Function of the actin nucleator mitoSPIRE1 in mitochondrial dynamics

Cells of all known living organisms require a constant energy supply in the form of adenosine triphosphate (ATP) for their survival and function. The intracellular transport and diffusion of ATP molecules are rather limited and thus, ATP producing mitochondria are directly transported to their site of action such as synaptic densities to provide all cellular compartments with an adequate amount of ATP. Fast and long-range transport of mitochondria along microtubule tracks via kinesin and dynein motor proteins is well established, whereas only little is known about actin / myosin functions in mitochondrial transport. There is growing evidence that actin filaments and myosin motor proteins play an essential role in the transport of mitochondria. A SPIRE formin actin nucleator complex facilitates actin filament generation. In addition, SPIRE proteins coordinate myosin 5 (MYO5) motor protein activation at vesicle and organelle membranes. Due to alternative splicing of the SPIRE1 gene, mammalian cells generate four SPIRE proteins from two SPIRE genes. The SPIRE1 actin / myosin organizer is targeted by the alternatively spliced exon 13 towards mitochondrial membranes and the corresponding protein is named ‘mitoSPIRE1’. The present thesis quantitatively addressed the expression of all SPIRE splice variants including mitoSPIRE1 in distinct organs, showing that the brain has the majority of SPIRE expression. In a previous study it was shown that SPIRE1 mutant mice lacking the expression of all SPIRE1 proteins, including mitoSPIRE1, have increased fear in both cued and contextual fear conditioning experiments. To dissect the vesicular and mitochondrial SPIRE functions in fear behavior and to analyze the function of the mitoSPIRE1 protein in more detail, we generated a novel knockout model by targeted deletion of the mouse SPIRE1 exon 13 - the mitoSPIRE1 knockout mouse. Fibroblasts of the novel mitoSPIRE1 knockout mouse and the SPIRE1 mutant mouse show increased mitochondrial motility in live cell fluorescence microscopy analysis. Colocalization studies unraveled that mitoSPIRE1 colocalizes with FMN subfamily formins and MYO5 proteins at mitochondria. Subsequent fluorescence-activated mitochondria sorting (FAMS) experiments confirmed that SPIRE proteins contribute to target MYO5 actin motor proteins towards mitochondrial membranes. Furthermore, FAMS and fluorescence microscopy revealed an influence of mitoSPIRE1 on mitochondrial morphology. SPIRE proteins did not influence mitochondrial oxygen consumption rate, which was analyzed by a Seahorse Mito Stress Test assay. The mentioned function of mitoSPIRE1 in the regulation of mitochondrial motility led us to speculate that mitoSPIRE1 might be involved in targeting mitochondria towards synaptic terminals and thereby influencing synaptic transmission and possibly fear behavior

The biogenesis and function of nucleosome arrays

Numerous chromatin remodeling enzymes position nucleosomes in eukaryotic cells. Aside from these factors, transcription, DNA sequence, and statistical positioning of nucleosomes also shape the nucleosome landscape. The precise contributions of these processes remain unclear due to their functional redundancy in vivo. By incisive genome engineering, we radically decreased their redundancy in Saccharomyces cerevisiae. The transcriptional machinery strongly disrupts evenly spaced nucleosomes. Proper nucleosome density and DNA sequence are critical for their biogenesis. The INO80 remodeling complex helps space nucleosomes in vivo and positions the first nucleosome over genes in an H2A.Z-independent fashion. INO80 requires its Arp8 subunit but unexpectedly not the Nhp10 module for spacing. Cells with irregularly spaced nucleosomes suffer from genotoxic stress including DNA damage, recombination and transpositions. We derive a model of the biogenesis of the nucleosome landscape and suggest that it evolved not only to regulate but also to protect the genome

Semi-Automated Location Planning for Urban Bike-Sharing Systems

Bike-sharing has developed into an established part of many urban transportation systems. However, new bikesharing systems (BSS) are still built and existing ones are extended. Particularly for large BSS, location planning is complex since factors determining potential usage are manifold. We propose a semi-automatic approach for creating or extending real-world sized BSS during general planning. Our approach optimizes locations such that the number of trips is maximized for a given budget respecting construction as well as operation costs. The approach consists of four steps: (1) collecting and preprocessing required data, (2) estimating a demand model, (3) calculating optimized locations considering estimated redistribution costs, and (4) presenting the solution to the planner in a visualization and planning front end. The full approach was implemented and evaluated positively with BSS and planning experts

The DNA binding CXC domain of MSL2 is required for faithful targeting the Dosage Compensation Complex to the X chromosome

Dosage compensation in Drosophila melanogaster involves the selective targeting of the male X chromosome by the dosage compensation complex (DCC) and the coordinate, ∼2-fold activation of most genes. The principles that allow the DCC to distinguish the X chromosome from the autosomes are not understood. Targeting presumably involves DNA sequence elements whose combination or enrichment mark the X chromosome. DNA sequences that characterize ‘chromosomal entry sites’ or ‘high-affinity sites’ may serve such a function. However, to date no DNA binding domain that could interpret sequence information has been identified within the subunits of the DCC. Early genetic studies suggested that MSL1 and MSL2 serve to recognize high-affinity sites (HAS) in vivo, but a direct interaction of these DCC subunits with DNA has not been studied. We now show that recombinant MSL2, through its CXC domain, directly binds DNA with low nanomolar affinity. The DNA binding of MSL2 or of an MSL2–MSL1 complex does not discriminate between different sequences in vitro, but in a reporter gene assay in vivo, suggesting the existence of an unknown selectivity cofactor. Reporter gene assays and localization of GFP-fusion proteins confirm the important contribution of the CXC domain for DCC targeting in vivo

Buffered fitness components: Antagonism between malnutrition and an insecticide in bumble bees.

Global insect biodiversity declines due to reduced fitness are linked to interactions between environmental stressors. In social insects, inclusive fitness depends on successful mating of reproductives, i.e. males and queens, and efficient collaborative brood care by workers. Therefore, interactive effects between malnutrition and environmental pollution on sperm and feeding glands (hypopharyngeal glands (HPGs)) would provide mechanisms for population declines, unless buffered against due to their fitness relevance. However, while negative effects for bumble bee colony fitness are known, the effects of malnutrition and insecticide exposure singly and in combination on individuals are poorly understood. Here we show, in a fully-crossed laboratory experiment, that malnutrition and insecticide exposure result in neutral or antagonistic interactions for spermatozoa and HPGs of bumble bees, Bombus terrestris, suggesting strong selection to buffer key colony fitness components. No significant effects were observed for mortality and consumption, but significant negative effects were revealed for spermatozoa traits and HPGs. The combined effects on these parameters were not higher than the individual stressor effects, which indicates an antagonistic interaction between both. Despite the clear potential for additive effects, due to the individual stressors impairing muscle quality and neurological control, simultaneous malnutrition and insecticide exposure surprisingly did not reveal an increased impact compared to individual stressors, probably due to key fitness traits being resilient. Our data support that stressor interactions require empirical tests on a case-by-case basis and need to be regarded in context to understand underlying mechanisms and so adequately mitigate the ongoing decline of the entomofauna

The Bank Vole (Clethrionomys glareolus) - Small Animal Model for Hepacivirus Infection

Many people worldwide suffer from hepatitis C virus (HCV) infection, which is frequently persistent. The lack of efficient vaccines against HCV and the unavailability of or limited compliance with existing antiviral therapies is problematic for health care systems worldwide. Improved small animal models would support further hepacivirus research, including development of vaccines and novel antivirals. The recent discovery of several mammalian hepaciviruses may facilitate such research. In this study, we demonstrated that bank voles (Clethrionomys glareolus) were susceptible to bank vole-associated Hepacivirus F and Hepacivirus J strains, based on the detection of hepaciviral RNA in 52 of 55 experimentally inoculated voles. In contrast, interferon α/β receptor deficient C57/Bl6 mice were resistant to infection with both bank vole hepaciviruses (BvHVs). The highest viral genome loads in infected voles were detected in the liver, and viral RNA was visualized by in situ hybridization in hepatocytes, confirming a marked hepatotropism. Furthermore, liver lesions in infected voles resembled those of HCV infection in humans. In conclusion, infection with both BvHVs in their natural hosts shares striking similarities to HCV infection in humans and may represent promising small animal models for this important human disease