SummaryEukaryotic cells store their chromosomes in a single nucleus. This is important to maintain genomic integrity, as chromosomes packaged into separate nuclei (micronuclei) are prone to massive DNA damage. During mitosis, higher eukaryotes disassemble their nucleus and release individualized chromosomes for segregation. How numerous chromosomes subsequently reform a single nucleus has remained unclear. Using image-based screening of human cells, we identified barrier-to-autointegration factor (BAF) as a key factor guiding membranes to form a single nucleus. Unexpectedly, nuclear assembly does not require BAF’s association with inner nuclear membrane proteins but instead relies on BAF’s ability to bridge distant DNA sites. Live-cell imaging and in vitro reconstitution showed that BAF enriches around the mitotic chromosome ensemble to induce a densely cross-bridged chromatin layer that is mechanically stiff and limits membranes to the surface. Our study reveals that BAF-mediated changes in chromosome mechanics underlie nuclear assembly with broad implications for proper genome function.

The filamentous fungus Aspergillus fumigatus is responsible for a lethal disease called invasive aspergillosis that affects immunocompromised patients. This disease, like other human fungal diseases, is generally treated by compounds targeting the primary fungal cell membrane sterol. Recently, glucan synthesis inhibitors were added to the limited antifungal arsenal and encouraged the search for novel targets in cell wall biosynthesis. Although galactomannan is a major component of the A. fumigatus cell wall and extracellular matrix, the biosynthesis and role of galactomannan are currently unknown. By a targeted gene deletion approach, we demonstrate that UDP-galactopyranose mutase, a key enzyme of galactofuranose metabolism, controls the biosynthesis of galactomannan and galactofuranose containing glycoconjugates. The glfA deletion mutant generated in this study is devoid of galactofuranose and displays attenuated virulence in a low-dose mouse model of invasive aspergillosis that likely reflects the impaired growth of the mutant at mammalian body temperature. Furthermore, the absence of galactofuranose results in a thinner cell wall that correlates with an increased susceptibility to several antifungal agents. The UDP-galactopyranose mutase thus appears to be an appealing adjunct therapeutic target in combination with other drugs against A. fumigatus. Its absence from mammalian cells indeed offers a considerable advantage to achieve therapeutic selectivity.

Polysaccharides (carbohydrates) are key regulators of a large number of cell biological processes. However, precise biochemical or genetic manipulation of these often complex structures is laborious and hampers experimental structure-function studies. Molecular Dynamics (MD) simulations provide a valuable alternative tool to generate and test hypotheses on saccharide function. Yet, currently used MD force fields often overestimate the aggregation propensity of polysaccharides, affecting the usability of those simulations. Here we tested MARTINI, a popular coarse-grained (CG) force field for biological macromolecules, for its ability to accurately represent molecular forces between saccharides. To this end, we calculated a thermodynamic solution property, the second virial coefficient of the osmotic pressure (B). Comparison with light scattering experiments revealed a nonphysical aggregation of a prototypical polysaccharide in MARTINI, pointing at an imbalance of the nonbonded solute-solute, solute-water, and water-water interactions. This finding also applies to smaller oligosaccharides which were all found to aggregate in simulations even at moderate concentrations, well below their solubility limit. Finally, we explored the influence of the Lennard-Jones (LJ) interaction between saccharide molecules and propose a simple scaling of the LJ interaction strength that makes MARTINI more reliable for the simulation of saccharides.

Galactofuranose (Galf) containing molecules have been described at the cell surface of several eukaryotes and shown to contribute to the virulence of the parasite Leishmania major and the fungus Aspergillus fumigatus. It is anticipated that a number of the surface glycoconjugates such as N-glycans or glycolipids are galactofuranosylated in the Golgi apparatus. This raises the question of how the substrate for galactofuranosylation reactions, UDP-Galf, which is synthesized in the cytosol, translocates into the organelles of the secretory pathway. Here we report the first identification of a Golgi-localized nucleotide sugar transporter, named GlfB, with specificity for a UDP-Galf. In vitro transport assays established binding of UDP-Galf toGlfB and excluded transport of several other nucleotide sugars. Furthermore, the implication of glfB in the galactofuranosylation of A. fumigatus glycoconjugates and galactomannan was demonstrated by a targeted gene deletion approach. Our data reveal a direct connection between galactomannan and the organelles of the secretory pathway that strongly suggests that the cell wall-bound polysaccharide originates from its glycosylphosphatidylinositol-anchored form.

Background: Understanding the mechanisms of cell wall biogenesis is important for development of antifungal strategies. Results: Biosynthesis of the fungal polysaccharide galactomannan requires import of GDP-mannose into the Golgi apparatus. Conclusion: It differs from the biosynthesis of other fungal cell wall polysaccharides.Significance: This provides a new paradigm for cell wall polysaccharide biosynthesis.

Glycoinositolphosphoceramides (GIPCs) are complex sphingolipids present at the plasma membrane of various eukaryotes with the important exception of mammals. In fungi, these glycosphingolipids commonly contain an α-mannose residue (Man) linked at position 2 of the inositol. However, several pathogenic fungi additionally synthesize zwitterionic GIPCs carrying an α-glucosamine residue (GlcN) at this position. In the human pathogen Aspergillus fumigatus, the GlcNα1,2IPC core (where IPC is inositolphosphoceramide) is elongated to Manα1,3Manα1,6GlcNα1,2IPC, which is the most abundant GIPC synthesized by this fungus. In this study, we identified an A. fumigatus N-acetylglucosaminyltransferase, named GntA, and demonstrate its involvement in the initiation of zwitterionic GIPC biosynthesis. Targeted deletion of the gene encoding GntA in A. fumigatus resulted in complete absence of zwitterionic GIPC; a phenotype that could be reverted by episomal expression of GntA in the mutant. The N-acetylhexosaminyltransferase activity of GntA was substantiated by production of N-acetylhexosamine-IPC in the yeast Saccharomyces cerevisiae upon GntA expression. Using an in vitro assay, GntA was furthermore shown to use UDP-N-acetylglucosamine as donor substrate to generate a glycolipid product resistant to saponification and to digestion by phosphatidylinositol-phospholipase C as expected for GlcNAcα1,2IPC. Finally, as the enzymes involved in mannosylation of IPC, GntA was localized to the Golgi apparatus, the site of IPC synthesis.

Three-dimensional protein structures are a key requisite for structure-based drug discovery. For many highly relevant targets, medicinal chemists are confronted with large numbers of target structures in their apo-forms or in complex with a wealth of different ligands. To exploit the full potential of such structure ensembles, in terms of aggregated knowledge that informs design, it is desirable to extract a manageable number of structures that provide a maximum of ligand design opportunities. Most commonly used structure comparison methods are largely based on atom positions and geometry-based metrics; medicinal chemists, however, seek ligand design opportunities and are interested in methods that allow such information to be distilled from structural data and guide them in an intuitive way. Here we present an approach for identifying nonobvious ligand design opportunities in protein conformation ensembles based on the information content in grid maps that represent, for example, binding hotspots. We use four different examples to show how this method can provide information orthogonal to established coordinatebased similarity methods. Furthermore, we demonstrate that ligand design opportunities can change substantially with very small structural variations. We expect that this approach will advance the identification of ligand design opportunities hidden in large collections of protein−ligand complex data that would otherwise have been missed.

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