Clathrin binding by the adaptor Ent5 promotes late stages of clathrin coat maturation

Clathrin is a ubiquitous protein that mediates membrane traffic at many locations. To function, clathrin requires clathrin adaptors that link it to transmembrane protein cargo. In addition to this cargo selection function, many adaptors also play mechanistic roles in the formation of the transport carrier. However, the full spectrum of these mechanistic roles is poorly understood. Here we report that Ent5, an endosomal clathrin adaptor in Saccharomyces cerevisiae, regulates the behavior of clathrin coats after the recruitment of clathrin. We show that loss of Ent5 disrupts clathrin-dependent traffic and prolongs the lifespan of endosomal structures that contain clathrin and other adaptors, suggesting a defect in coat maturation at a late stage. We find that the direct binding of Ent5 with clathrin is required for its role in coat behavior and cargo traffic. Surprisingly, the interaction of Ent5 with other adaptors is dispensable for coat behavior but not cargo traffic. These findings support a model in which Ent5 clathrin binding performs a mechanistic role in coat maturation, whereas Ent5 adaptor binding promotes cargo incorporation

Regulating polarity by directing traffic: Cdc42 prevents adherens junctions from Crumblin' aPart

The GTPase Cdc42 was among the original genes identified with roles in cell polarity, and interest in its cellular roles from yeast to humans remains high. Cdc42 is a well-known regulator of the actin cytoskeleton, but also plays important roles in vesicular trafficking. In this issue, Harris and Tepass (Harris, K.P, and U. Tepass. 2008. J. Cell. Biol. 183:1129–1143) provide new insights into how Cdc42 and Par proteins work together to modulate cell adhesion and polarity during embryonic morphogenesis by regulating the traffic of key cell junction proteins

Exomer complex regulates protein traffic at the TGN through differential interactions with cargos and clathrin adaptor complexes

[EN] Protein sorting at the trans-Golgi network (TGN) usually requires the assistance of cargo adaptors. However, it remains to be examined how the same complex can mediate both the export and retention of different proteins or how sorting complexes interact among themselves. In Saccharomyces cerevisiae, the exomer complex is involved in the polarized transport of some proteins from the TGN to the plasma membrane (PM). Intriguingly, exomer and its cargos also show a sort of functional relationship with TGN clathrin adaptors that is still unsolved. Here, using a wide range of techniques, including time-lapse and BIFC microscopy, we describe new molecular implications of the exomer complex in protein sorting and address its different layers of functional interaction with clathrin adaptor complexes. Exomer mutants show impaired amino acid uptake because it facilitates not only the polarized delivery of amino acid permeases to the PM but also participates in their endosomal traffic. We propose a model for exomer where it modulates the recruitment of TGN clathrin adaptors directly or indirectly through the Arf1 function. Moreover, we describe an in vivo competitive relationship between the exomer and AP-1 complexes for the model cargo Chs3. These results highlight a broad role for exomer in regulating protein sorting at the TGN that is complementary to its role as cargo adaptor and present a model to understand the complexity of TGN protein sorting.Ministerio de Economía, Industria y Competitividad, Gobierno de España (Ministeri d'Economia, Indústria i Competitivitat), Grant/Award Number: CICYT/FEDER BFU2017-84508-P; Consejería de Educación, Junta de Castilla y León (Ministry of Education, Government of Castile-Leon), Grant/Award Number: SA116G19; Ministerio de Economía, Industria y Competitividad, Gobierno de España (Ministeri d'Economia, Indústria i Competitivitat), Grant/Award Number: RTC-2017-6468-2-AR; Ministerio de Economía, Industria y Competitividad, Gobierno de España (Ministeri d'Economia, Indústria i Competitivitat), Grant/Award Number: BIO2016-77776-P; Foundation for the National Institutes of Health (FNIH), Grant/Award Number: R01 GM092741Anton-Plagaro, C.; Sánchez, N.; Valle, R.; Mulet, JM.; Duncan, MC.; Roncero, C. (2021). Exomer complex regulates protein traffic at the TGN through differential interactions with cargos and clathrin adaptor complexes. The FASEB Journal. 35(6):1-26. https://doi.org/10.1096/fj.202002610R12635

Energy metabolism regulates clathrin adaptors at the trans -Golgi network and endosomes

Localization of endosomal clathrin adaptors is regulated by glucose availability via an unknown mechanism. Studies in intact and permeabilized cells show that clathrin adaptor localization is precisely tuned by cellular ATP concentration. These data provide evidence for how membrane traffic is coordinated with overall proliferation rates.Glucose is a master regulator of cell behavior in the yeast Saccharomyces cerevisiae. It acts as both a metabolic substrate and a potent regulator of intracellular signaling cascades. Glucose starvation induces the transient delocalization and then partial relocalization of clathrin adaptors at the trans-Golgi network and endosomes. Although these localization responses are known to depend on the protein kinase A (PKA) signaling pathway, the molecular mechanism of this regulation is unknown. Here we demonstrate that PKA and the AMP-regulated kinase regulate adaptor localization through changes in energy metabolism. We show that genetic and chemical manipulation of intracellular ATP levels cause corresponding changes in adaptor localization. In permeabilized cells, exogenous ATP is sufficient to induce adaptor localization. Furthermore, we reveal distinct energy-dependent steps in adaptor localization: a step that requires the ADP-ribosylation factor ARF, an ATP-dependent step that requires the phosphatidyl-inositol-4 kinase Pik1, and third ATP-dependent step for which we provide evidence but for which the mechanism is unknown. We propose that these energy-dependent mechanisms precisely synchronize membrane traffic with overall proliferation rates and contribute a crucial aspect of energy conservation during acute glucose starvation

The actin-microtubule cross-linking activity of Drosophila Short stop is regulated by intramolecular inhibition

Actin and microtubule dynamics must be precisely coordinated during cell migration, mitosis, and morphogenesis—much of this coordination is mediated by proteins that physically bridge the two cytoskeletal networks. We have investigated the regulation of the Drosophila actin-microtubule cross-linker Short stop (Shot), a member of the spectraplakin family. Our data suggest that Shot's cytoskeletal cross-linking activity is regulated by an intramolecular inhibitory mechanism. In its inactive conformation, Shot adopts a “closed” conformation through interactions between its NH2-terminal actin-binding domain and COOH-terminal EF-hand-GAS2 domain. This inactive conformation is targeted to the growing microtubule plus end by EB1. On activation, Shot binds along the microtubule through its COOH-terminal GAS2 domain and binds to actin with its NH2-terminal tandem CH domains. We propose that this mechanism allows Shot to rapidly cross-link dynamic microtubules in response to localized activating signals at the cell cortex

Adaptor Autoregulation Promotes Coordinated Binding within Clathrin Coats

Membrane traffic is an essential process that allows protein and lipid exchange between the endocytic, lysosomal, and secretory compartments. Clathrin-mediated traffic between the trans-Golgi network and endosomes mediates responses to the environment through the sorting of biosynthetic and endocytic protein cargo. Traffic through this pathway is initiated by the controlled assembly of a clathrin-adaptor protein coat on the cytosolic surface of the originating organelle. In this process, clathrin is recruited by different adaptor proteins that act as a bridge between clathrin and the transmembrane cargo proteins to be transported. Interactions between adaptors and clathrin and between different types of adaptors lead to the formation of a densely packed protein network within the coat. A key unresolved issue is how the highly complex adaptor-clathrin interaction and adaptor-adaptor interaction landscape lead to the correct spatiotemporal assembly of the clathrin coat. Here we report the discovery of a new autoregulatory motif within the clathrin adaptor Gga2 that drives synergistic binding of Gga2 to clathrin and the adaptor Ent5. This autoregulation influences the temporal and/or spatial location of the Gga2-Ent5 interaction. We propose that this synergistic binding provides built-in regulation to ensure the correct assembly of clathrin coats

The 31 Deg2^2 Release of the Stripe 82 X-ray Survey: The Point Source Catalog

We release the next installment of the Stripe 82 X-ray survey point-source catalog, which currently covers 31.3 deg$^2$ of the Sloan Digital Sky Survey (SDSS) Stripe 82 Legacy field. In total, 6181 unique X-ray sources are significantly detected with {\it XMM-Newton} ($>5\sigma$) and {\it Chandra} ($>4.5\sigma$). This catalog release includes data from {\it XMM-Newton} cycle AO 13, which approximately doubled the Stripe 82X survey area. The flux limits of the Stripe 82X survey are $8.7\times10^{-16}$ erg s$^{-1}$ cm$^{-2}$, $4.7\times10^{-15}$ erg s$^{-1}$ cm$^{-2}$, and $2.1\times10^{-15}$ erg s$^{-1}$ cm$^{-2}$ in the soft (0.5-2 keV), hard (2-10 keV), and full bands (0.5-10 keV), respectively, with approximate half-area survey flux limits of $5.4\times10^{-15}$ erg s$^{-1}$ cm$^{-2}$, $2.9\times10^{-14}$ erg s$^{-1}$ cm$^{-2}$, and $1.7\times10^{-14}$ erg s$^{-1}$ cm$^{-2}$. We matched the X-ray source lists to available multi-wavelength catalogs, including updated matches to the previous release of the Stripe 82X survey; 88\% of the sample is matched to a multi-wavelength counterpart. Due to the wide area of Stripe 82X and rich ancillary multi-wavelength data, including coadded SDSS photometry, mid-infrared {\it WISE} coverage, near-infrared coverage from UKIDSS and VHS, ultraviolet coverage from {\it GALEX}, radio coverage from FIRST, and far-infrared coverage from {\it Herschel}, as well as existing $\sim$30\% optical spectroscopic completeness, we are beginning to uncover rare objects, such as obscured high-luminosity AGN at high-redshift. The Stripe 82X point source catalog is a valuable dataset for constraining how this population grows and evolves, as well as for studying how they interact with the galaxies in which they live.Comment: accepted for publication in ApJ; 23 pages (emulateapj

Glucose Starvation Inhibits Autophagy via Vacuolar Hydrolysis and Induces Plasma Membrane Internalization by Down-regulating Recycling

Cellular energy influences all aspects of cellular function. Although cells can adapt to a gradual reduction in energy, acute energy depletion poses a unique challenge. Because acute depletion hampers the transport of new energy sources into the cell, the cell must use endogenous substrates to replenish energy after acute depletion. In the yeast Saccharomyces cerevisiae, glucose starvation causes an acute depletion of intracellular energy that recovers during continued glucose starvation. However, how the cell replenishes energy during the early phase of glucose starvation is unknown. In this study, we investigated the role of pathways that deliver proteins and lipids to the vacuole during glucose starvation. We report that in response to glucose starvation, plasma membrane proteins are directed to the vacuole through reduced recycling at the endosomes. Furthermore, we found that vacuolar hydrolysis inhibits macroautophagy in a target of rapamycin complex 1-dependent manner. Accordingly, we found that endocytosis and hydrolysis are required for survival in glucose starvation, whereas macroautophagy is dispensable. Together, these results suggest that hydrolysis of components delivered to the vacuole independent of autophagy is the cell survival mechanism used by S. cerevisiae in response to glucose starvation

The Hunt for Red Quasars : Luminous Obscured Black Hole Growth Unveiled in the Stripe 82 X-Ray Survey

We present results of a ground-based near-infrared campaign with Palomar TripleSpec, Keck NIRSPEC, and Gemini GNIRS to target two samples of reddened active galactic nucleus (AGN) candidates from the 31 deg(2) Stripe 82 X-ray survey. One sample, which is similar to 89% complete to K 4, Vega). The fainter sample (K > 17, Vega) represents a pilot program to follow-up four sources from a parent sample of 34 that are not detected in the single-epoch SDSS catalog and have WISE quasar colors. All 12 sources are broad-line AGNs (at least one permitted emission line has an FWHM exceeding 1300 km s(-1)) and span a redshift range 0.59 0.5), and a greater percentage have high X-ray luminosities (L-X,L- full > 10(44) erg s(-1)). Such outflows and high luminosities may be consistent with the paradigm that reddened broad-line AGNs represent a transitory phase in AGN evolution as described by the major merger model for black hole growth. Results from our pilot program demonstrate proof of concept that our selection technique is successful in discovering reddened quasars at z > 1 missed by optical surveys.Peer reviewe

High-Frequency Oscillatory Ventilation Use and Severe Pediatric ARDS in the Pediatric Hematopoietic Cell Transplant Recipient

INTRODUCTION: The effectiveness of high-frequency oscillatory ventilation (HFOV) in the pediatric hematopoietic cell transplant patient has not been established. We sought to identify current practice patterns of HFOV, investigate parameters during HFOV and their association with mortality, and compare the use of HFOV to conventional mechanical ventilation in severe pediatric ARDS. METHODS: This is a retrospective analysis of a multi-center database of pediatric and young adult allogeneic hematopoietic cell transplant subjects requiring invasive mechanical ventilation for critical illness from 2009 through 2014. Twelve United States pediatric centers contributed data. Continuous variables were compared using a Wilcoxon rank-sum test or a Kruskal-Wallis analysis. For categorical variables, univariate analysis with logistic regression was performed. RESULTS: The database contains 222 patients, of which 85 subjects were managed with HFOV. Of this HFOV cohort, the overall pediatric ICU survival was 23.5% (n = 20). HFOV survivors were transitioned to HFOV at a lower oxygenation index than nonsurvivors (25.6, interquartile range 21.1-36.8, vs 37.2, interquartile range 26.5-52.2, P = .046). Survivors were transitioned to HFOV earlier in the course of mechanical ventilation, (day 0 vs day 2, P = .002). No subject survived who was transitioned to HFOV after 1 week of invasive mechanical ventilation. We compared subjects with severe pediatric ARDS treated only with conventional mechanical ventilation versus early HFOV (within 2 d of invasive mechanical ventilation) versus late HFOV. There was a trend toward difference in survival (conventional mechanical ventilation 24%, early HFOV 30%, and late HFOV 9%, P = .08). CONCLUSIONS: In this large database of pediatric allogeneic hematopoietic cell transplant subjects who had acute respiratory failure requiring invasive mechanical ventilation for critical illness with severe pediatric ARDS, early use of HFOV was associated with improved survival compared to late implementation of HFOV, and the subjects had outcomes similar to those treated only with conventional mechanical ventilation