Stone, M. R. L. | Search bioRxiv

bioRxiv2022

Activity-Based Imaging of Lipid Environments Targeted by Peroxynitrite in Biomimetic Vesicles and Live Cells

Lipid environments can be chemically impacted by peroxynitrite (ONOO-), a reactive species generated under nitrative stress. Molecular tools used for investigating ONOO- reactivity in biological membranes remain underdeveloped, available probes lack the ability of subcellular localization, and the standard methods for detecting ONOO- in vivo are indirect. Here we investigated ONOO- in diverse lipid environments (biomimetic giant vesicles, live mammalian cells, and within the lung lining) using a biocompatible and membrane-localized phospholipid named DPPC-TC-ONOO-. This designer lipid and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine self-assemble to giant vesicles that respond to ONOO- by generating fluorescence. These vesicles remain intact after sensing ONOO- and exhibit excellent selectivity against other redox species. We delivered DPPC-TC-ONOO- into live HeLa and RAW cells via lipid nanoparticles (LNPs). Cytokine-induced nitrative stress led to enhanced fluorescence of the lipid clusters, primarily in the endoplasmic reticulum. These LNPs allowed the detection of ONOO- reactivity and nitrative stress around bronchioles within precision cut lung slices in response to acute lung injury (ALI). Furthermore, the use of the LNPs allowed for the detection of pulmonary macrophages from bronchoalveolar lavage following ALI in C57BL6/J but not in Nos2-/- mice. These investigations revealed significant advantages of DPPC-TC-ONOO- over its non-amphiphilic analog. Our work presents (i) an unprecedented function for biomimetic membranes, (ii) the potential of LNPs for delivering designer lipids into cells and tissues, (iii) real-time imaging of endogenous ONOO- at the organelle level in mammalian cells, and (iv) a direct method of studying nitrative stress due to ALI ex vivo and in vivo.

biochemistry↗

bioRxiv2021

CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage

The Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 [A] resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.

biophysics↗