RESUMEN
Curvature-generating proteins that direct membrane trafficking assemble on the surface of lipid bilayers to bud transport intermediates, which move protein and lipid cargoes from one cellular compartment to another. However, it remains unclear what controls the overall shape of the membrane bud once curvature induction has begun. In vitro experiments showed that excessive concentrations of the COPII protein Sar1 promoted the formation of membrane tubules from synthetic vesicles, while COPII-coated transport intermediates in cells are generally more spherical or lobed in shape. To understand the origin of these morphological differences, we employ atomistic, coarse-grained (CG), and continuum mesoscopic simulations of membranes in the presence of multiple curvature-generating proteins. We first characterize the membrane-bending ability of amphipathic peptides derived from the amino terminus of Sar1, as a function of interpeptide angle and concentration using an atomistic bicelle simulation protocol. Then, we employ CG simulations to reveal that Sec23 and Sec24 control the relative spacing between Sar1 protomers and form the inner-coat unit through an attachment with Sar1. Finally, using dynamical triangulated surface simulations based on the Helfrich Hamiltonian, we demonstrate that the uniform distribution of spacer molecules among curvature-generating proteins is crucial to the spherical budding of the membrane. Overall, our analyses suggest a new role for Sec23, Sec24, and cargo proteins in COPII-mediated membrane budding process in which they act as spacers to preserve a dispersed arrangement of Sar1 protomers and help determine the overall shape of the membrane bud.
RESUMEN
Coat protein complex II (COPII) governs the initial steps of biosynthetic secretory protein transport from the endoplasmic reticulum (ER), facilitating the movement of a wide variety of cargoes. Here, we demonstrate that Trk-fused gene (TFG) regulates the rate at which inner COPII coat proteins are concentrated at ER subdomains. Specifically, in cells lacking TFG, the GTPase-activating protein (GAP) Sec23 accumulates more rapidly at budding sites on the ER as compared with control cells, potentially altering the normal timing of GTP hydrolysis on Sar1. Under these conditions, anterograde trafficking of several secretory cargoes is delayed, irrespective of their predicted size. We propose that TFG controls the local, freely available pool of Sec23 during COPII coat formation and limits its capacity to prematurely destabilize COPII complexes on the ER. This function of TFG enables it to act akin to a rheostat, promoting the ordered recruitment of Sec23, which is critical for efficient secretory cargo export.
Asunto(s)
Vesículas Cubiertas por Proteínas de Revestimiento , Retículo Endoplásmico , Proteínas de Unión al GTP Monoméricas , Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Transporte Vesicular , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Activadoras de GTPasa/genética , Aparato de Golgi/metabolismoRESUMEN
Gene-editing technologies promise to create a new class of therapeutics that can achieve permanent correction with a single intervention. Besides eliminating mutant alleles in familial disease, gene-editing can also be used to favorably manipulate upstream pathophysiologic events and alter disease-course in wider patient populations, but few such feasible therapeutic avenues have been reported. Here we use CRISPR-Cas9 to edit the last exon of amyloid precursor protein (App), relevant for Alzheimer's disease (AD). Our strategy effectively eliminates an endocytic (YENPTY) motif at APP C-terminus, while preserving the N-terminus and compensatory APP-homologues. This manipulation favorably alters events along the amyloid-pathway - inhibiting toxic APP-ß-cleavage fragments (including Aß) and upregulating neuroprotective APP-α-cleavage products. AAV-driven editing ameliorates neuropathologic, electrophysiologic, and behavioral deficits in an AD knockin mouse model. Effects persist for many months, and no abnormalities are seen in WT mice even after germline App-editing; underlining overall efficacy and safety. Pathologic alterations in the glial-transcriptome of App-KI mice, as seen by single nuclei RNA-sequencing (sNuc-Seq), are also normalized by App C-terminus editing. Our strategy takes advantage of innate transcriptional rules that render terminal exons insensitive to nonsense-decay, and the upstream manipulation is expected to be effective for all forms of AD. These studies offer a path for a one-time disease-modifying treatment for AD.
RESUMEN
Co-assembly of the multilayered coat protein complex II (COPII) with the Sar1 GTPase at subdomains of the endoplasmic reticulum (ER) enables secretory cargoes to be concentrated efficiently within nascent transport intermediates, which subsequently deliver their contents to ER-Golgi intermediate compartments. Here, we define the spatiotemporal accumulation of native COPII subunits and secretory cargoes at ER subdomains under differing nutrient availability conditions using a combination of CRISPR/Cas9-mediated genome editing and live cell imaging. Our findings demonstrate that the rate of inner COPII coat recruitment serves as a determinant for the pace of cargo export, irrespective of COPII subunit expression levels. Moreover, increasing inner COPII coat recruitment kinetics is sufficient to rescue cargo trafficking deficits caused by acute nutrient limitation. Our findings are consistent with a model in which the rate of inner COPII coat addition acts as an important control point to regulate cargo export from the ER.
Asunto(s)
Retículo Endoplásmico , Proteínas , Transporte de Proteínas/fisiología , Transporte Biológico , Proteínas/metabolismo , Retículo Endoplásmico/metabolismo , Nutrientes , Aparato de Golgi/metabolismoRESUMEN
Sympathetic innervation influences homeostasis, repair, and pathology in the cardiac ventricles; in contrast, parasympathetic innervation is considered to have minimal contribution and influence in the ventricles. Here, we use genetic models, whole-mount imaging, and three-dimensional modeling to define cardiac nerve architecture during development, disease, and regeneration. Our approach reveals that parasympathetic nerves extensively innervate the cardiac ventricles. Furthermore, we identify that parasympathetic and sympathetic axons develop synchronously and are bundled throughout the ventricles. We further investigate cardiac nerve remodeling in the regenerative neonatal and the non-regenerative postnatal mouse heart. Our results show that the regenerating myocardium undergoes a unique process of physiological reinnervation, where proper nerve distribution and architecture is reestablished, in stark contrast to the non-regenerating heart. Mechanistically, we demonstrate that physiological reinnervation during regeneration is dependent on collateral artery formation. Our results reveal clinically significant insights into cardiac nerve plasticity which can identify new therapies for cardiac disease.
RESUMEN
Coat protein complex II (COPII) plays an integral role in the packaging of secretory cargoes within membrane-enclosed transport carriers that leave the endoplasmic reticulum (ER) from discrete subdomains. Lipid bilayer remodeling necessary for this process is driven initially by membrane penetration mediated by the Sar1 GTPase and further stabilized by assembly of a multilayered complex of several COPII proteins. However, the relative contributions of these distinct factors to transport carrier formation and protein trafficking remain unclear. Here, we demonstrate that anterograde cargo transport from the ER continues in the absence of Sar1, although the efficiency of this process is dramatically reduced. Specifically, secretory cargoes are retained nearly five times longer at ER subdomains when Sar1 is depleted, but they ultimately remain capable of being translocated to the perinuclear region of cells. Taken together, our findings highlight alternative mechanisms by which COPII promotes transport carrier biogenesis.
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GTP Fosfohidrolasas , Proteínas de Transporte Vesicular , GTP Fosfohidrolasas/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Retículo Endoplásmico/metabolismo , Transporte de Proteínas , Transporte Biológico , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismoRESUMEN
Co-assembly of the multilayered coat protein complex II (COPII) with the Sari GTPase at subdomains of the endoplasmic reticulum (ER) enables secretory cargoes to be concentrated efficiently within nascent transport intermediates, which subsequently deliver their contents to ER-Golgi intermediate compartments. Here, we define the spatiotemporal accumulation of native COPII subunits and secretory cargoes at ER subdomains under differing nutrient availability conditions using a combination of CRISPR/Cas9-mediated genome editing and live cell imaging. Our findings demonstrate that the rate of inner COPII coat assembly serves as a determinant for the pace of cargo export, irrespective of COPII subunit expression levels. Moreover, increasing inner COPII coat assembly kinetics is sufficient to rescue cargo trafficking deficits caused by acute nutrient limitation in a manner dependent on Sar1 GTPase activity. Our findings are consistent with a model in which the rate of inner COPII coat formation acts as an important control point to regulate cargo export from the ER.
RESUMEN
The Sar1 GTPase initiates coat protein II (COPII)-mediated protein transport by generating membrane curvature at subdomains on the endoplasmic reticulum, where it is activated by the guanine nucleotide exchange factor (GEF) Sec12. Crystal structures of GDP- and GTP-bound forms of Sar1 suggest that it undergoes a conformational switch in which GTP binding enhances the exposure of an amino-terminal amphipathic helix necessary for efficient membrane penetration. However, key residues in the amino terminus were not resolved in crystal structures, and experimental studies have suggested that the amino terminus of Sar1 is solvent-exposed in the absence of a membrane, even in the GDP-bound state. Therefore, the molecular mechanism by which GTP binding activates the membrane-remodeling activity of Sar1 remains unclear. Using atomistic molecular dynamics simulations, we compare the membrane-binding and curvature generation activities of Sar1 in its GDP- and GTP-bound states. We show that in the GTP-bound state, Sar1 inserts into the membrane with its complete (residues 1 to 23) amphipathic amino-terminal helix, while Sar1-GDP binds to the membrane only through its first 12 residues. Such differential membrane-binding modes translate into significant differences in the protein volume inserted into the membrane. As a result, Sar1-GTP generates positive membrane curvature 10 to 20 times higher than Sar1-GDP. Dimerization of the GTP-bound form of Sar1 further amplifies curvature generation. Taken together, our results present a detailed molecular mechanism for how the nucleotide-bound state of Sar1 regulates its membrane-binding and remodeling activities in a concentration-dependent manner, paving the way toward a better understanding COPII-mediated membrane transport.
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Proteínas de Unión al GTP Monoméricas , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Dimerización , Guanosina Trifosfato/metabolismo , Transporte de Proteínas , Factores de Intercambio de Guanina Nucleótido/metabolismoRESUMEN
Cardiac nerves regulate neonatal mouse heart regeneration and are susceptible to pathological remodeling following adult injury. Understanding cardiac nerve remodeling can lead to new strategies to promote cardiac repair. Our current understanding of cardiac nerve architecture has been limited to two-dimensional analysis. Here, we use genetic models, whole-mount imaging, and three-dimensional modeling tools to define cardiac nerve architecture and neurovascular association during development, disease, and regeneration. Our results demonstrate that cardiac nerves sequentially associate with coronary veins and arteries during development. Remarkably, our results reveal that parasympathetic nerves densely innervate the ventricles. Furthermore, parasympathetic and sympathetic nerves develop synchronously and are intertwined throughout the ventricles. Importantly, the regenerating myocardium reestablishes physiological innervation, in stark contrast to the non-regenerating heart. Mechanistically, reinnervation during regeneration is dependent on collateral artery formation. Our results reveal how defining cardiac nerve remodeling during homeostasis, disease, and regeneration can identify new therapies for cardiac disease.
RESUMEN
LIM-domain-containing repeat (LCR) proteins are recruited to strained actin filaments within stress fibers in cultured cells,1,2,3 but their roles at cell-cell junctions in living organisms have not been extensively studied. Here, we show that the Caenorhabditis elegans LCR proteins TES-1/Tes and ZYX-1/Zyxin are recruited to apical junctions during embryonic elongation when junctions are under tension. In genetic backgrounds in which embryonic elongation fails, junctional recruitment is severely compromised. The two proteins display complementary patterns of expression: TES-1 is expressed in lateral (seam) epidermal cells, whereas ZYX-1 is expressed in dorsal and ventral epidermal cells. tes-1 and zyx-1 mutant embryos display junctional F-actin defects. The loss of either protein strongly enhances morphogenetic defects in hypomorphic mutant backgrounds for cadherin/catenin complex (CCC) components. The LCR regions of TES-1 and ZYX-1 are recruited to stress fiber strain sites (SFSSs) in cultured vertebrate cells. Together, these data establish TES-1 and ZYX-1 as components of a multicellular, tension-sensitive system that stabilizes the junctional actin cytoskeleton during embryonic morphogenesis.
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Actinas , Caenorhabditis elegans , Animales , Actinas/genética , Caenorhabditis elegans/genéticaRESUMEN
Increased Aurora B protein expression, which is common in cancers, is expected to increase Aurora B kinase activity, yielding elevated phosphorylation of Aurora B substrates. In contrast, here we show that elevated expression of Aurora B reduces phosphorylation of six different Aurora B substrates across three species and causes defects consistent with Aurora B inhibition. Complexes of Aurora B and its binding partner INCENP autophosphorylate in trans to achieve full Aurora B activation. Increased expression of Aurora B mislocalizes INCENP, reducing the local concentration of Aurora B:INCENP complexes at the inner centromere/kinetochore. Co-expression of INCENP rescues Aurora B kinase activity and mitotic defects caused by elevated Aurora B. However, INCENP expression is not elevated in concert with Aurora B in breast cancer, and increased expression of Aurora B causes resistance rather than hypersensitivity to Aurora B inhibitors. Thus, increased Aurora B expression reduces, rather than increases, Aurora B kinase activity.
RESUMEN
Membrane remodeling mediated by heteropolymeric filaments composed of ESCRT-III subunits is an essential process that occurs at a variety of organelles to maintain cellular homeostasis. Members of the evolutionarily conserved Lgd/CC2D1 protein family have been suggested to regulate ESCRT-III polymer assembly, although their specific roles, particularly in vivo, remain unclear. Using the Caenorhabditis elegans early embryo as a model system, we show that Lgd/CC2D1 localizes to endosomal membranes, and its loss impairs endolysosomal cargo sorting and degradation. At the ultrastructural level, the absence of Lgd/CC2D1 results in the accumulation of enlarged endosomal compartments that contain a reduced number of intralumenal vesicles (ILVs). However, unlike aberrant endosome morphology caused by depletion of other ESCRT components, ILV size is only modestly altered in embryos lacking Lgd/CC2D1. Instead, loss of Lgd/CC2D1 impairs normal accumulation of ESCRT-III on endosomal membranes, likely slowing the kinetics of ILV formation. Together, our findings suggest a role for Lgd/CC2D1 in the recruitment and/or stable assembly of ESCRT-III subunits on endosomal membranes to facilitate efficient ILV biogenesis.
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Endosomas , Cuerpos Multivesiculares , Animales , Cuerpos Multivesiculares/metabolismo , Endosomas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Membranas Intracelulares/metabolismo , Transporte de Proteínas/fisiología , Caenorhabditis elegans/metabolismoRESUMEN
Molecular pathways that intrinsically regulate neuronal maintenance are poorly understood, but rare pathogenic mutations that underlie neurodegenerative disease can offer important insights into the mechanisms that facilitate lifelong neuronal function. Here, we leverage a rat model to demonstrate directly that the TFG p.R106C variant implicated previously in complicated forms of hereditary spastic paraplegia (HSP) underlies progressive spastic paraparesis with accompanying ventriculomegaly and thinning of the corpus callosum, consistent with disease phenotypes identified in adolescent patients. Analyses of primary cortical neurons obtained from CRISPR-Cas9-edited animals reveal a kinetic delay in biosynthetic secretory protein transport from the endoplasmic reticulum (ER), in agreement with prior induced pluripotent stem cell-based studies. Moreover, we identify an unexpected role for TFG in the trafficking of Rab4A-positive recycling endosomes specifically within axons and dendrites. Impaired TFG function compromises the transport of at least a subset of endosomal cargoes, which we show results in down-regulated inhibitory receptor signaling that may contribute to excitation-inhibition imbalances. In contrast, the morphology and trafficking of other organelles, including mitochondria and lysosomes, are unaffected by the TFG p.R106C mutation. Our findings demonstrate a multifaceted role for TFG in secretory and endosomal protein sorting that is unique to cells of the central nervous system and highlight the importance of these pathways to maintenance of corticospinal tract motor neurons.
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Endosomas , Neuronas Motoras , Transporte de Proteínas , Animales , Ratas , Retículo Endoplásmico/metabolismo , Endosomas/metabolismo , Neuronas Motoras/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Proteínas/metabolismo , Paraplejía Espástica Hereditaria/metabolismoRESUMEN
OBJECTIVES: In an effort to exploit the elevated need for phospholipids displayed by cancer cells relative to normal cells, we have developed tumor-targeted alkylphosphocholines (APCs) as broad-spectrum cancer imaging and therapy agents. Radioactive APC analogs have exhibited selective uptake and prolonged tumor retention in over 50 cancer types in preclinical models, as well as over 15 cancer types in over a dozen clinical trials. To push the structural limits of this platform, we recently added a chelating moiety capable of binding gadolinium and many other metals for cancer-targeted magnetic resonance imaging (MRI), positron emission tomography imaging, and targeted radionuclide therapy. The aim of this work was to synthesize, characterize, and validate the tumor selectivity of a new broad-spectrum, tumor-targeted, macrocyclic MRI chelate, Gd-NM600, in xenograft and orthotopic tumor models. A secondary aim was to identify and track the in vivo chemical speciation and spatial localization of this new chelate Gd-NM600 in order to assess its Gd deposition properties. MATERIALS AND METHODS: T1 relaxivities of Gd-NM600 were characterized in water and plasma at 1.5 T and 3.0 T. Tumor uptake and subcellular localization studies were performed using transmission electron microscopy. We imaged 8 different preclinical models of human cancer over time and compared the T1-weighted imaging results to that of a commercial macrocyclic Gd chelate, Gd-DOTA. Finally, matrix-assisted laser desorption and ionization-mass spectrometry imaging was used to characterize and map the tissue distribution of the chemical species of Gd-NM600. RESULTS: Gd-NM600 exhibits high T1 relaxivity (approximately 16.4 s-1/mM at 1.5 T), excellent tumor uptake (3.95 %ID/g at 48 hours), prolonged tumor retention (7 days), and MRI conspicuity. Moreover, minimal tumor uptake saturability of Gd-NM600 was observed. Broad-spectrum tumor-specific uptake was demonstrated in 8 different human cancer models. Cancer cell uptake of Gd-NM600 via endosomal internalization and processing was revealed with transmission electron microscopy. Importantly, tissue mass spectrometry imaging successfully interrogated the spatial localization and chemical speciation of Gd compounds and also identified breakdown products of Gd species. CONCLUSIONS: We have introduced a new macrocyclic cancer-targeted Gd chelate that achieves broad-spectrum tumor uptake and prolonged retention. Furthermore, we have demonstrated in vivo stability of Gd-NM600 by ultrahigh resolution MS tissue imaging. A tumor-targeted contrast agent coupled with the enhanced imaging resolution of MRI relative to positron emission tomography may transform oncologic imaging.
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Medios de Contraste , Neoplasias , Quelantes , Medios de Contraste/química , Gadolinio , Humanos , Imagen por Resonancia Magnética , Neoplasias/diagnóstico por imagenRESUMEN
The late-acting endosomal sorting complex required for transport (ESCRT) machinery has been implicated in facilitating the resealing of the nuclear envelope (NE) after mitosis, enabling compartmentalization of the genome away from the cytoplasm. Here, we leverage the stereotypic first division of the C. elegans embryo to identify additional functions of the ESCRT machinery in maintaining the structure of the inner nuclear membrane. Specifically, impaired ESCRT function results in a defect in the pruning of inner nuclear membrane invaginations, which arise normally during NE reformation and expansion. Additionally, in combination with a hypomorphic mutation that interferes with assembly of the underlying nuclear lamina, inhibition of ESCRT function significantly perturbs NE architecture and increases chromosome segregation defects, resulting in penetrant embryonic lethality. Our findings highlight links between ESCRT-mediated inner nuclear membrane remodeling, maintenance of nuclear envelope morphology, and the preservation of the genome during early development.
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Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Mitosis/fisiología , Membrana Nuclear/metabolismo , Animales , Caenorhabditis elegansRESUMEN
Using the endosomal sorting complex required for transport (ESCRT)-III membrane remodeling complex as an example, we analyze three popular coarse-grained models (the regular MARTINI, polarizable MARTINI (POL-MARTINI), and big multipole water MARTINI (BMW-MARTINI)) for the description of membrane curvature sensing and generation activities of peripheral proteins. Although the three variants of the MARTINI model provide consistent descriptions for the protein-protein interface in a linear filament model of ESCRT-III, they differ considerably in terms of protein-membrane interface and therefore membrane curvature sensing and generation behaviors. In particular, BMW-MARTINI provides the most consistent description of the protein-membrane interface as compared to all-atom simulations, whereas the regular MARTINI is most consistent with atomistic simulations in terms of the qualitative sign of membrane curvature sensing and generation. With POL-MARTINI, the ESCRT-III model interacts weakly with the membrane and therefore does not exhibit any curvature-sensitive activities. Analysis suggests that the incorrect membrane curvature activities predicted by BMW-MARTINI are due to overestimated insertion depth of an amphipathic helix and incorrect sign for the spontaneous curvature of anionic lipids. These results not only point to ways that coarse-grained models can be improved but also explicitly highlight local lipid composition and insertion depth of protein motifs as essential regulatory factors for membrane curvature sensing and generation.
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Simulación de Dinámica Molecular , Proteínas , Membrana Dobles de Lípidos , AguaRESUMEN
Nε-lysine acetylation in the ER is an essential component of the quality control machinery. ER acetylation is ensured by a membrane transporter, AT-1/SLC33A1, which translocates cytosolic acetyl-CoA into the ER lumen, and two acetyltransferases, ATase1 and ATase2, which acetylate nascent polypeptides within the ER lumen. Dysfunctional AT-1, as caused by gene mutation or duplication events, results in severe disease phenotypes. Here, we used two models of AT-1 dysregulation to investigate dynamics of the secretory pathway: AT-1 sTg, a model of systemic AT-1 overexpression, and AT-1S113R/+, a model of AT-1 haploinsufficiency. The animals displayed reorganization of the ER, ERGIC, and Golgi apparatus. In particular, AT-1 sTg animals displayed a marked delay in Golgi-to-plasma membrane protein trafficking, significant alterations in Golgi-based N-glycan modification, and a marked expansion of the lysosomal network. Collectively our results indicate that AT-1 is essential to maintain proper organization and engagement of the secretory pathway.
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Acetilcoenzima A/genética , Acetiltransferasas/genética , Retículo Endoplásmico/genética , Proteínas de Transporte de Membrana/genética , Acetilcoenzima A/metabolismo , Acetilación , Autofagia/genética , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica/genética , Aparato de Golgi/genética , Aparato de Golgi/patología , Haploinsuficiencia/genética , Humanos , Lisosomas/genética , Mutación/genética , Procesamiento Proteico-Postraduccional/genética , Transporte de Proteínas/genética , Vías Secretoras/genéticaRESUMEN
The emergence of drug-resistant fungi has prompted an urgent threat alert from the US Centers for Disease Control (CDC). Biofilm assembly by these pathogens further impairs effective therapy. We recently identified an antifungal, turbinmicin, that inhibits the fungal vesicle-mediated trafficking pathway and demonstrates broad-spectrum activity against planktonically growing fungi. During biofilm growth, vesicles with unique features play a critical role in the delivery of biofilm extracellular matrix components. As these components are largely responsible for the drug resistance associated with biofilm growth, we explored the utility of turbinmicin in the biofilm setting. We found that turbinmicin disrupted extracellular vesicle (EV) delivery during biofilm growth and that this impaired the subsequent assembly of the biofilm matrix. We demonstrated that elimination of the extracellular matrix rendered the drug-resistant biofilm communities susceptible to fungal killing by turbinmicin. Furthermore, the addition of turbinmicin to otherwise ineffective antifungal therapy potentiated the activity of these drugs. The underlying role of vesicles explains this dramatic activity and was supported by phenotype reversal with the addition of exogenous biofilm EVs. This striking capacity to cripple biofilm assembly mechanisms reveals a new approach to eradicating biofilms and sheds light on turbinmicin as a promising anti-biofilm drug.
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Benzopiranos/farmacología , Biopelículas/efectos de los fármacos , Candida/fisiología , Vesículas Extracelulares/metabolismo , Isoquinolinas/farmacología , Biopelículas/crecimiento & desarrolloRESUMEN
New antifungal drugs are urgently needed to address the emergence and transcontinental spread of fungal infectious diseases, such as pandrug-resistant Candida auris. Leveraging the microbiomes of marine animals and cutting-edge metabolomics and genomic tools, we identified encouraging lead antifungal molecules with in vivo efficacy. The most promising lead, turbinmicin, displays potent in vitro and mouse-model efficacy toward multiple-drug-resistant fungal pathogens, exhibits a wide safety index, and functions through a fungal-specific mode of action, targeting Sec14 of the vesicular trafficking pathway. The efficacy, safety, and mode of action distinct from other antifungal drugs make turbinmicin a highly promising antifungal drug lead to help address devastating global fungal pathogens such as C. auris.
Asunto(s)
Antifúngicos/farmacología , Benzopiranos/farmacología , Candida/efectos de los fármacos , Candidiasis Invasiva/tratamiento farmacológico , Farmacorresistencia Fúngica Múltiple , Isoquinolinas/farmacología , Micromonospora/química , Urocordados/microbiología , Animales , Antifúngicos/química , Antifúngicos/uso terapéutico , Benzopiranos/química , Benzopiranos/uso terapéutico , Modelos Animales de Enfermedad , Proteínas Fúngicas/metabolismo , Isoquinolinas/química , Isoquinolinas/uso terapéutico , Ratones , Microbiota , Proteínas de Transferencia de Fosfolípidos/metabolismoRESUMEN
The nuclear permeability barrier depends on closure of nuclear envelope (NE) holes. Here, we investigate closure of the NE opening surrounding the meiotic spindle in C. elegans oocytes. ESCRT-III components accumulate at the opening but are not required for nuclear closure on their own. 3D analysis revealed cytoplasmic membranes directly adjacent to NE holes containing meiotic spindle microtubules. We demonstrate that the NE protein phosphatase, CNEP-1/CTDNEP1, controls de novo glycerolipid synthesis through lipin to prevent invasion of excess ER membranes into NE holes and a defective NE permeability barrier. Loss of NE adaptors for ESCRT-III exacerbates ER invasion and nuclear permeability defects in cnep-1 mutants, suggesting that ESCRTs restrict excess ER membranes during NE closure. Restoring glycerolipid synthesis in embryos deleted for CNEP-1 and ESCRT components rescued NE permeability defects. Thus, regulating the production and feeding of ER membranes into NE holes together with ESCRT-mediated remodeling is required for nuclear closure.