Highly multiplexed profiling of single-cell effector functions reveals deep functional heterogeneity in response to pathogenic ligands
Yao Lu, Qiong Xue, Markus R. Eisele, Endah S. Sulistijo, Kara Brower, Lin Han, El-ad David Amir, Dana Pe’er, Kathryn Miller-Jensen, and Rong Fan
We demonstrated (pp. E607–E615) codetection of 42 immune effector proteins in single cells, representing the highest multiplexing recorded to date for a single-cell secretion assay. Using this platform to profile differentiated macrophages stimulated with lipopolysaccharide reveals previously unobserved deep functional heterogeneity and varying levels of pathogenic activation, which is conserved throughout the cell activation process and prevails as it is extended to other Toll-like receptor (TLR) ligands and to primary human macrophages. The results indicate that the phenotypically similar cell population could still exhibit a large degree of intrinsic heterogeneity at the cell function level. This technology enables full-spectrum dissection of immune functional states in response to pathogenic stimulation and allows for more comprehensive and accurate monitoring of cellular immunity.
Mitochondrial peroxiredoxin functions as crucial chaperone reservoir in Leishmania infantum
Filipa Teixeira, Helena Castro, Tânia Cruz, Eric Tse, Philipp Koldewey, Daniel R. Southworth, Ana M. Tomás, and Ursula Jakob
Peroxiredoxins (Prxs) are highly abundant proteins, which serve two seemingly mutually exclusive roles as peroxidases and molecular chaperones. Little is known about the precise mechanism of Prxs’ activation as chaperone and the physiological significance of this second function. Here (pp. E616–E624) we demonstrate that in Leishmania infantum, reduced Prx provides a crucial, stress-specific chaperone reservoir, which is activated rapidly upon exposure to unfolding stress conditions. Once activated, Prx protects a wide range of different clients against protein unfolding. Clients are bound in the center of the decameric ring, providing experimental evidence for previous claims that Prxs serve as likely ancestors of chaperonins. Interference with client binding impairs Leishmania infectivity, providing compelling evidence for the in vivo importance of Prx’s chaperone function.
Distinct activation of an E2 ubiquitin-conjugating enzyme by its cognate E3 ligases
Itamar Cohen, Reuven Wiener, Yuval Reiss, and Tommer Ravid
Ubiquitin (Ub) conjugation triggers protein degradation by the proteasome. Here (pp. E625–E632) we describe an unexplored feature of the Ub conjugation system that entails differential activation of an E2-conjugating enzyme by its cognate E3 Ub ligases. In vitro and in vivo analyses of activity-reducing mutants of the yeast Ub-conjugating (Ubc) enzyme, Ubc7, demonstrated selective activation by one of its two cognate E3 ligases, Hrd1, but not by the other, Doa10. Supported by structural modeling of the RING:Ubc7∼Ub complex, these findings are consistent with a model whereby the E2∼Ub transition state depends on noncovalent interactions between helix α2 of Ubc7 and Ub that are differentially stabilized by the two E3 RING domains. This differential activation represents a previously unidentified mechanism for regulating protein ubiquitylation.
Archaeal replicative primases can perform translesion DNA synthesis
Stanislaw K. Jozwiakowski, Farimah Borazjani Gholami, and Aidan J. Doherty
DNA replicases stall at lesions during replication, potentially leading to genome instability. However, cells use specialized lesion bypass polymerases to restart stalled replisomes. Although most organisms possess these damage tolerance polymerases, capable of traversing blocking DNA lesions, many appear to lack these enzymes. We have discovered (pp. E633–E638) that replicative primases from archaea, previously considered to be solely involved in priming replication, are also capable of performing translesion DNA synthesis. This discovery has major implications for our understanding of additional roles of DNA primases during replication and the subsequent evolution of related lesion bypass pathways in eukaryotic organisms.
A conserved amphipathic helix is required for membrane tubule formation by Yop1p
Jacob P. Brady, Jolyon K. Claridge, Peter G. Smith, and Jason R. Schnell
The first structural studies, to our knowledge, of a reticulon homology domain (RHD), which is essential for maintaining smooth endoplasmic reticulum (ER) tubules and the edges of ER sheets, are described. We show here (pp. E639–E648) that the RHD of the protein Yop1p from the YOP1 gene has hydrophobic helices long enough to cross the membrane fully but contains a previously uncharacterized amphipathic helix (APH) that is necessary for membrane tubule formation. The APH is highly conserved in its amino acid properties and its location relative to the RHD both in the DP1 (deleted in polyposis) and reticulon families. These results place the DP1/reticulon proteins into the large and growing class of membrane-remodeling proteins that use APHs to influence membrane curvature.
CryoEM and mutagenesis reveal that the smallest capsid protein cements and stabilizes Kaposi's sarcoma-associated herpesvirus capsid
Xinghong Dai, Danyang Gong, Yuchen Xiao, Ting-Ting Wu, Ren Sun, and Z. Hong Zhou
Kaposi's sarcoma-associated herpesvirus (KSHV) and EBV are cancer-causing human herpesviruses. Their smallest capsid proteins (SCPs) were shown to be required for capsid assembly and are potential drug targets for curbing viral infections, but how they work is unclear. By cryoEM and genetic engineering, we determine the structures of KSHV capsids bearing full-length or truncated SCPs and localize regions of SCP that are important for capsid assembly. We show (pp. E649–E656) that a long kinked helix of SCP cross-links neighboring subunits of the major capsid protein of hexons to stabilize the capsid. Our results explain how SCP, acting like a cementing protein found in bacterial viruses, facilitates tumor herpesvirus capsid assembly and viral maturation.
Autoinhibition and relief mechanism for Polo-like kinase 4
Joseph E. Klebba, Daniel W. Buster, Tiffany A. McLamarrah, Nasser M. Rusan, and Gregory C. Rogers
Polo-like kinases (Plks) are a conserved family of enzymes that function as master regulators for the process of cell division. Among their duties, Plks control the assembly of centrosomes, tiny organelles that facilitate mitotic spindle assembly and maintain the fidelity of chromosome inheritance. Plks are overexpressed in cancer, and therefore it is critical to unravel the normal regulation of these kinases. Here (pp. E657–E666), we studied Plk4 regulation whose activity controls centrosome number. We showed that, as do other Plks, Plk4 autoinhibits its kinase activity. However, Plk4 is unique in its ability to relieve autoinhibition through a third Polo box domain not present in other Plk family members. Moreover, autoinhibition controls Plk4 oligomerization, which ultimately governs its stability and thus centrosome duplication.
Aged insulin granules display reduced microtubule-dependent mobility and are disposed within actin-positive multigranular bodies
Peter Hoboth, Andreas Müller, Anna Ivanova, Hassan Mziaut, Jaber Dehghany, Anke Sönmez, Martina Lachnit, Michael Meyer-Hermann, Yannis Kalaidzidis, and Michele Solimena
Insulin is key for control of glucose homeostasis in vertebrates. Insufficient insulin secretion relative to metabolic needs causes diabetes. Pancreatic beta cells store insulin into secretory granules (SGs), which release insulin extracellularly upon fusion with the plasma membrane. SGs exist in different functional pools, with newly generated SGs being preferentially secreted. Here (pp. E667–E676) we show that aged SGs display reduced competence for glucose-stimulated microtubule-mediated transport and are disposed within actin-positive multigranular bodies. These data highlight the link between SG age and mobility and thus are relevant for better understanding insulin secretion in health and diabetes.
Genome-wide targeting of the epigenetic regulatory protein CTCF to gene promoters by the transcription factor TFII-I
Rodrigo Peña-Hernández, Maud Marques, Khalid Hilmi, Teijun Zhao, Amine Saad, Moulay A. Alaoui-Jamali, Sonia V. del Rincon, Todd Ashworth, Ananda L. Roy, Beverly M. Emerson, and Michael Witcher
CCCTC-binding factor (CTCF) is an epigenetic regulatory protein that is not only functionally diverse, but is also targeted to highly diverse DNA binding sites. CTCF cooperates with accessory proteins to achieve various functional outputs. Further evidence in Drosophila shows that CTCF may also be targeted to chromatin via accessory proteins. The identity of such mammalian proteins remains elusive. Herein (pp. E677–E686), we describe evidence that the transcription factor general transcription factor II-I (TFII-I) targets CTCF binding to metabolism-related genes across the genome. We find that TFII-I regulates the transcription of genes within this network on the level of initiation via RNA polymerase II phosphorylation. These results provide a starting point for understanding a biological network communicating information between chromatin architecture, transcription, and metabolism.
The autophagic machinery ensures nonlytic transmission of mycobacteria
Lilli Gerstenmaier, Rachel Pilla, Lydia Herrmann, Hendrik Herrmann, Monica Prado, Geno J. Villafano, Margot Kolonko, Rudolph Reimer, Thierry Soldati, Jason S. King, and Monica Hagedorn
Pathogenic mycobacteria can be transmitted by direct ejection from one host cell to another. However, the mechanism of ejection, and how lysing the host cell is prevented are unknown. This study (pp. E687–E692) explains how the host cell remains intact and alive while Mycobacterium marinum breaks through its plasma membrane during ejection. We show that a membraneous cup is specifically recruited to the distal pole of ejecting M. marinum. We demonstrate that these membranes are formed by the canonical autophagic pathway, though they do not mature to autophagolysosomes. Disruption of autophagy causes the host cells to become leaky and die during ejection. This dramatically reduces cell-to-cell transmission of the infection, demonstrating an important and unexpected role for autophagy in maintaining plasma membrane integrity during mycobacterial infection.
Bacterial proteins pinpoint a single eukaryotic root
Romain Derelle, Guifré Torruella, Vladimír Klimeš, Henner Brinkmann, Eunsoo Kim, Čestmír Vlček, B. Franz Lang, and Marek Eliáš
The root of eukaryote phylogeny formally represents the last eukaryotic common ancestor (LECA), but its position has remained controversial. Using new genome sequences, we revised and expanded (pp. E693–E699) two datasets of eukaryotic proteins of bacterial origin, which previously yielded conflicting views on the eukaryotic root. Analyses using state-of-the-art phylogenomic methodology revealed that both expanded datasets now support the same root position. Our results justify a new nomenclature for the two main eukaryotic groups and provide a robust phylogenetic framework to investigate the early evolution of the eukaryotic cell.
PTEN regulates natural killer cell trafficking in vivo
Jeffrey W. Leong, Stephanie E. Schneider, Ryan P. Sullivan, Bijal A. Parikh, Bryan A. Anthony, Anvita Singh, Brea A. Jewell, Timothy Schappe, Julia A. Wagner, Daniel C. Link, Wayne M. Yokoyama, and Todd A. Fehniger
Natural killer (NK) cells are critical players in the response to viruses and transformed cells, but the molecular mechanisms controlling their functions are incompletely understood. A major pathway leading to NK cell activation is the phosphoinositide 3-kinase pathway. However, the impact of phosphatase and tensin homolog (PTEN), a key phosphatase opposing this pathway, on NK cells has not been reported. We generated a previously unreported NK cell-intrinsic PTEN-deletion mouse model to evaluate its role in NK cell biology. In contrast to other lymphocytes, we demonstrate (pp. E700–E709) that the primary effects of PTEN loss are marked perturbation in NK cell trafficking and distribution during both homeostasis and malignancy. These findings indicate that PTEN plays an essential role in NK cell localization in vivo.
Suppression of systemic autoimmunity by the innate immune adaptor STING
Shruti Sharma, Allison M. Campbell, Jennie Chan, Stefan A. Schattgen, Gregory M. Orlowski, Ribhu Nayar, Annie H. Huyler, Kerstin Nündel, Chandra Mohan, Leslie J. Berg, Mark J. Shlomchik, Ann Marshak-Rothstein, and Katherine A. Fitzgerald
Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease that presents with a diverse array of clinical symptoms and afflicts over 1.5 million Americans. Current treatments involve immunosuppressive regimens associated with debilitating and adverse effects. With the description of a role for innate signaling in SLE, safe and efficient therapies that block Toll-like receptors also have been stymied by the relative short in vivo half lives of known inhibitors and the dangerous outcome of complete MyD88 blockade. Key natural regulators of the disease process are not well described but are more likely to provide disease-specific therapeutics with fewer adverse effects. In this study (pp. E710–E717), we have identified a novel function for Stimulator of interferon genes as a suppressor of disease and a target for future SLE therapeutics.
The SWI/SNF chromatin remodeling complex regulates germinal center formation by repressing Blimp-1 expression
Jinwook Choi, Shin Jeon, Seungjin Choi, Kyungsoo Park, and Rho Hyun Seong
Germinal center (GC) response is central for generation of memory B cells and plasma cells that produce high-affinity antibodies, which are crucial for protective immunity against various foreign antigens. Even though key genetic factors for the GC formation are known, it is largely unknown how this process is controlled by epigenetic factors. Here (pp. E718–E727) we have demonstrated that the activity of SWI/SNF chromatin remodeling complex is required for the development of both GC B cells and follicular helper T cells. The SWI/SNF complex modulates Bcl-6–mediated Blimp-1 repression, and thus plays as a key mediator in the GC response. Our results provide fundamental insights into the epigenetic regulation in the GC reaction.
Overlapping hotspots in CDRs are critical sites for V region diversification
Lirong Wei, Richard Chahwan, Shanzhi Wang, Xiaohua Wang, Phuong T. Pham, Myron F. Goodman, Aviv Bergman, Matthew D. Scharff, and Thomas MacCarthy
The somatic hypermutation of immunoglobulin (Ig) variable (V) regions is required to produce high-affinity protective antibodies. Activation-induced deaminase (AID) initiates the accumulation of mutations in the V regions at frequencies that are a million times higher than the normal mutation rates in non-Ig genes. On the basis of the in vivo pattern of mutations in the highly used IGHV3-23*01 human V region and the manipulation of DNA sequence motifs in that V region in a mutating B-cell line and biochemically, we have concluded (pp. E728–E737) that particular DNA sequence motifs focus and influence AID activity on those parts of the V region that affect antigen binding and should be considered in vaccine strategies.
Adverse childhood experiences and physiological wear-and-tear in midlife: Findings from the 1958 British birth cohort
Cristina Barboza Solís, Michelle Kelly-Irving, Romain Fantin, Muriel Darnaudéry, Jérôme Torrisani, Thierry Lang, and Cyrille Delpierre
The role of early life experiences on health is of major concern to research. Recent studies have shown that chronic stress may “get under the skin” to alter human developmental processes and impact later health. Our findings (pp. E738–E746) suggest that early negative circumstances during childhood, collected prospectively in a British birth cohort, could be associated with physiological wear-and-tear in midlife as measured by allostatic load. This relationship was largely explained by health behaviors, body mass index, and socioeconomic status in adulthood, but not entirely. These results suggest that a biological link between adverse childhood exposures and adult health may be plausible. Our findings contribute to the development of more adapted public health interventions, both at a societal and individual level.
An HD-domain phosphodiesterase mediates cooperative hydrolysis of c-di-AMP to affect bacterial growth and virulence
TuAnh Ngoc Huynh, Shukun Luo, Daniel Pensinger, John-Demian Sauer, Liang Tong, and Joshua J. Woodward
The small nucleotide cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) recently emerged as a ubiquitous signaling molecule among bacteria, with essential roles in both bacterial physiology and host–pathogen interactions. Bacterial mutants with abnormal c-di-AMP levels exhibit growth and virulence defects, reflecting the importance of regulating c-di-AMP synthesis and degradation for normal signal transduction and adaptation to changing environments. Previously documented phosphodiesterases hydrolyze c-di-AMP via the DHH-DHHA1 domain, but they are not present in all c-di-AMP synthesizing species. We identified (pp. E747–E756) a previously unrecognized class of His-Asp -domain phosphodiesterases that are widespread across several taxonomic groups. Furthermore, for the bacterial pathogen Listeria monocytogenes, phosphodiesterase mutants exhibit enhanced host inflammation, growth defects inside host cells, and significantly attenuated virulence in a murine model of infection.
Novel mixed-linkage β-glucan activated by c-di-GMP in Sinorhizobium meliloti
Daniel Pérez-Mendoza, Miguel Ángel Rodríguez-Carvajal, Lorena Romero-Jiménez, Gabriela de Araujo Farias, Javier Lloret, María Trinidad Gallegos, and Juan Sanjuán
We report (pp. E757–E765) a novel linear mixed-linkage (1→3)(1→4)-β-glucan produced by the bacterium Sinorhizobium meliloti upon raising cyclic diguanylate (c-di-GMP) intracellular levels. This unique bacterial polysaccharide resembles (1→3)(1→4)-β-glucans found in cereals and certain lichens but has a distinctive primary structure. Genes, proteins, and regulatory pathways for producing this new polymer are described. Our findings open the possibility of using bacteria to produce (1→3)(1→4)-β-glucans, which are receiving increasing interest as bioactive compounds, and provide new elements to disclose molecular mechanisms of c-di-GMP regulation as well as for investigating the evolution, activity, and specificity of glycosyl transferases.
Differential RNA-seq of Vibrio cholerae identifies the VqmR small RNA as a regulator of biofilm formation
Kai Papenfort, Konrad U. Förstner, Jian-Ping Cong, Cynthia M. Sharma, and Bonnie L. Bassler
To our knowledge, this work (pp. E766–E775) describes the first genome-wide annotation of transcriptional start sites in Vibrio cholerae and the discovery and characterization of a regulatory RNA, named VqmR, which controls collective behaviors in this major human pathogen. We show that VqmR is activated by the VqmA transcriptional regulator. VqmR represses expression of multiple mRNA targets including those encoding the Rtx (repeats in toxin) toxin and VpsT, which is required for biofilm formation. Indeed, we show that VqmR controls biofilm formation through repression of vpsT.
Two-photon brightness of azobenzene photoswitches designed for glutamate receptor optogenetics
Elizabeth C. Carroll, Shai Berlin, Joshua Levitz, Michael A. Kienzler, Zhe Yuan, Dorte Madsen, Delmar S. Larsen, and Ehud Y. Isacoff
MAGs (maleimide-azobenzene-glutamate) are photoswitches that covalently bind to genetically engineered glutamate receptors (GluRs) and, under the control of light, mimic or block the action of the excitatory neurotransmitter glutamate. However the blue and near-UV light that optimally photoswitch MAGs do not penetrate well into the brain. In this paper (pp. E776–E785), we show how MAGs can instead be photoswitched by two-photon (2P) absorption of near-infrared light, which penetrates deeper into tissue. We demonstrate 2P control of MAG-dependent ionic currents in neurons, and synthesize a new MAG photoswitch to enable 2P activation of a G protein coupled receptor signaling cascade through a metabotropic GluR. These optogenetic tools bring exceptional spatiotemporal resolution and pharmacological specificity to the study of synaptic transmission and plasticity in intact neural circuits.
Regulator of G protein signaling 6 is a critical mediator of both reward-related behavioral and pathological responses to alcohol
Adele Stewart, Biswanath Maity, Simon P. Anderegg, Chantal Allamargot, Jianqi Yang, and Rory A. Fisher
Almost 20% of women and 40% of men in the United States abuse alcohol or have experienced alcohol dependence in their lifetime. Though accidents, traffic fatalities, and violent crimes account for the majority of alcohol-involved mortalities, excessive, chronic drinking also causes often irreversible heart and liver damage. We identify (pp. E786–E795) regulator of G protein signaling 6 (RGS6) as a novel drug target with substantive potential clinical utility in the treatment of alcoholism and amelioration of the resultant hepatic and cardiac toxicity. Mice lacking RGS6 exhibit a reduction in voluntary alcohol consumption, conditioned reward and withdrawal. In addition, RGS6−/− mice are largely protected from alcohol-induced cardiomyopathy, hepatic steatosis, gastrointestinal barrier dysfunction, and endotoxemia. Thus, targeting RGS6 could reduce alcohol cravings while simultaneously protecting the heart and liver from damage.
Potassium channelopathy-like defect underlies early-stage cerebrovascular dysfunction in a genetic model of small vessel disease
Fabrice Dabertrand, Christel Krøigaard, Adrian D. Bonev, Emmanuel Cognat, Thomas Dalsgaard, Valérie Domenga-Denier, David C. Hill-Eubanks, Joseph E. Brayden, Anne Joutel, and Mark T. Nelson
Small vessel disease (SVD) of the brain refers to a group of pathological processes leading to cerebral lesions, cognitive decline, and stroke. Despite the importance of SVD, there is no specific treatment, mainly due to a limited understanding of the disease pathogenesis. Using a recently developed mouse model of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, a hereditary form of SVD, we determined (pp. E796–E805) the basis of altered brain artery function at an early stage of disease progression. We found that cerebrospecific up-regulation of the voltage-gated potassium channel, KV1, prevents intracerebral arterioles from constricting in response to physiological levels of intraluminal pressure. This impairment of a fundamental vascular function is expected to impact cerebral blood flow autoregulation and local dilation in response to neuronal activity (functional hyperemia).
An early secretory pathway mediated by GNOM-LIKE 1 and GNOM is essential for basal polarity establishment in Arabidopsis thaliana
Siamsa M. Doyle, Ash Haeger, Thomas Vain, Adeline Rigal, Corrado Viotti, Małgorzata Łangowska, Qian Ma, Jiří Friml, Natasha V. Raikhel, Glenn R. Hicks, and Stéphanie Robert
Within plants, controlled gradients of the hormone auxin are essential for development. These gradients are achieved through intracellular polar positioning of auxin transporter proteins, such as PIN-FORMED proteins (PINs), at the plasma membrane, thus guiding the direction of auxin transport. The establishment and maintenance of PIN polarity is controlled within each cell by complicated trafficking pathways of the endomembrane system. In the model plant Arabidopsis, it has long been known that endocytosis and recycling trafficking routes play roles in PIN polarity. In this study (pp. E806–E815), we reveal the role of a secretory route in this process. Our evidence shows that this secretory trafficking route selectively controls basal (rootward) but not apical (shootward) PIN polarity.