SFB1535_MibiNet

Microtubule-dependent endosomal transport is crucial for polar growth, ensuring the precise distribution of cellular cargos such as proteins and mRNAs. However, the molecular mechanism linking mRNAs to the endosomal surface remains poorly understood. Here, we present a structural analysis of the key RNA-binding protein Rrm4 from Ustilago maydis. Our findings reveal a different type of MademoiseLLE domain (MLLE) featuring a seven-helical bundle that provides a distinct binding interface. A comparative analysis with the canonical MademoiseLLE domain of the poly(A)-binding protein Pab1 disclosed unique characteristics of both domains. Deciphering the MLLE binding code enabled prediction and verification of previously unknown Rrm4 interactors containing short linear motifs. Importantly, we demonstrated that the human MLLE domains, such as those of PABPC1 and UBR5, employed a similar principle to distinguish among interaction partners. Thus, our study provides detailed mechanistic insights into how structural variations in the widely distributed MLLE domain facilitate mRNA attachment during endosomal transport.

The trace element iron is essential for life, but elevated levels can rapidly cause cellular damage through oxidative stress. Bacteria, like Corynebacterium glutamicum, tightly regulate iron and heme homeostasis via the global regulators DtxR and HrrA. This study provides the first analysis of the genome-wide binding patterns of these two regulators demonstrating significant differences in binding dependent on the tested iron regimes. Overall, we identified 25 new DtxR targets and 210 previously unknown HrrA targets, including genes with crucial roles in central metabolism and DNA repair. Notably, DtxR was shown to link iron metabolism to methionine synthesis, which might be important to protect the cell from oxidative stress. Our findings highlight the interconnected nature of DtxR and HrrA networks and underscore the value of condition-specific analysis to deepen the understanding of how bacteria adapt to environmental changes. 

This ARC presents diverse data from MibiNet members to illustrate the application of MibiNet-specific SWATE templates and showcase examples of metadata annotation.

Fungal pathogens depend on sophisticated gene expression programs for successful infection. A crucial component is RNA regulation mediated by RNA-binding proteins (RBPs). However, little is known about the spatiotemporal RNA control mechanisms during fungal pathogenicity. Here, we discover that the RBP Khd4 defines a distinct mRNA regulon to orchestrate membrane trafficking during pathogenic development of Ustilago maydis. By establishing hyperTRIBE for fungal RBPs, we generated a comprehensive transcriptome-wide map of Khd4 interactions in vivo. We identify a defined set of target mRNAs enriched for regulatory proteins involved, e.g., in GTPase signaling. Khd4 controls the stability of target mRNAs via its cognate regulatory element AUACCC present in their 3′ untranslated regions. Studying individual examples reveals a unique link between Khd4 and vacuole maturation. Thus, we uncover a distinct role for an RNA stability factor defining a specific mRNA regulon for membrane trafficking during pathogenicity.

Quantification of cell growth is central to any study of photoautotrophic microorganisms. However, cellular self-shading and limited CO2 control in conventional photobioreactors lead to heterogeneous conditions that obscure distinct correlations between the environment and cellular physiology. Here we present a microfluidic cultivation platform that enables precise analysis of cyanobacterial growth with spatio-temporal resolution. Since cyanobacteria are cultivated in monolayers, cellular self-shading does not occur, allowing homogeneous illumination and precise knowledge of the photon-flux density at single-cell resolution. A single chip contains multiple channels, each connected to several hundred growth chambers. In combination with an externally applied light gradient, this setup enables high-throughput multi-parameter analysis in short time. In addition, the multilayered microfluidic design allows continuous perfusion of defined gas mixtures. Transversal CO2 diffusion across the intermediate polydimethylsiloxane membrane results in homogeneous CO2 supply, with a unique exchange-surface to cultivation-volume ratio. Three cyanobacterial model strains were examined under various, static and dynamic environmental conditions. Phase-contrast and chlorophyll fluorescence images were recorded by automated time-lapse microscopy. Deep-learning trained cell segmentation was used to efficiently analyse large image stacks, thereby generating statistically reliable data. Cell division was highly synchronized, and growth was robust under continuous illumination but stopped rapidly upon initiating dark phases. CO2-Limitation, often a limiting factor in photobioreactors, was only observed when the device was operated under reduced CO2 between 50 and 0 ppm. Here we provide comprehensive and precise data on cyanobacterial growth at single-cell resolution, accessible for further growth studies and modeling.