Molecular Biology of the Cell最新文献

The maintenance of healthy mitochondria is essential to neuronal homeostasis. Mitophagy is a critical mechanism that degrades damaged mitochondria, and disruption of this process is associated with neurodegenerative disease. Previous work has shown that mammalian optineurin (OPTN), a gene mutated in familial forms of amyotrophic lateral sclerosis (ALS) and glaucoma, is an adaptor to recruit autophagy machinery to mitochondria for ubiquitin-dependent mitophagy in cultured cells. However, OPTN's role in neuronal mitophagy in vivo remains largely unknown. Here, we demonstrate that the Drosophila autophagy adaptor gene Kenny, a homologue of OPTN, mediates the recruitment of the phagophore to mitochondria undergoing ubiquitin-dependent mitophagy. We find that Kenny colocalizes with ubiquitinated mitochondria targeted for autophagic degradation in larval motoneurons, and is concentrated on the mitochondrial surface in areas opposed to the phagophore. Removal of Kenny in conditions of induced mitophagy eliminates the recruitment of the phagophore to ubiquitinated mitochondria and decreases mitophagic flux. In basal conditions, loss of Kenny causes accumulation of ubiquitinated mitochondria in neurons, indicative of stalled mitophagy. These phenotypes were reproduced in Kenny mutants, ablating the LC3-interacting region domain. Overall, this work establishes Kenny as a functional homologue of OPTN in flies and a mediator of neuronal mitophagy in vivo.

Mitochondria have a fascinating array of tools in their armory for maintaining cellular homeostasis, of which the formation of Mitochondrial-Derived Vesicles (MDVs) is the least energy-intensive. MDVs have become the "go-to" vesicles for mitochondria to perform functions such as ferrying damaged mitochondrial proteins to lysosomes and regulating peroxisomal morphology. In a corollary to the increasing number of MDV functions, the discovery of MDV subsets has also increased. However, all the known MDV communications have been from mitochondria to other organelles. Using purified mitochondria from rat liver, we show that MDVs can be generated in vitro, and proteomic analyses reveal that liver MDVs are enriched in metabolic proteins mirroring the liver's metabolic hub status. Intriguingly, live cell imaging studies in HepG2 cells reveal a new subset of MDVs that are TOMM70+ve but TOMM20-ve. This subset of MDVs harbors metabolic enzymes, such as ALDH7A1, an aldehyde dehydrogenase. Remarkably, this class of MDVs facilitates communication between mitochondria, revealing a previously unknown communication channel.

Activation of the cGAS-STING pathway stimulates innate immune signaling as well as LC3B lipidation and ubiquitylation at Golgi-related vesicles upon STING trafficking. Although ubiquitylation at these subcellular sites has been associated with regulating NF-κB-related innate immune signaling, the mechanisms of Golgi-localized polyubiquitin chain regulation of immune signaling are not well understood. We report here that the ubiquitin- and LC3B-binding proteins, TNIP1 and autophagy receptors p62, NBR1, NDP52, TAX1BP1, and OPTN, associate with STING-induced ubiquitin and LC3B-labeled vesicles, and that p62 and NBR1 act redundantly in spatial clustering of the LC3B-labeled vesicles in the perinuclear region. We also find that while TBK1 kinase activity is not required for the recruitment of TNIP1 and the autophagy receptors, it plays a role in the sequestration of the LC3B-labeled vesicles. The ubiquitin binding domains, rather than the LC3-interacting regions, of TNIP1 and OPTN are specifically important for their recruitment to Ub/LC3B-associated perinuclear vesicles, and OPTN is also recruited through a TBK1-dependent mechanism. Functionally, we find that TNIP1 plays a role in STING-mediated innate immune signaling, acting as a negative regulator of IRF3-mediated gene expression. Together, these results highlight autophagy-independent mechanisms of autophagy receptors and TNIP1 with unanticipated roles in regulating STING-mediated innate immunity.

Zinc finger (ZNF) proteins are widely studied as DNA-binding transcriptional regulators, yet many remain functionally uncharacterized, particularly in the context of repetitive genomic regions. Here, we report that ZNF787 specifically binds a conserved 9-bp core motif within mammalian-wide interspersed repeats (MIRs) and interacts with the nucleosome remodeling and deacetylase (NuRD) complex. Depletion of ZNF787 leads to de-repression of nearby genes accompanied by increased local H3K27ac levels. Genetic rescue experiments confirm that the C2H2 zinc-finger domain of ZNF787 is essential for this repression. Thus, our research identifies ZNF787 as a repressor that binds MIR elements and interacts with the NuRD complex to mediate repeat-directed transcriptional silencing. This finding expands the functional repertoire of ZNF proteins and illustrates how specific repetitive sequences can encode local epigenetic regulatory information.

Motor protein tails, long considered flexible and disordered linkers that mediate transient cargo interactions, are increasingly recognized as dynamic hubs containing context-dependent structural motifs. Advances in cryo-EM single-particle analysis, flexible refinement tools, and in situ cryo-ET now allow visualization of partially ordered elements within these flexible regions. Recent studies of kinesin reveal that local folding events regulate adaptor binding, cargo recognition, and motor activation, challenging the traditional view of the kinesin tail as mostly disordered. This emerging perspective highlights motor tails as regulatory platforms where intrinsic disorder coexists with hidden structure, reshaping our understanding of transport regulation.

Promyelocytic leukemia (PML) protein isoform II is a component of PML nuclear bodies (PML NBs) that also forms patches on nuclear lipid droplets (nLDs) and the inner nuclear membrane (INM). Here we tested whether different metabolic treatments that induce membrane curvature elastic stress (CES) in the INM, detected by recruitment of CTP:phosphocholine cytidylyltransferase α (CCTα) and a nuclear diacylglycerol (DAG) biosensor, are a precondition for PML-II membrane association. We found that treatment of U2OS cells with unsaturated 18-carbon fatty acids and DAG acyltransferase inhibitors caused the rapid formation of PML patches on the INM that coincided with DAG enrichment and the recruitment and stabilization of CCTα, all of which were reversed upon removal of the CES stimulus. PML patches were depleted of canonical PML NB-associated proteins, occurred at sites of lamin depletion, were specific for the PML-II isoform, and occurred in cells regardless of their capacity to assemble nLDs. Induction of INM curvature stress by knockout of the terminal enzymes of the CDP-choline pathway or lipid activators of CCTα also promoted PML patches as well as stabilization of CCTα on the INM. We conclude that CES in the INM promotes the reversible assembly of PML-II-dependent membrane-associated patches.

Paralogs engage in biological processes through both redundant and nonredundant functions. In the yeast Saccharomyces cerevisiae, approximately one-fifth of the genome consists of paralogs, with their encoded proteins involved in multiple pathways. However, the unique contributions of individual paralogs have remained poorly defined. Here, we undertook a systematic examination of eight paralog pairs in the glucose-sensing pathways, deleting each component and measuring the resulting changes in gene expression. To that end, we established a new transcription reporter system to monitor the response to glucose as well as to nonpreferred sugars in single cells. Focusing on the PKA catalytic subunits, comprised of the paralogs Tpk1 and Tpk3 as well as the isomorphic kinase Tpk2, we employed mass spectrometry to identify their contribution to cellular metabolism, used a GFP-based sensor to follow changes in cytosolic pH, and used BioID to identify unique and shared candidate binding partners. Our data reveal that paralogs in the glucose-sensing pathway contribute in multiple and unique ways to signal transduction, and establish potential mechanisms driving the preservation of these and other duplicated genes throughout long periods of evolution.

Syncytia provide a unique system in which to understand the mechanisms of cellular organization. Two dramatic features of syncytial cells are the number of nuclei and the positioning of nuclei within a shared cytoplasm. Whether the formation of the syncytia and the organization of the syncytia are linked is not known. We have characterized the subperineurial glial cells (SPG), which form the most restrictive layer of the Drosophila blood-brain barrier. We found that disruption of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, Kinesin, or cytoplasmic Dynein affected both SPG cell development and general brain development. Specifically, the brains were smaller in each case, and the SPG cells were smaller when the LINC complex or cytoplasmic Dynein were disrupted. The number of nuclei per cell was increased when Kinesin was disrupted and decreased when cytoplasmic Dynein was disrupted. Finally, the positions of nuclei relative to their nearest neighbor were decreased when the expression of each gene was disrupted, and nuclei were closer to the cell edge when either Kinesin or cytoplasmic Dynein was disrupted. Together, these data illustrate that the formation of SPG cells and the organization of SPG cells are dependent on microtubule motors and the LINC complex.

We describe a noncanonical, membrane receptor-like regulation of the human copper transporter-1 (CTR1) in response to copper stimuli. CTR1 is the sole high-affinity trimeric plasma-membrane copper-importing channel that self-regulates by undergoing endocytosis to limit copper uptake. We observed that preceding copper-induced endocytosis, CTR1 forms clusters on the plasma membrane, a phenomenon that is typically observed in membrane receptors. We deciphered the mechanism of CTR1 clustering and studied its ramifications on the physical properties of plasma membranes harboring these clusters that could favor endocytosis. Membrane tension and fluctuation are fundamental regulators of pre- and post-endocytic events. Using coarse-grain molecular dynamics (MD)-simulations and coupled interference reflection microscopy-total internal reflection fluorescence microscopy (TIRF) we demonstrated that CTR1 clusters induce positive membrane curvature, an increase in local membrane tension, and a decrease in local membrane fluctuation; alterations that favor the formation of endocytic pits. Clustering is facilitated by copper-sequestering methionine-rich extracellular amino-terminus of CTR1. MD-simulations and IRM-TIRF imaging revealed that CTR1 clustering is facilitated by membrane cholesterol, depletion of which delays CTR1 endocytosis. CTR1 clustering promotes clathrin-coated pit formation that engages recruitment of adaptor protein AP-2. To summarize, we report a hitherto unknown "pre-endocytic" "receptor-like" phenomenon of ligand-induced clustering of a metal channel, which in turn regulates self-endocytosis by modulating membrane properties.

The vascular endothelium serves as a selectively permeable barrier, tightly regulating transendothelial transport of small molecules, macromolecules, and immune cells. In the context of cardiovascular and inflammatory pathologies, the integrity of this barrier is often markedly compromised, contributing to tissue dysfunction, multi-organ failure, and, ultimately, mortality. Therapeutic strategies aimed at mitigating endothelial barrier dysfunction primarily focus on enhancing the stability of endothelial cell-cell junctions. However, the precise molecular mechanisms governing junctional stability remain incompletely understood. In this study, we demonstrate that the N-terminal domain of the Rho guanine nucleotide exchange factor Trio (TrioN) enhances endothelial junctional stability by promoting the formation of tensile F-actin bundles at intercellular junctions, thereby inducing a linear junctional architecture and packaging of junctional proteins. This structural reorganization leads to an increase in endothelial barrier function. The small GTPase Rap1 is responsible for junctional tension, as depletion of Rap1 results in reduced junctional tension, loss of linearity, and increased permeability. In vivo TrioN gain-of-function experiments reveal enhanced local mechanical tension and a concomitant improvement in vascular barrier integrity within atherosclerotic vein grafts in murine models. These findings position TrioN as a promising therapeutic target for the restoration and reinforcement of endothelial barrier function in vascular disease.