The early insect embryo develops as a multinucleated cell distributing the genome uniformly to the cell cortex. Mechanistic insight for nuclear positioning beyond cytoskeletal requirements is missing. Contemporary hypotheses propose actomyosin-driven cytoplasmic movement transporting nuclei or repulsion of neighbor nuclei driven by microtubule motors. Here, we show that microtubule cross-linking by Feo and Klp3A is essential for nuclear distribution and internuclear distance maintenance in Drosophila. Germline knockdown causes irregular, less-dense nuclear delivery to the cell cortex and smaller distribution in ex vivo embryo explants. A minimal internuclear distance is maintained in explants from control embryos but not from Feo-inhibited embryos, following micromanipulation-assisted repositioning. A dimerization-deficient Feo abolishes nuclear separation in embryo explants, while the full-length protein rescues the genetic knockdown. We conclude that Feo and Klp3A cross-linking of antiparallel microtubule overlap generates a length-regulated mechanical link between neighboring microtubule asters. Enabled by a novel experimental approach, our study illuminates an essential process of embryonic multicellularity.

Gram-positive bacteria homeostasis and antibiotic resistance mechanisms are dependent on the intricate architecture of the cell wall, where amidated peptidoglycan plays an important role. The amidation reaction is carried out by the bi-enzymatic complex MurT-GatD, for which biochemical and structural information is very scarce. In this work, we report the first crystal structure of the glutamine amidotransferase member of this complex, GatD from Staphylococcus aureus, at 1.85 Å resolution. A glutamine molecule is found close to the active site funnel, hydrogen-bonded to the conserved R128. In vitro functional studies using 1H-NMR spectroscopy showed that S. aureus MurT-GatD complex has glutaminase activity even in the absence of lipid II, the MurT substrate. In addition, we produced R128A, C94A and H189A mutants, which were totally inactive for glutamine deamidation, revealing their essential role in substrate sequestration and catalytic reaction. GatD from S. aureus and other pathogenic bacteria share high identity to enzymes involved in cobalamin biosynthesis, which can be grouped in a new sub-family of glutamine amidotransferases. Given the ubiquitous presence of GatD, these results provide significant insights into the molecular basis of the so far undisclosed amidation mechanism, contributing to the development of alternative therapeutics to fight infections.

The influence of the protein staining used to visualize protein bands, after in-gel protein separation, for the correct identification of proteins by peptide mass fingerprint (PMF) after application of the ultrasonic in-gel protein protocol was studied. Coomassie brilliant blue and silver nitrate, both visible stains, and the fluorescent dyes Sypro Red and Sypro Orange were evaluated. Results obtained after comparison with the overnight in-gel protocol showed that good results, in terms of protein sequence coverage and number of peptides matched, can be obtained with anyone of the four stains studied. Two minutes of enzymatic digestion time was enough for proteins stained with coomassie blue, while 4 min was necessary when silver or Sypro stainings were employed in order to reach equivalent results to those obtained for the overnigh in-gel protein protocol. For the silver nitrate stain, the concentration of silver present in the staining solution must be 0.09% (w/v) to minimize background in the MALDI mass spectra.

13The early insect embryo develops as multinucleated cell distributing genomes uniformly to the cell 14 cortex. Mechanistic insight for nuclear positioning beyond cytoskeletal requirements is missing to 15 date. Contemporary hypotheses propose actomyosin driven cytoplasmic movement transporting 16 nuclei, or repulsion of neighbor nuclei driven by microtubule motors. Here, we show that 17 microtubule crosslinking by Feo and Klp3A is essential for nuclear distribution and internuclear 18 distance maintenance in Drosophila. RNAi knockdown in the germline causes irregular, less dense 19 nuclear delivery to the embryo cortex and smaller distribution in ex vivo embryo explants. A 20 minimal internuclear distance is maintained in explants from control embryos but not from Feo 21 depleted embryos, following micromanipulation assisted repositioning. A dominant-negative Feo 22 protein abolishes nuclear separation in embryo explants while the full-length protein rescues the 23 genetic knockdown. We conclude that antiparallel microtubule overlap crosslinking by Feo and 24Klp3A generates a length-regulated mechanical link between neighboring microtubule asters. 25Enabled by a novel experimental approach, our study illuminates an essential process of embryonic 26 multicellularity. 27 28 spindle midzone 29,37-40 . One of these motors is Klp3A, a Kinesin-4 homolog, a microtubule 63 depolymerase with chromatin binding affinity 35,[41][42][43][44] . PRC1 and Kinesin-4 are sufficient to form a 64 stable microtubule overlap in vitro 35 . Kinesin-5 is able to reduce overlapping, antiparallel 65 microtubules crosslinked by PRC1 in vitro 36 , which was proposed to contribute to force balance in 66 the spindle midzone during anaphase B 30 . Here, we investigated whether these three proteins are 67 required for nuclear separation, lending support to an aster-aster interaction model 15,45 . We 68 performed a combination of gene knockdown, micromanipulation and perturbation by exogenous 69 protein addition in embryo explants which enable time-lapse visualization of nuclear and 70 cytoskeletal dynamics previously unachieved. 71

Amidation of peptidoglycan is an essential feature in Staphylococcus aureus that is necessary for resistance to β-lactams and lysozyme. GatD, a 27 kDa type I glutamine amidotransferase-like protein, together with MurT ligase, catalyses the amidation reaction of the glutamic acid residues of the peptidoglycan of S. aureus. The native and the selenomethionine-derivative proteins were crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol, sodium acetate and calcium acetate. The crystals obtained diffracted beyond 1.85 and 2.25 Å, respectively, and belonged to space group P212121. X-ray diffraction data sets were collected at Diamond Light Source (on beamlines I02 and I04) and were used to obtain initial phases.

Membrane organelle function, localization, and proper partitioning upon cell division depend on interactions with the cytoskeleton. Whether membrane organelles also impact the function of cytoskeletal elements remains less clear. Here, we show that acute disruption of the ER around spindle poles affects mitotic spindle size and function inDrosophilasyncytial embryos. Acute ER disruption was achieved through the inhibition of ER membrane fusion by the dominant-negative cytoplasmic domain of atlastin. We reveal that when centrosome-proximal ER membranes are disrupted, specifically at metaphase, mitotic spindles become smaller, despite no significant changes in microtubule dynamics. These smaller spindles are still able to mediate sister chromatid separation, yet with decreased velocity. Furthermore, by inducing mitotic exit, we found that nuclear separation and distribution are affected by ER disruption. Our results suggest that ER integrity around spindle poles is crucial for the maintenance of mitotic spindle shape and pulling forces. In addition, ER integrity also ensures nuclear spacing during syncytial divisions.

Membrane organelle function, localization, and proper partitioning upon cell division depend on interactions with the cytoskeleton. Whether, reciprocally, membrane organelles also impact on the function of cytoskeletal elements remains less clear. Here, we show that acute disruption of the Endoplasmic Reticulum (ER) around spindle poles affects mitotic spindle size and function in Drosophila syncytial embryos. Acute ER disruption was achieved through the inhibition of ER membrane fusion by the dominant-negative cytoplasmic domain of Atlastin. We reveal that when the ER is disrupted specifically at metaphase, mitotic spindles become smaller, despite no significant changes in microtubule dynamics. These smaller spindles are still able to mediate sister chromatid separation, yet with decreased velocity. Furthermore, by inducing mitotic exit, we found that nuclear separation and distribution are affected upon ER disruption. Our results suggest that ER integrity around spindle poles is crucial for the maintenance of mitotic spindle shape and pulling forces. Additionally, ER integrity also ensures nuclear spacing during syncytial divisions.

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