According to a new survey of residents living near two major national parks in Panama, jaguars deserve increased protection. Nature and wildlife are considered national […]

The post Jaguar conservation depends on neighbor attitudes appeared first on Smithsonian Insider.

In the past 50 years, the amount of water in the open ocean with zero oxygen has increased more than fourfold. In coastal water bodies, […]

The post Earth’s oceans are losing their breath. Here’s the global scope appeared first on Smithsonian Insider.

In 1854, a curious-looking spider was found preserved in 50 million-year-old amber. With an elongated neck-like structure and long mouthparts that protruded from the “head” […]

The post These newly discovered pelican spiders will make you want to visit Madagascar appeared first on Smithsonian Insider.

Flitting swiftly through the darkness above the tropical forest canopy in Central and South America, a group of cute little bats with dog-like faces have […]

The post More sky puppies! Scientists discover two new species of dog-faced bat appeared first on Smithsonian Insider.

“Somber” is the adjective Smithsonian beetle expert Terry Erwin uses to describe the insects he collects on the forest floor in Peru and Ecuador. “They […]

The post Meet the newest New World canopy beetle species. ‘Gazillions’ await discovery. appeared first on Smithsonian Insider.

There are currently more than 3,500 confirmed known exoplanets thanks to the remarkable sensitivity of the Kepler spacecraft and to technological advances in space and […]

The post Astronomers detect comets transiting distant stars appeared first on Smithsonian Insider.

Some of Hong Kong’s most disturbed and pristine marine environments were the recent focus of an intense 10-day MarineGEO survey of living organisms by a […]

The post MarineGEO survey conducted in Hong Kong appeared first on Smithsonian Insider.

The Sun glows with a surface temperature of about 5,500 degrees Celsius (9,932 degrees Fahrenheit). On the other hand its hot outer layer, the corona, […]

The post Magnetic reconnection in the sun appeared first on Smithsonian Insider.

Every day, astronomers at the Harvard-Smithsonian Center for Astrophysics depend on computers to help them solve the mysteries of the universe, just as they did […]

The post Underpaid women “computers” mapped the universe in the 19th century appeared first on Smithsonian Insider.

This winter has brought many intense and powerful storms, with cold fronts sweeping across much of the United States. On a much grander scale, astronomers […]

The post Scientists surprised by relentless cosmic cold front appeared first on Smithsonian Insider.

Animal species can become endangered, but what about languages? Around 80 percent of the world’s population speaks just 20 percent of its 7,000 languages. That […]

The post Why languages become endangered, and how we can keep them alive appeared first on Smithsonian Insider.

When a natural disaster strikes, it devastates lives and homes, and can even destroy a culture’s identity and history. After a disaster, humanitarian response is […]

The post Protecting Puerto Rico’s heritage from another disaster appeared first on Smithsonian Insider.

Very early on, cockroach moms found out maternal care gave their offspring a better chance at survival. The cockroach parenting method—which includes feeding, guarding and […]

The post Helicopter cockroach moms have protected their young for millions of years appeared first on Smithsonian Insider.

Five new species of snail-eating snake, from a group of snakes affectionately known to scientists as “goo-eaters,” have been discovered by a team working in […]

The post Newly discovered snakes use curved teeth to pry snails from their shells appeared first on Smithsonian Insider.

When many people think of the Chesapeake Bay, one of the first creatures that comes to mind is the iconic blue crab. But parts of […]

The post Smithsonian scientists become shark detectives to track species in the Chesapeake Bay appeared first on Smithsonian Insider.

l-Hydroxyproline (l-Hyp) is a nonstandard amino acid that is present in certain proteins, in some antibiotics and in the cell-wall components of plants. l-Hyp is the product of the post-translational modification of protein prolines by prolyl hydroxylase enzymes, and the isomers trans-3-hydroxy-l-proline (T3LHyp) and trans-4-hydroxy-l-proline (T4LHyp) are major components of mammalian collagen. T4LHyp follows two distinct degradation pathways in bacteria and mammals, while T3LHyp is metabolized by a two-step metabolic pathway that is conserved in bacteria and mammals, which involves a T3LHyp dehydratase and a Δ1-pyrroline-2-carboxylate (Pyr2C) reductase. In order to shed light on the structure and catalysis of the enzyme involved in the second step of the T3LHyp degradation pathway, the crystal structure of Pyr2C reductase from the archaeon Thermococcus litoralis DSM 5473 complexed with NADH and l-proline is presented. The model allows the mapping of the residues involved in cofactor and product binding and represents a valid model for rationalizing the catalysis of Pyr2C reductases.

The biological processes related to protein homeostasis in Mycobacterium tuberculosis, the etiologic agent of tuberculosis, have recently been established as critical pathways for therapeutic intervention. Proteins of particular interest are ClpC1 and the ClpC1–ClpP1–ClpP2 proteasome complex. The structure of the potent antituberculosis macrocyclic depsipeptide ecumicin complexed with the N-terminal domain of ClpC1 (ClpC1-NTD) is presented here. Crystals of the ClpC1-NTD–ecumicin complex were monoclinic (unit-cell parameters a = 80.0, b = 130.0, c = 112.0 Å, β = 90.07°; space group P21; 12 complexes per asymmetric unit) and diffracted to 2.5 Å resolution. The structure was solved by molecular replacement using the self-rotation function to resolve space-group ambiguities. The new structure of the ecumicin complex showed a unique 1:2 (target:ligand) stoichiometry exploiting the intramolecular dyad in the α-helical fold of the target N-terminal domain. The structure of the ecumicin complex unveiled extensive interactions in the uniquely extended N-terminus, a critical binding site for the known cyclopeptide complexes. This structure, in comparison with the previously reported rufomycin I complex, revealed unique features that could be relevant for understanding the mechanism of action of these potential antituberculosis drug leads. Comparison of the ecumicin complex and the ClpC1-NTD-L92S/L96P double-mutant structure with the available structures of rufomycin I and cyclomarin A complexes revealed a range of conformational changes available to this small N-terminal helical domain and the minor helical alterations involved in the antibiotic-resistance mechanism. The different modes of binding and structural alterations could be related to distinct modes of action.

The bond-valence method has been used for valence calculations of FeMo/V cofactors in FeMo/V proteins using 51 crystallographic data sets of FeMo/V proteins from the Protein Data Bank. The calculations show molybdenum(III) to be present in MoFe7S9C(Cys)(HHis)[R-(H)homocit] (where H4homocit is homocitric acid, HCys is cysteine and HHis is histidine) in FeMo cofactors, while vanadium(III) with a more reduced iron complement is obtained for FeV cofactors. Using an error analysis of the calculated valences, it was found that in FeMo cofactors Fe1, Fe6 and Fe7 can be unambiguously assigned as iron(III), while Fe2, Fe3, Fe4 and Fe5 show different degrees of mixed valences for the individual Fe atoms. For the FeV cofactors in PDB entry 5n6y, Fe4, Fe5 and Fe6 correspond to iron(II), iron(II) and iron(III), respectively, while Fe1, Fe2, Fe3 and Fe7 exhibit strongly mixed valences. Special situations such as CO-bound and selenium-substituted FeMo cofactors and O(N)H-bridged FeV cofactors are also discussed and suggest rearrangement of the electron configuration on the substitution of the bridging S atoms.

Mycoplasma hyopneumoniae is a prokaryotic pathogen that colonizes the respiratory ciliated epithelial cells in swine. Infected animals suffer respiratory lesions, causing major economic losses in the porcine industry. Characterization of the immunodominant membrane-associated proteins from M. hyopneumoniae may be instrumental in the development of new therapeutic approaches. Here, the crystal structure of P46, one of the main surface-antigen proteins, from M. hyopneumoniae is presented and shows N- and C-terminal α/β domains connected by a hinge. The structures solved in this work include a ligand-free open form of P46 (3.1 Å resolution) and two ligand-bound structures of P46 with maltose (2.5 Å resolution) and xylose (3.5 Å resolution) in open and closed conformations, respectively. The ligand-binding site is buried in the cleft between the domains at the hinge region. The two domains of P46 can rotate with respect to each other, giving open or closed alternative conformations. In agreement with this structural information, sequence analyses show similarities to substrate-binding members of the ABC transporter superfamily, with P46 facing the extracellular side as a functional subunit. In the structure with xylose, P46 was also bound to a high-affinity (Kd = 29 nM) Fab fragment from a monoclonal antibody, allowing the characterization of a structural epitope in P46 that exclusively involves residues from the C-terminal domain. The Fab structure in the complex with P46 shows only small conformational rearrangements in the six complementarity-determining regions (CDRs) with respect to the unbound Fab (the structure of which is also determined in this work at 1.95 Å resolution). The structural information that is now available should contribute to a better understanding of sugar nutrient intake by M. hyopneumoniae. This information will also allow the design of protocols and strategies for the generation of new vaccines against this important swine pathogen.

Leucocyte common antigen-related protein (LAR) is a post-synaptic type I transmembrane receptor protein that is important for neuronal functionality and is genetically coupled to neuronal disorders such as attention deficit hyperactivity disorder (ADHD). To understand the molecular function of LAR, structural and biochemical studies of protein fragments derived from the ectodomain of human LAR have been performed. The crystal structure of a fragment encompassing the first four FNIII domains (LARFN1–4) showed a characteristic L shape. SAXS data suggested limited flexibility within LARFN1–4, while rigid-body refinement of the SAXS data using the X-ray-derived atomic model showed a smaller angle between the domains defining the L shape compared with the crystal structure. The capabilities of the individual LAR fragments to interact with heparin was examined using microscale thermophoresis and heparin-affinity chromatography. The results showed that the three N-terminal immunoglobulin domains (LARIg1–3) and the four C-terminal FNIII domains (LARFN5–8) both bound heparin, while LARFN1–4 did not. The low-molecular-weight heparin drug Innohep induced a shift in hydrodynamic volume as assessed by size-exclusion chromatography of LARIg1–3 and LARFN5–8, while the chemically defined pentameric heparin drug Arixtra did not. Together, the presented results suggest the presence of an additional heparin-binding site in human LAR.

The new book Subsistence Economies of Indigenous North American Societies provides a comprehensive and in-depth documentation of how Native American societies met the challenges of […]

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Only the Wing is a new book by Russell Lee that recounts Horten's epic quest to stabalize and control the all-wing aircraft.

The post New Book: “Only the Wing: Reimar Horten’s Epic Quest to Stabilize and Control the All-Wing Aircraft” appeared first on Smithsonian Insider.

Despite the importance of seasonally dry forests, little is known of their ecology. Now, a new book The Ecology and Conservation of Seasonally Dry Forests in Asia, published by Smithsonian Institution Scholarly Press, explores these unique ecosystems, its animals, plants, and the people that inhabit them.

The post New book: The Ecology and Conservation of Seasonally Dry Forests in Asia appeared first on Smithsonian Insider.

Between the world wars of the early Twentieth Century, an age of adventure travel and cultural exploration flourished when newly developed transport and recording technologies–particularly […]

The post New Book: “Recreating First Contact: Expeditions, Anthropology, and Popular Culture” appeared first on Smithsonian Insider.

Eric Peterman, Mindaugas Valius, and Rytis Prekeris

During mitotic cell division, the actomyosin cytoskeleton undergoes several dynamic changes that play key roles in progression through mitosis. While the regulators of cytokinetic ring formation and contraction are well-established, proteins that regulate cortical stability during anaphase and telophase have been understudied. Here, we describe a role for CLIC4 in regulating actin and actin-regulators at the cortex and cytokinetic cleavage furrow during cytokinesis. We first describe CLIC4 as a new component of the cytokinetic cleavage furrow that is required for successful completion of mitotic cell division. We also demonstrate that CLIC4 regulates the remodeling of sub-plasma membrane actomyosin network within the furrow by recruiting MST4 kinase and regulating ezrin phosphorylation. This work identifies and characterizes new molecular players involved in regulating cortex stiffness and blebbing during late stages of cytokinetic furrowing.

Laura O'Regan, Giancarlo Barone, Rozita Adib, Chang Gok Woo, Hui Jeong Jeong, Emily L. Richardson, Mark W. Richards, Patricia A.J. Muller, Spencer J. Collis, Dean A. Fennell, Jene Choi, Richard Bayliss, and Andrew M. Fry

EML4-ALK is an oncogenic fusion present in ~5% non-small cell lung cancers. However, alternative breakpoints in the EML4 gene lead to distinct variants with different patient outcomes. Here, we show in cell models that EML4-ALK variant 3 (V3), which is linked to accelerated metastatic spread, causes microtubule stabilization, formation of extended cytoplasmic protrusions and increased cell migration. It also recruits the NEK9 and NEK7 kinase to microtubules via the N-terminal EML4 microtubule-binding region. Overexpression of wild-type EML4 as well as constitutive activation of NEK9 also perturb cell morphology and accelerate migration in a microtubule-dependent manner that requires the downstream kinase NEK7 but not ALK activity. Strikingly, elevated NEK9 expression is associated with reduced progression-free survival in EML4-ALK patients. Hence, we propose that EML4-ALK V3 promotes microtubule stabilization through NEK9 and NEK7 leading to increased cell migration. This represents a novel actionable pathway that could drive metastatic disease progression in EML4-ALK lung cancer.

Osiris Martinez-Guzman, Mathilda M. Willoughby, Arushi Saini, Jonathan V. Dietz, Iryna Bohovych, Amy E. Medlock, Oleh Khalimonchuk, and Amit R. Reddi

Heme is a cofactor and signaling molecule that is essential for much of aerobic life. All heme-dependent processes in eukaryotes require that heme is trafficked from its site of synthesis in the mitochondria to hemoproteins located throughout the cell. However, the mechanisms governing the mobilization of heme out of the mitochondria, and the spatio-temporal dynamics of these processes, are poorly understood. Herein, using genetically encoded fluorescent heme sensors, we developed a live cell assay to monitor heme distribution dynamics between the mitochondrial inner-membrane, where heme is synthesized, and the mitochondrial matrix, cytosol, and nucleus. Surprisingly, heme trafficking to the nucleus is ~25% faster than to the cytosol or mitochondrial matrix, which are nearly identical, potentially supporting a role for heme as a mitochondrial-nuclear retrograde signal. Moreover, we discovered that the heme synthetic enzyme, 5-aminolevulinic acid synthase (ALAS), and GTPases in control of the mitochondrial dynamics machinery, Mgm1 and Dnm1, and ER contact sites, Gem1, regulate the flow of heme between the mitochondria and nucleus. Overall, our results indicate that there are parallel pathways for the distribution of bioavailable heme.

Christian Renz, Veronique Albanese, Vera Tröster, Thomas K. Albert, Olivier Santt, Susan C. Jacobs, Anton Khmelinskii, Sebastien Leon, and Helle D. Ulrich

Polyubiquitin chains linked via lysine (K) 63 play an important role in endocytosis and membrane trafficking. Their primary source is the ubiquitin protein ligase (E3) Rsp5/NEDD4, which acts as a key regulator of membrane protein sorting. The heterodimeric ubiquitin-conjugating enzyme (E2), Ubc13-Mms2, catalyses K63-specific polyubiquitylation in genome maintenance and inflammatory signalling. In budding yeast, the only ubiquitin protein ligase (E3) known to cooperate with Ubc13-Mms2 so far is a nuclear RING finger protein, Rad5, involved in the replication of damaged DNA. We now report a contribution of Ubc13-Mms2 to the sorting of membrane proteins to the yeast vacuole via the multivesicular body (MVB) pathway. In this context, Ubc13-Mms2 cooperates with Pib1, a FYVE-RING finger protein associated with internal membranes. Moreover, we identified a family of membrane-associated FYVE-(type)-RING finger proteins as cognate E3s for Ubc13-Mms2 in several species, and genetic analysis indicates that the contribution of Ubc13-Mms2 to membrane trafficking in budding yeast goes beyond its cooperation with Pib1. Thus, our results widely implicate Ubc13-Mms2 as an Rsp5-independent source of K63-linked polyubiquitin chains in the regulation of membrane protein sorting.

Hem Sapkota, Jonathan D. Wren, and Gary J. Gorbsky

Centrosomes focus microtubules to promote mitotic spindle bipolarity, a critical requirement for balanced chromosome segregation. Comprehensive understanding of centrosome function and regulation requires a complete inventory of components. While many centrosome components have been identified, others may yet remain undiscovered. We have used a bioinformatics approach, based on "guilt by association" expression to identify novel mitotic components among the large group of predicted human proteins that have yet to be functionally characterized. Here we identify Chondrosarcoma-Associated Gene 1 (CSAG1) in maintaining centrosome integrity during mitosis. Depletion of CSAG1 disrupts centrosomes and leads to multipolar spindles more effectively in cells with compromised p53 function. Thus, CSAG1 may reflect a class of "mitotic addiction" genes whose expression is more essential in transformed cells.

Aline A. Alves, Heloisa B. Gabriel, Maria J. R. Bezerra, Wanderley de Souza, Sue Vaughan, Narcisa L. Cunha-e-Silva, and Jack D. Sunter

Eukaryotic flagella are complex microtubule based organelles and in many organisms there are extra-axonemal structures present, including the outer dense fibres of mammalian sperm and the paraflagellar rod (PFR) of trypanosomes. Flagellum assembly is a complex process occurring across three main compartments, the cytoplasm, the transition fibre-transition zone, and the flagellum. It begins with translation of protein components, followed by their sorting and trafficking into the flagellum, transport to the assembly site and then incorporation. Flagella are formed from over 500 proteins; the principles governing axonemal component assembly are relatively clear. However, the coordination and sites of extra-axonemal structure assembly processes are less clear.

We have discovered two cytoplasmic proteins in T. brucei that are required for PFR formation, PFR assembly factors 1 and 2. Deletion of either PFR-AF1 or PFR-AF2 dramatically disrupted PFR formation and caused a reduction in the amount of major PFR proteins. The presence of cytoplasmic factors required for PFR formation aligns with the concept of processes occurring across multiple compartments to facilitate axoneme assembly and this is likely a common theme for extra-axonemal structure assembly.

Su Ming Sun, Amandine Batte, Mireille Tittel-Elmer, Sophie van der Horst, Tibor van Welsem, Gordon Bean, Trey Ideker, Fred van Leeuwen, and Haico van Attikum

Eukaryotic chromosomes are replicated in interphase and the two newly duplicated sister chromatids are held together by the cohesin complex and several cohesin auxiliary factors. Sister chromatid cohesion is essential for accurate chromosome segregation during mitosis, yet has also been implicated in other processes, including DNA damage repair, transcription and DNA replication. To assess how cohesin and associated factors functionally interconnect and coordinate with other cellular processes, we systematically mapped genetic interactions of 17 cohesin genes centered on quantitative growth measurements of >52,000 gene pairs in budding yeast. Integration of synthetic genetic interactions unveiled a cohesin functional map that constitutes 373 genetic interactions, revealing novel functional connections with post-replication repair, microtubule organization and protein folding. Accordingly, we show that the microtubule-associated protein Irc15 and the prefoldin complex members Gim3, Gim4 and Yke2 are new factors involved in sister chromatid cohesion. Our genetic interaction map thus provides a unique resource for further identification and functional interrogation of cohesin proteins. Since mutations in cohesin proteins have been associated with cohesinopathies and cancer, it may also identify cohesin interactions relevant in disease etiology.

Tara L. Mastro, Vishnu P. Tripathi, and Susan L. Forsburg

Translesion synthesis polymerases (TLSPs) are non-essential error-prone enzymes that ensure cell survival by facilitating DNA replication in the presence of DNA damage. In addition to their role in bypassing lesions, TLSPs have been implicated in meiotic double strand break repair in several systems. Here we examine the joint contribution of four TLS polymerases to meiotic progression in the fission yeast S. pombe. We observed the dramatic loss of spore viability in fission yeast lacking all four TLSPs which is accompanied by disruptions in chromosome segregation during meiosis I and II. Rec8 cohesin dynamics are altered in the absence of the TLSPs. These data suggest that the TLSPs contribute to multiple aspects of meiotic chromosome dynamics.

Karolina Losenkova, Mariachiara Zuccarini, Marika Karikoski, Juha Laurila, Detlev Boison, Sirpa Jalkanen, and Gennady G. Yegutkin

Extracellular adenosine mediates diverse anti-inflammatory, angiogenic and vasoactive effects and becomes an important therapeutic target for cancer, which has been translated into clinical trials. This study was designed to comprehensively assess adenosine metabolism in prostate and breast cancer cells. We identified cellular adenosine turnover as a complex cascade, comprised of (a) the ectoenzymatic breakdown of ATP via sequential nucleotide pyrophosphatase/phosphodiesterase-1, ecto-5’-nucleotidase/CD73 and adenosine deaminase reactions, and ATP re-synthesis through counteracting adenylate kinase and nucleoside diphosphokinase; (b) the uptake of nucleotide-derived adenosine via equilibrative nucleoside transporters; and (c) the intracellular adenosine phosphorylation into ATP by adenosine kinase and other nucleotide kinases. The exposure of cancer cells to 1% O₂ for 24 hours triggered ~2-fold up-regulation of CD73, without affecting nucleoside transporters, adenosine kinase activity and cellular ATP content. The ability of adenosine to inhibit the tumor-initiating potential of breast cancer cells via receptor-independent mechanism was confirmed in vivo using a xenograft mouse model. The existence of redundant pathways controlling extracellular and intracellular adenosine provides a sufficient justification for reexamination of the current concepts of cellular purine homeostasis and signaling in cancer.

Laurence Haren, Dorian Farache, Laurent Emorine, and Andreas Merdes

-tubulin is a major protein involved in the nucleation of microtubules in all eukaryotes. It forms two different complexes with proteins of the GCP family (gamma-tubulin complex proteins): -tubulin small complexes (TuSCs), containing -tubulin and GCPs 2 and 3, and -tubulin ring complexes (TuRCs), containing multiple TuSCs, in addition to GCPs 4, 5, and 6. Whereas the structure and assembly properties of TuSCs have been intensively studied, little is known about the assembly of TuRCs, and about the specific roles of GCPs 4, 5, and 6. Here, we demonstrate that two copies of GCP4 and one copy each of GCP5 and GCP6 form a salt-resistant sub-complex within the TuRC that assembles independently of the presence of TuSCs. Incubation of this sub-complex with cytoplasmic extracts containing TuSCs leads to the reconstitution of TuRCs that are competent to nucleate microtubules. In addition, we investigate sequence extensions and insertions that are specifically found at the amino-terminus of GCP6, and between the GCP6 grip1 and grip2 motifs, and we demonstrate that these are involved in the assembly or stabilization of the TuRC.

Juan Martin D'Ambrosio, Veronique Albanese, Nicolas-Frederic Lipp, Lucile Fleuriot, Delphine Debayle, Guillaume Drin, and Alenka Copic

Osh6 and Osh7 are lipid transfer proteins (LTPs) that move phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM). High PS level at the PM is key for many cellular functions. Intriguingly, Osh6/7 localize to ER-PM contact sites, although they lack membrane-targeting motifs, in contrast to multidomain LTPs that both bridge membranes and convey lipids. We show that Osh6 localization to contact sites depends on its interaction with the cytosolic tail of the ER-PM tether Ist2, a homologue of TMEM16 proteins. We identify a motif in the Ist2 tail, conserved in yeasts, as the Osh6-binding region, and we map an Ist2-binding surface on Osh6. Mutations in the Ist2 tail phenocopy osh6 osh7 deletion: they decrease cellular PS levels, and block PS transport to the PM. Our study unveils an unexpected partnership between a TMEM16-like protein and a soluble LTP, which together mediate lipid transport at contact sites.

Kelly L. Dunlevy, Valentina Medvedeva, Jade E. Wilson, Mohammed Hoque, Trinity Pellegrin, Adam Maynard, Madison M. Kremp, Jason S. Wasserman, Andrey Poleshko, and Richard A. Katz

A large fraction of epigenetically silent heterochromatin is anchored to the nuclear periphery via "tethering proteins" that function to bridge heterochromatin and the nuclear membrane or nuclear lamina. We identified previously a human tethering protein, PRR14, that binds heterochromatin through an N-terminal domain, but the mechanism and regulation of nuclear lamina association remained to be investigated. Here we identify an evolutionarily conserved PRR14 nuclear lamina binding domain (LBD) that is both necessary and sufficient for positioning of PRR14 at the nuclear lamina. We also show that PRR14 associates dynamically with the nuclear lamina, and provide evidence that such dynamics are regulated through phosphorylation-dephosphorylation of the LBD. Furthermore, we identified a PP2A phosphatase recognition motif within the evolutionarily conserved PRR14 C-terminal Tantalus domain. Disruption of this motif affected PRR14 localization to the nuclear lamina. The overall findings demonstrate a heterochromatin anchoring mechanism whereby the PRR14 tether simultaneously binds heterochromatin and the nuclear lamina through two separable, modular domains. The findings also describe an optimal PRR14 LBD fragment that could be used for efficient targeting of fusion proteins to the nuclear lamina.

Samantha-Su Z. Taylor, Nicole L. Jacobsen, Tasha K. Pontifex, Paul Langlais, and Janis M. Burt

Connexin 37 (Cx37) expression profoundly suppresses proliferation of rat insulinoma (Rin) cells in a manner dependent on gap junction channel (GJCh) functionality and the presence and phosphorylation status of its carboxyl-terminus (CT). In Rin cells growth arrested by induced Cx37 expression, serine 319 (S319) is frequently phosphorylated. Preventing phosphorylation at this site (alanine substitution; S319A) relieved Cx37 of its growth suppressive effect whereas mimicking phosphorylation at this site (aspartate substitution; S319D) enhanced Cx37's growth suppressive properties. Like Cx37-WT, -S319D GJChs and hemichannels (HChs) preferred the closed state, rarely opening fully, and gated slowly. In contrast, Cx37-S319A channels preferred open states, opened fully, and gated rapidly. These data indicate that phosphorylation-dependent conformational differences in Cx37 protein and channel function underlie Cx37-induced growth arrest vs. growth permissive phenotypes. That the closed state of -WT and Cx37-S319D GJChs and HChs favors growth arrest suggests that rather than specific permeants mediating cell cycle arrest, the closed conformation instead supports interaction of Cx37 with growth regulatory proteins that result in growth arrest.

Westley Heydeck, Brian A. Bayless, Alexander J. Stemm-Wolf, Eileen T. O'Toole, Amy S. Fabritius, Courtney Ozzello, Marina Nguyen, and Mark Winey

Basal bodies (BBs) are microtubule-based organelles that template and stabilize cilia at the cell surface. Centrins ubiquitously associate with BBs and function in BB assembly, maturation, and stability. Human POC5 (hPOC5) is a highly conserved centrin-binding protein that binds centrins through Sfi1p-like repeats and is required for building full-length, mature centrioles. Here, we use the BB-rich cytoskeleton of Tetrahymena thermophila to characterize Poc5 BB functions. Tetrahymena Poc5 (TtPoc5) uniquely incorporates into assembling BBs and is then removed from mature BBs prior to ciliogenesis. Complete genomic knockout of TtPOC5 leads to a significantly increased production of BBs yet a markedly reduced ciliary density, both of which are rescued by reintroduction of TtPoc5. A second Tetrahymena POC5-like gene, SFR1, is similarly implicated in modulating BB production. When TtPOC5 and SFR1 are co-deleted, cell viability is compromised, and levels of BB overproduction are exacerbated. Overproduced BBs display defective transition zone formation and a diminished capacity for ciliogenesis. This study uncovers a requirement for Poc5 in building mature BBs, providing a possible functional link between hPOC5 mutations and impaired cilia.

Thomas O'Loughlin, Antonina J. Kruppa, Andre L. R. Ribeiro, James R. Edgar, Abdulaziz Ghannam, Andrew M. Smith, and Folma Buss

Optineurin (OPTN) is a multifunctional protein involved in autophagy, secretion as well as NF-B and IRF3 signalling and OPTN mutations are associated with several human diseases. Here we show that, in response to viral RNA, OPTN translocates to foci in the perinuclear region, where it negatively regulates NF-B and IRF3 signalling pathways and downstream pro-inflammatory cytokine secretion. These OPTN foci consist of a tight cluster of small membrane vesicles, which are positive for ATG9A. Disease mutations linked to POAG cause aberrant foci formation in the absence of stimuli, which correlates with the ability of OPTN to inhibit signalling. Using proximity labelling proteomics, we identify the LUBAC complex, CYLD and TBK1 as part of the OPTN interactome and show that these proteins are recruited to this OPTN-positive perinuclear compartment. Our work uncovers a crucial role for OPTN in dampening NF-B and IRF3 signalling through the sequestration of LUBAC and other positive regulators in this viral RNA-induced compartment leading to altered pro-inflammatory cytokine secretion.

Stephen Kershaw, David J. Morgan, James Boyd, David G. Spiller, Gareth Kitchen, Egor Zindy, Mudassar Iqbal, Magnus Rattray, Chris M. Sanderson, Andrew Brass, Claus Jorgensen, Tracy Hussell, Laura C. Matthews, and David W. Ray

Glucocorticoids (GCs) act through the glucocorticoid receptor (GR) to regulate immunity, energy metabolism, and tissue repair. Upon ligand binding, activated GR mediates cellular effects by regulating gene expression, but some GR effects can occur rapidly without new transcription. We show GCs rapidly inhibit cell migration, in response to both GR agonist and antagonist ligand binding. The inhibitory effect on migration is prevented by GR knockdown with siRNA, confirming GR specificity, but not by actinomycin D treatment, suggesting a non-transcriptional mechanism. We identified a rapid onset increase in microtubule polymerisation following glucocorticoid treatment, identifying cytoskeletal stabilisation as the likely mechanism of action. HDAC6 overexpression, but not knockdown of αTAT1, rescued the GC effect, implicating HDAC6 as the GR effector. Consistent with this hypothesis, ligand-dependent cytoplasmic interaction between GR and HDAC6 was demonstrated by quantitative imaging. Taken together, we propose that activated GR inhibits HDAC6 function and thereby increases the stability of the microtubule network to reduce cell motility. We therefore report a novel, non-transcriptional mechanism whereby GCs impair cell motility through inhibition of HDAC6 and rapid reorganization of the cell architecture.