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OmpA, a predominant outer membrane (OM) protein in Escherichia coli, affects virulence, adhesion, and bacterial OM integrity. However, despite more than 50 y of research, the molecular basis for the role of OmpA has remained elusive. In this study, we demonstrate that OmpA organizes the OM protein lattice and mechanically connects it to the cell wall (CW). Using gene fusions, atomic force microscopy, simulations, and microfluidics, we show that the β-barrel domain of OmpA is critical for maintaining the permeability barrier, but both the β-barrel and CW-binding domains are necessary to enhance the cell envelope's strength. OmpA integrates the compressive properties of the OM protein lattice with the tensile strength of the CW, forming a mechanically robust composite that increases overall integrity. This coupling likely underpins the ability of the entire envelope to function as a cohesive, resilient structure, critical for the survival of bacteria. [ABSTRACT FROM AUTHOR]

Multiplexed proteomics has become a powerful tool for investigating biological systems. Using balancer-peptide conjugates (e.g., TMTproC complementary ions) in the MS2 spectra for quantification circumvents the ratio distortion problem inherent in multiplexed proteomics. However, TMTproC quantification scans require long Orbitrap transients and extended ion injection times to achieve sufficient ion statistics and spectral resolution. Real-Time Search (RTS) algorithms have demonstrated increased speed and sensitivity by selectively informing precursor peak quantification. Here, we combine complementary ion quantification with Real-Time Search (TMTproC-RTS) to enhance sensitivity while maintaining accuracy and precision in quantitative proteomics at the MS2 level. We demonstrate the utility of this method by quantifying protein dynamics during the embryonic development of Drosophila melanogaster (fly), Ciona robusta (sea squirt), and Xenopus laevis (frog). We quantify 7.8k, 8.6k, and 12.7k proteins in each organism, which is an improvement of 12%, 13%, and 14%, respectively, compared to naive TMTproC analysis. For all three organisms, the newly acquired data outperform previously published datasets and provides a diverse, deep, and accurate database of protein dynamics during embryogenesis which will advance the study of evolutionary comparison in early embryogenesis., Competing Interests: Conflict of interest statement G.M., W.B., J.C., J.H., R. M., and V.Z. are employees of Thermo Fisher Scientific. Thermo Fisher provides limited support to M.W. laboratory under a collaborative research agreement with Princeton University., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Extracellular-signal-regulated kinase (ERK) signaling controls development and homeostasis and is genetically deregulated in human diseases, including neurocognitive disorders and cancers. Although the list of ERK functions is vast and steadily growing, the full spectrum of processes controlled by any specific ERK activation event remains unknown. Here, we show how ERK functions can be systematically identified using targeted perturbations and global readouts of ERK activation. Our experimental model is the Drosophila embryo, where ERK signaling at the embryonic poles has thus far only been associated with the transcriptional patterning of the future larva. Through a combination of live imaging and phosphoproteomics, we demonstrated that ERK activation at the poles is also critical for maintaining the speed and synchrony of embryonic cleavages. The presented approach to interrogating phosphorylation networks identifies a hidden function of a well-studied signaling event and sets the stage for similar studies in other organisms., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)