High‐Content Image‐Based Screening and Deep Learning for the Detection of Anti‐Inflammatory Drug Leads

We developed a high-content image-based screen that utilizes the pro-inflammatory stimulus lipopolysaccharide (LPS) and murine macrophages (RAW264.7) with the goal of enabling the identification of novel anti-inflammatory lead compounds. We screened 2,259 bioactive compounds with annotated mechanisms of action (MOA) to identify compounds that block the LPS-induced phenotype in macrophages. We utilized a set of seven fluorescence microscopy probes to generate images that were used to train and optimize a deep neural network classifier to distinguish between unstimulated and LPS-stimulated macrophages. The top hits from the deep learning classifier were validated using a linear classifier trained on individual cells and subsequently investigated in a multiplexed cytokine secretion assay. All 12 hits significantly modulated the expression of at least one cytokine upon LPS stimulation. Seven of these were allosteric inhibitors of the mitogen-activated protein kinase kinase (MEK1/2) and showed similar effects on cytokine expression. This deep learning morphological assay identified compounds that modulate the innate immune response to LPS and may aid in identifying new anti-inflammatory drug leads

The interactome of intact mitochondria by cross-linking mass spectrometry provides evidence for coexisting respiratory supercomplexes

Mitochondria exert an immense amount of cytophysiological functions, but the structural basis of most of these processes is still poorly understood. Here we use cross-linking mass spectrometry to probe the organization of proteins in native mouse heart mitochondria. Our approach provides the largest survey of mitochondrial protein interactions reported so far. In total, we identify 3,322 unique residue-to-residue contacts involving half of the mitochondrial proteome detected by bottom-up proteomics. The obtained mitochondrial protein interactome gives insights in the architecture and submitochondrial localization of defined protein assemblies, and reveals the mitochondrial localization of four proteins not yet included in the MitoCarta database. As one of the highlights, we show that the oxidative phosphorylation complexes I-V exist in close spatial proximity, providing direct evidence for supercomplex assembly in intact mitochondria. The specificity of these contacts is demonstrated by comparative analysis of mitochondria after high salt treatment, which disrupts the native supercomplexes and substantially changes the mitochondrial interactome

Genetic Analysis of Lysosomal Trafficking in Caenorhabditis elegans

The intestinal cells of Caenorhabditis elegans embryos contain prominent, birefringent gut granules that we show are lysosome-related organelles. Gut granules are labeled by lysosomal markers, and their formation is disrupted in embryos depleted of AP-3 subunits, VPS-16, and VPS-41. We define a class of gut granule loss (glo) mutants that are defective in gut granule biogenesis. We show that the glo-1 gene encodes a predicted Rab GTPase that localizes to lysosome-related gut granules in the intestine and that glo-4 encodes a possible GLO-1 guanine nucleotide exchange factor. These and other glo genes are homologous to genes implicated in the biogenesis of specialized, lysosome-related organelles such as melanosomes in mammals and pigment granules in Drosophila. The glo mutants thus provide a simple model system for the analysis of lysosome-related organelle biogenesis in animal cells

glo-3, a Novel Caenorhabditis elegans Gene, Is Required for Lysosome-Related Organelle Biogenesis

Gut granules are specialized lysosome-related organelles that act as sites of fat storage in Caenorhabditis elegans intestinal cells. We identified mutations in a gene, glo-3, that functions in the formation of embryonic gut granules. Some glo-3(−) alleles displayed a complete loss of embryonic gut granules, while other glo-3(−) alleles had reduced numbers of gut granules. A subset of glo-3 alleles led to mislocalization of gut granule contents into the intestinal lumen, consistent with a defect in intracellular trafficking. glo-3(−) embryos lacking gut granules developed into adults containing gut granules, indicating that glo-3(+) function may be differentially required during development. We find that glo-3(+) acts in parallel with or downstream of the AP-3 complex and the PGP-2 ABC transporter in gut granule biogenesis. glo-3 encodes a predicted membrane-associated protein that lacks obvious sequence homologs outside of nematodes. glo-3 expression initiates in embryonic intestinal precursors and persists almost exclusively in intestinal cells through adulthood. GLO-3∷GFP localizes to the gut granule membrane, suggesting it could play a direct role in the trafficking events at the gut granule. smg-1(−) suppression of glo-3(−) nonsense alleles indicates that the C-terminal half of GLO-3, predicted to be present in the cytoplasm, is not necessary for gut granule formation. Our studies identify GLO-3 as a novel player in the formation of lysosome-related organelles

<em>C. elegans</em> BLOC-1 Functions in Trafficking to Lysosome-Related Gut Granules

<div><p>The human disease Hermansky-Pudlak syndrome results from defective biogenesis of lysosome-related organelles (LROs) and can be caused by mutations in subunits of the BLOC-1 complex. Here we show that <em>C. elegans glo-2</em> and <em>snpn-1</em>, despite relatively low levels of amino acid identity, encode Pallidin and Snapin BLOC-1 subunit homologues, respectively. BLOC-1 subunit interactions involving Pallidin and Snapin were conserved for GLO-2 and SNPN-1. Mutations in <em>glo-2</em> and <em>snpn-1</em>,or RNAi targeting 5 other BLOC-1 subunit homologues in a genetic background sensitized for <em>glo-2</em> function, led to defects in the biogenesis of lysosome-related gut granules. These results indicate that the BLOC-1 complex is conserved in <em>C. elegans</em>. To address the function of <em>C. elegans</em> BLOC-1, we assessed the intracellular sorting of CDF-2::GFP, LMP-1, and PGP-2 to gut granules. We validated their utility by analyzing their mislocalization in intestinal cells lacking the function of AP-3, which participates in an evolutionarily conserved sorting pathway to LROs. BLOC-1(−) intestinal cells missorted gut granule cargo to the plasma membrane and conventional lysosomes and did not have obviously altered function or morphology of organelles composing the conventional lysosome protein sorting pathway. Double mutant analysis and comparison of AP-3(−) and BLOC-1(−) phenotypes revealed that BLOC-1 has some functions independent of the AP-3 adaptor complex in trafficking to gut granules. We discuss similarities and differences of BLOC-1 activity in the biogenesis of gut granules as compared to mammalian melanosomes, where BLOC-1 has been most extensively studied for its role in sorting to LROs. Our work opens up the opportunity to address the function of this poorly understood complex in cell and organismal physiology using the genetic approaches available in <em>C. elegans</em>.</p> </div

Gut granule formation in adults.

<p>In wild-type adults, autofluorescent gut granules accumulated markers of acidification (A–B), hydrophobicity (C–D), terminal endocytic compartments (E–F) and contained PGP-2::GFP (G–H). <i>glo-2(zu455)</i> adults had substantially reduced numbers of autofluorescent compartments (I). The majority of these organelles were stained with LysoTracker Red (I-J), Nile Red (K–L) and contained PGP-2::GFP (O–P). (M–N) In contrast, few of the autofluorescent compartments accumulated TRITC-Dextran and those that did localized the marker to a subdomain within the organelle. In all panels, white arrows identify autofluorescent compartments that contained the gut granule marker. The black arrows denote the location of the intestinal lumen.</p

Analyzing the identity of LMP-1::GFP compartments in <i>glo-2(</i>

<p>−<b><i>)</i></b><b> embryos.</b> The majority of LMP-1::GFP containing compartments (marked with white arrows in all panels) lacked RAB-5 and RAB-7 in wild type (A–F) and <i>glo-2(zu455)</i> (J–O), however a subset of LMP-1::GFP colocalized with RAB-7 (black arrows). In both wild type (G–I) and <i>glo-2(zu455</i>) (P-R), the lysosomal hydrolase F11E6.1::mCherry localized to LMP-1::GFP containing organelles. In all panels, black arrowheads flank the intestine of 1.5-fold stage embryos.</p

Analysis of LMP-1 trafficking.

<p>(A–C) In intestinal cells LMP-1::GFP localized to the basolateral plasma membrane (white arrow) and apically enriched organelles (black arrow) that did not overlap with PGP-2 containing gut granules (white arrowhead). (D–F) Endogenous LMP-1 localized to apical CNTS-1::GFP containing conventional lysosomes (white arrows) and more basally localized compartments (black arrow) lacking CNTS-1::GFP. (G–I) In the basal region of the intestine, LMP-1 localized to PGP-2 containing gut granules (black arrows). (J–L) In a mutant lacking AP-3 function, gut granules containing PGP-2 (white arrowhead) lacked LMP-1, which was restricted to apical compartments (black arrows). (M–O) PGP-2 containing organelles were lacking in <i>glo-2(zu455)</i> embryos and LMP-1 was enriched on apically localized organelles (black arrows). In all panels, black arrowheads flank the intestine. In A–C a pretzel stage embryo and in D–O 1.5-fold stage embryos are shown.</p

<i>C. elegans</i> BLOC-1 subunits.

<p>The gene name of each <i>C. elegans</i> BLOC-1 subunit and its human and <i>D. melanogaster</i> orthologues are listed. The amino acid identity of the <i>C. elegans</i> protein with each orthologue was derived from pairwise sequence alignments with the full length <i>C. elegans</i> protein.</p