survival time distributions
Matthias Reisser, Johannes Hettich, Timo Kuhn, View ORCID ProfileJ. Christof M. Gebhardt
Actions of molecular species, for example binding of transcription factors to chromatin, are intrinsically stochastic and may comprise several mutually exclusive pathways. Inverse Laplace transformation in principle resolves the rate constants and frequencies of superimposed reaction processes, however current approaches are challenged by single molecule fluorescence time series prone to photobleaching. Here, we present a genuine rate identification method (GRID) that infers the quantity, rates and frequencies of dissociation processes from single molecule fluorescence survival time distributions using a dense grid of possible decay rates. In particular, GRID is able to resolve broad clusters of rate constants not accessible to common models of one to three exponential decay rates. We validate GRID by simulations and apply it to the problem of in-vivo TF-DNA dissociation, which recently gained interest due to novel single molecule imaging technologies. We consider dissociation of the transcription factor CDX2 from chromatin. GRID resolves distinct, decay rates and identifies residence time classes overlooked by other methods. We confirm that such sparsely distributed decay rates are compatible with common models of TF sliding on DNA.
bioRxiv, posted June 21, 2019, 1-12
Synaptic FUS Localization During Motoneuron Development and Its Accumulation in Human ALS Synapses
Dhruva Deshpande, Julia Higelin, Michael Schoen, Thomas Vomhof, Tobias M. Boeckers, Maria Demestre and Jens Michaelis
Mutations in the fused in Sarcoma (FUS) gene induce cytoplasmic FUS aggregations, contributing to the neurodegenerative disease amyotrophic lateral sclerosis (ALS) in certain cases. While FUS is mainly a nuclear protein involved in transcriptional processes with limited cytoplasmic functions, it shows an additional somatodendritic localization in neurons. In this study we analyzed the localization of FUS in motoneuron synapses, these being the most affected neurons in ALS, using super-resolution microscopy to distinguish between the pre- and postsynaptic compartments. We report a maturation-based variation of FUS localization in rodent synapses where a predominantly postsynaptic FUS was observed in the early stages of synaptic development, while in mature synapses the protein was entirely localized in the axonal terminal. Likewise, we also show that at the synapse of human motoneurons derived from induced pluripotent stem cells of a healthy control, FUS is mainly postsynaptic in the early developmental stages. In motoneurons derived from ALS patients harboring a very aggressive juvenile FUS mutation, increased synaptic accumulation of mutated FUS was observed. Moreover increased aggregation of other synaptic proteins Bassoon and Homer1 was also detected in these abnormal synapses. Having demonstrated changes in the FUS localization during synaptogenesis, a role of synaptic FUS in both dendritic and axonal cellular compartments is probable, and we propose a gain-of-toxic function due to the synaptic aggregation of mutant FUS in ALS.
Front. Cell. Neurosci., June 2019, Volume 13, Article 256
Mitotic chromosome binding predicts transcription factor properties in interphase
Mahé Raccaud, Elias T. Friman, Andrea B. Alber, Harsha Agarwa, Cédric Deluz, Timo Kuhn, J. Christof M. Gebhardt & David M. Suter
Mammalian transcription factors (TFs) differ broadly in their nuclear mobility and sequence-specific/non-specific DNA binding. How these properties affect their ability to occupy specific genomic sites and modify the epigenetic landscape is unclear. The association of TFs with mitotic chromosomes observed by fluorescence microscopy is largely mediated by non-specific DNA interactions and differs broadly between TFs. Here we combine quantitative measurements of mitotic chromosome binding (MCB) of 501 TFs, TF mobility measurements by fluorescence recovery after photobleaching, single molecule imaging of DNA binding, and mapping of TF binding and chromatin accessibility. TFs associating to mitotic chromosomes are enriched in DNA-rich compartments in interphase and display slower mobility in interphase and mitosis. Remarkably, MCB correlates with relative TF on-rates and genome-wide specific site occupancy, but not with TF residence times. This suggests that non-specific DNA binding properties of TFs regulate their search efficiency and occupancy of specific genomic sites.
Nat Commun. 2019 Jan 30;10(1):487