TY - JOUR
AB - The GNOM (GN) Guanine nucleotide Exchange Factor for ARF small GTPases (ARF-GEF) is among the best studied trafficking regulators in plants, playing crucial and unique developmental roles in patterning and polarity. The current models place GN at the Golgi apparatus (GA), where it mediates secretion/recycling, and at the plasma membrane (PM) presumably contributing to clathrin-mediated endocytosis (CME). The mechanistic basis of the developmental function of GN, distinct from the other ARF-GEFs including its closest homologue GNOM-LIKE1 (GNL1), remains elusive. Insights from this study largely extend the current notions of GN function. We show that GN, but not GNL1, localizes to the cell periphery at long-lived structures distinct from clathrin-coated pits, while CME and secretion proceed normally in gn knockouts. The functional GN mutant variant GNfewerroots, absent from the GA, suggests that the cell periphery is the major site of GN action responsible for its developmental function. Following inhibition by Brefeldin A, GN, but not GNL1, relocates to the PM likely on exocytic vesicles, suggesting selective molecular associations en route to the cell periphery. A study of GN-GNL1 chimeric ARF-GEFs indicates that all GN domains contribute to the specific GN function in a partially redundant manner. Together, this study offers significant steps toward the elucidation of the mechanism underlying unique cellular and development functions of GNOM.
AU - Adamowski, Maciek
AU - Matijevic, Ivana
AU - Friml, Jiří
ID - 15033
JF - eLife
KW - General Immunology and Microbiology
KW - General Biochemistry
KW - Genetics and Molecular Biology
KW - General Medicine
KW - General Neuroscience
SN - 2050-084X
TI - Developmental patterning function of GNOM ARF-GEF mediated from the cell periphery
VL - 13
ER -
TY - JOUR
AB - Mosaic analysis with double markers (MADM) technology enables the generation of genetic mosaic tissue in mice and high-resolution phenotyping at the individual cell level. Here, we present a protocol for isolating MADM-labeled cells with high yield for downstream molecular analyses using fluorescence-activated cell sorting (FACS). We describe steps for generating MADM-labeled mice, perfusion, single-cell suspension, and debris removal. We then detail procedures for cell sorting by FACS and downstream analysis. This protocol is suitable for embryonic to adult mice.
For complete details on the use and execution of this protocol, please refer to Contreras et al. (2021).1
AU - Amberg, Nicole
AU - Cheung, Giselle T
AU - Hippenmeyer, Simon
ID - 14683
IS - 1
JF - STAR Protocols
KW - General Immunology and Microbiology
KW - General Biochemistry
KW - Genetics and Molecular Biology
KW - General Neuroscience
SN - 2666-1667
TI - Protocol for sorting cells from mouse brains labeled with mosaic analysis with double markers by flow cytometry
VL - 5
ER -
TY - JOUR
AB - Immune responses rely on the rapid and coordinated migration of leukocytes. Whereas it is well established that single-cell migration is often guided by gradients of chemokines and other chemoattractants, it remains poorly understood how these gradients are generated, maintained, and modulated. By combining experimental data with theory on leukocyte chemotaxis guided by the G protein–coupled receptor (GPCR) CCR7, we demonstrate that in addition to its role as the sensory receptor that steers migration, CCR7 also acts as a generator and a modulator of chemotactic gradients. Upon exposure to the CCR7 ligand CCL19, dendritic cells (DCs) effectively internalize the receptor and ligand as part of the canonical GPCR desensitization response. We show that CCR7 internalization also acts as an effective sink for the chemoattractant, dynamically shaping the spatiotemporal distribution of the chemokine. This mechanism drives complex collective migration patterns, enabling DCs to create or sharpen chemotactic gradients. We further show that these self-generated gradients can sustain the long-range guidance of DCs, adapt collective migration patterns to the size and geometry of the environment, and provide a guidance cue for other comigrating cells. Such a dual role of CCR7 as a GPCR that both senses and consumes its ligand can thus provide a novel mode of cellular self-organization.
AU - Alanko, Jonna H
AU - Ucar, Mehmet C
AU - Canigova, Nikola
AU - Stopp, Julian A
AU - Schwarz, Jan
AU - Merrin, Jack
AU - Hannezo, Edouard B
AU - Sixt, Michael K
ID - 14274
IS - 87
JF - Science Immunology
KW - General Medicine
KW - Immunology
SN - 2470-9468
TI - CCR7 acts as both a sensor and a sink for CCL19 to coordinate collective leukocyte migration
VL - 8
ER -
TY - JOUR
AB - With more than 80 members worldwide, the Orthobunyavirus genus in the Peribunyaviridae family is a large genus of enveloped RNA viruses, many of which are emerging pathogens in humans and livestock. How orthobunyaviruses (OBVs) penetrate and infect mammalian host cells remains poorly characterized. Here, we investigated the entry mechanisms of the OBV Germiston (GERV). Viral particles were visualized by cryo-electron microscopy and appeared roughly spherical with an average diameter of 98 nm. Labeling of the virus with fluorescent dyes did not adversely affect its infectivity and allowed the monitoring of single particles in fixed and live cells. Using this approach, we found that endocytic internalization of bound viruses was asynchronous and occurred within 30-40 min. The virus entered Rab5a+ early endosomes and, subsequently, late endosomal vacuoles containing Rab7a but not LAMP-1. Infectious entry did not require proteolytic cleavage, and endosomal acidification was sufficient and necessary for viral fusion. Acid-activated penetration began 15-25 min after initiation of virus internalization and relied on maturation of early endosomes to late endosomes. The optimal pH for viral membrane fusion was slightly below 6.0, and penetration was hampered when the potassium influx was abolished. Overall, our study provides real-time visualization of GERV entry into host cells and demonstrates the importance of late endosomal maturation in facilitating OBV penetration.
AU - Windhaber, Stefan
AU - Xin, Qilin
AU - Uckeley, Zina M.
AU - Koch, Jana
AU - Obr, Martin
AU - Garnier, Céline
AU - Luengo-Guyonnot, Catherine
AU - Duboeuf, Maëva
AU - Schur, Florian KM
AU - Lozach, Pierre-Yves
ID - 10639
IS - 5
JF - Journal of Virology
KW - virology
KW - insect science
KW - immunology
KW - microbiology
SN - 0022-538X
TI - The Orthobunyavirus Germiston enters host cells from late endosomes
VL - 96
ER -
TY - JOUR
AB - Several promising strategies based on combining or cycling different antibiotics have been proposed to increase efficacy and counteract resistance evolution, but we still lack a deep understanding of the physiological responses and genetic mechanisms that underlie antibiotic interactions and the clinical applicability of these strategies. In antibiotic-exposed bacteria, the combined effects of physiological stress responses and emerging resistance mutations (occurring at different time scales) generate complex and often unpredictable dynamics. In this Review, we present our current understanding of bacterial cell physiology and genetics of responses to antibiotics. We emphasize recently discovered mechanisms of synergistic and antagonistic drug interactions, hysteresis in temporal interactions between antibiotics that arise from microbial physiology and interactions between antibiotics and resistance mutations that can cause collateral sensitivity or cross-resistance. We discuss possible connections between the different phenomena and indicate relevant research directions. A better and more unified understanding of drug and genetic interactions is likely to advance antibiotic therapy.
AU - Römhild, Roderich
AU - Bollenbach, Mark Tobias
AU - Andersson, Dan I.
ID - 10812
JF - Nature Reviews Microbiology
KW - General Immunology and Microbiology
KW - Microbiology
KW - Infectious Diseases
SN - 1740-1526
TI - The physiology and genetics of bacterial responses to antibiotic combinations
VL - 20
ER -
TY - JOUR
AB - Studies of protein fitness landscapes reveal biophysical constraints guiding protein evolution and empower prediction of functional proteins. However, generalisation of these findings is limited due to scarceness of systematic data on fitness landscapes of proteins with a defined evolutionary relationship. We characterized the fitness peaks of four orthologous fluorescent proteins with a broad range of sequence divergence. While two of the four studied fitness peaks were sharp, the other two were considerably flatter, being almost entirely free of epistatic interactions. Mutationally robust proteins, characterized by a flat fitness peak, were not optimal templates for machine-learning-driven protein design – instead, predictions were more accurate for fragile proteins with epistatic landscapes. Our work paves insights for practical application of fitness landscape heterogeneity in protein engineering.
AU - Gonzalez Somermeyer, Louisa
AU - Fleiss, Aubin
AU - Mishin, Alexander S
AU - Bozhanova, Nina G
AU - Igolkina, Anna A
AU - Meiler, Jens
AU - Alaball Pujol, Maria-Elisenda
AU - Putintseva, Ekaterina V
AU - Sarkisyan, Karen S
AU - Kondrashov, Fyodor
ID - 11448
JF - eLife
KW - General Immunology and Microbiology
KW - General Biochemistry
KW - Genetics and Molecular Biology
KW - General Medicine
KW - General Neuroscience
SN - 2050-084X
TI - Heterogeneity of the GFP fitness landscape and data-driven protein design
VL - 11
ER -
TY - JOUR
AB - Empirical essays of fitness landscapes suggest that they may be rugged, that is having multiple fitness peaks. Such fitness landscapes, those that have multiple peaks, necessarily have special local structures, called reciprocal sign epistasis (Poelwijk et al. in J Theor Biol 272:141–144, 2011). Here, we investigate the quantitative relationship between the number of fitness peaks and the number of reciprocal sign epistatic interactions. Previously, it has been shown (Poelwijk et al. in J Theor Biol 272:141–144, 2011) that pairwise reciprocal sign epistasis is a necessary but not sufficient condition for the existence of multiple peaks. Applying discrete Morse theory, which to our knowledge has never been used in this context, we extend this result by giving the minimal number of reciprocal sign epistatic interactions required to create a given number of peaks.
AU - Saona Urmeneta, Raimundo J
AU - Kondrashov, Fyodor
AU - Khudiakova, Kseniia
ID - 11447
IS - 8
JF - Bulletin of Mathematical Biology
KW - Computational Theory and Mathematics
KW - General Agricultural and Biological Sciences
KW - Pharmacology
KW - General Environmental Science
KW - General Biochemistry
KW - Genetics and Molecular Biology
KW - General Mathematics
KW - Immunology
KW - General Neuroscience
SN - 0092-8240
TI - Relation between the number of peaks and the number of reciprocal sign epistatic interactions
VL - 84
ER -
TY - JOUR
AB - Intravascular neutrophils and platelets collaborate in maintaining host integrity, but their interaction can also trigger thrombotic complications. We report here that cooperation between neutrophil and platelet lineages extends to the earliest stages of platelet formation by megakaryocytes in the bone marrow. Using intravital microscopy, we show that neutrophils “plucked” intravascular megakaryocyte extensions, termed proplatelets, to control platelet production. Following CXCR4-CXCL12-dependent migration towards perisinusoidal megakaryocytes, plucking neutrophils actively pulled on proplatelets and triggered myosin light chain and extracellular-signal-regulated kinase activation through reactive oxygen species. By these mechanisms, neutrophils accelerate proplatelet growth and facilitate continuous release of platelets in steady state. Following myocardial infarction, plucking neutrophils drove excessive release of young, reticulated platelets and boosted the risk of recurrent ischemia. Ablation of neutrophil plucking normalized thrombopoiesis and reduced recurrent thrombosis after myocardial infarction and thrombus burden in venous thrombosis. We establish neutrophil plucking as a target to reduce thromboischemic events.
AU - Petzold, Tobias
AU - Zhang, Zhe
AU - Ballesteros, Iván
AU - Saleh, Inas
AU - Polzin, Amin
AU - Thienel, Manuela
AU - Liu, Lulu
AU - Ul Ain, Qurrat
AU - Ehreiser, Vincent
AU - Weber, Christian
AU - Kilani, Badr
AU - Mertsch, Pontus
AU - Götschke, Jeremias
AU - Cremer, Sophie
AU - Fu, Wenwen
AU - Lorenz, Michael
AU - Ishikawa-Ankerhold, Hellen
AU - Raatz, Elisabeth
AU - El-Nemr, Shaza
AU - Görlach, Agnes
AU - Marhuenda, Esther
AU - Stark, Konstantin
AU - Pircher, Joachim
AU - Stegner, David
AU - Gieger, Christian
AU - Schmidt-Supprian, Marc
AU - Gärtner, Florian R
AU - Almendros, Isaac
AU - Kelm, Malte
AU - Schulz, Christian
AU - Hidalgo, Andrés
AU - Massberg, Steffen
ID - 12119
IS - 12
JF - Immunity
KW - Infectious Diseases
KW - Immunology
KW - Immunology and Allergy
SN - 1074-7613
TI - Neutrophil “plucking” on megakaryocytes drives platelet production and boosts cardiovascular disease
VL - 55
ER -
TY - JOUR
AB - Replication-incompetent adenoviral vectors have been extensively used as a platform for vaccine design, with at least four anti-COVID-19 vaccines authorized to date. These vaccines elicit neutralizing antibody responses directed against SARS-CoV-2 Spike protein and confer significant level of protection against SARS-CoV-2 infection. Immunization with adenovirus-vectored vaccines is known to be accompanied by the production of anti-vector antibodies, which may translate into reduced efficacy of booster or repeated rounds of revaccination. Here, we used blood samples from patients who received an adenovirus-based Gam-COVID-Vac vaccine to address the question of whether anti-vector antibodies may influence the magnitude of SARS-CoV-2-specific humoral response after booster vaccination. We observed that rAd26-based prime vaccination with Gam-COVID-Vac induced the development of Ad26-neutralizing antibodies, which persisted in circulation for at least 9 months. Our analysis further indicates that high pre-boost Ad26 neutralizing antibody titers do not appear to affect the humoral immunogenicity of the Gam-COVID-Vac boost. The titers of anti-SARS-CoV-2 RBD IgGs and antibodies, which neutralized both the wild type and the circulating variants of concern of SARS-CoV-2 such as Delta and Omicron, were independent of the pre-boost levels of Ad26-neutralizing antibodies. Thus, our results support the development of repeated immunization schedule with adenovirus-based COVID-19 vaccines.
AU - Byazrova, Maria G.
AU - Astakhova, Ekaterina A.
AU - Minnegalieva, Aygul
AU - Sukhova, Maria M.
AU - Mikhailov, Artem A.
AU - Prilipov, Alexey G.
AU - Gorchakov, Andrey A.
AU - Filatov, Alexander V.
ID - 12131
JF - npj Vaccines
KW - Pharmacology (medical)
KW - Infectious Diseases
KW - Pharmacology
KW - Immunology
KW - SARS-COV-2
KW - COVID
SN - 2059-0105
TI - Anti-Ad26 humoral immunity does not compromise SARS-COV-2 neutralizing antibody responses following Gam-COVID-Vac booster vaccination
VL - 7
ER -
TY - JOUR
AB - Polygenic adaptation is thought to be ubiquitous, yet remains poorly understood. Here, we model this process analytically, in the plausible setting of a highly polygenic, quantitative trait that experiences a sudden shift in the fitness optimum. We show how the mean phenotype changes over time, depending on the effect sizes of loci that contribute to variance in the trait, and characterize the allele dynamics at these loci. Notably, we describe the two phases of the allele dynamics: The first is a rapid phase, in which directional selection introduces small frequency differences between alleles whose effects are aligned with or opposed to the shift, ultimately leading to small differences in their probability of fixation during a second, longer phase, governed by stabilizing selection. As we discuss, key results should hold in more general settings and have important implications for efforts to identify the genetic basis of adaptation in humans and other species.
AU - Hayward, Laura
AU - Sella, Guy
ID - 12157
JF - eLife
KW - General Immunology and Microbiology
KW - General Biochemistry
KW - Genetics and Molecular Biology
KW - General Medicine
KW - General Neuroscience
TI - Polygenic adaptation after a sudden change in environment
VL - 11
ER -
TY - JOUR
AB - Dose–response relationships are a general concept for quantitatively describing biological systems across multiple scales, from the molecular to the whole-cell level. A clinically relevant example is the bacterial growth response to antibiotics, which is routinely characterized by dose–response curves. The shape of the dose–response curve varies drastically between antibiotics and plays a key role in treatment, drug interactions, and resistance evolution. However, the mechanisms shaping the dose–response curve remain largely unclear. Here, we show in Escherichia coli that the distinctively shallow dose–response curve of the antibiotic trimethoprim is caused by a negative growth-mediated feedback loop: Trimethoprim slows growth, which in turn weakens the effect of this antibiotic. At the molecular level, this feedback is caused by the upregulation of the drug target dihydrofolate reductase (FolA/DHFR). We show that this upregulation is not a specific response to trimethoprim but follows a universal trend line that depends primarily on the growth rate, irrespective of its cause. Rewiring the feedback loop alters the dose–response curve in a predictable manner, which we corroborate using a mathematical model of cellular resource allocation and growth. Our results indicate that growth-mediated feedback loops may shape drug responses more generally and could be exploited to design evolutionary traps that enable selection against drug resistance.
AU - Angermayr, Andreas
AU - Pang, Tin Yau
AU - Chevereau, Guillaume
AU - Mitosch, Karin
AU - Lercher, Martin J
AU - Bollenbach, Mark Tobias
ID - 12261
IS - 9
JF - Molecular Systems Biology
KW - Applied Mathematics
KW - Computational Theory and Mathematics
KW - General Agricultural and Biological Sciences
KW - General Immunology and Microbiology
KW - General Biochemistry
KW - Genetics and Molecular Biology
KW - Information Systems
TI - Growth‐mediated negative feedback shapes quantitative antibiotic response
VL - 18
ER -
TY - JOUR
AB - The COVID−19 pandemic not only resulted in a global crisis, but also accelerated vaccine development and antibody discovery. Herein we report a synthetic humanized VHH library development pipeline for nanomolar-range affinity VHH binders to SARS-CoV-2 variants of concern (VoC) receptor binding domains (RBD) isolation. Trinucleotide-based randomization of CDRs by Kunkel mutagenesis with the subsequent rolling-cycle amplification resulted in more than 1011 diverse phage display library in a manageable for a single person number of electroporation reactions. We identified a number of nanomolar-range affinity VHH binders to SARS-CoV-2 variants of concern (VoC) receptor binding domains (RBD) by screening a novel synthetic humanized antibody library. In order to explore the most robust and fast method for affinity improvement, we performed affinity maturation by CDR1 and CDR2 shuffling and avidity engineering by multivalent trimeric VHH fusion protein construction. As a result, H7-Fc and G12x3-Fc binders were developed with the affinities in nM and pM range respectively. Importantly, these affinities are weakly influenced by most of SARS-CoV-2 VoC mutations and they retain moderate binding to BA.4\5. The plaque reduction neutralization test (PRNT) resulted in IC50 = 100 ng\ml and 9.6 ng\ml for H7-Fc and G12x3-Fc antibodies, respectively, for the emerging Omicron BA.1 variant. Therefore, these VHH could expand the present landscape of SARS-CoV-2 neutralization binders with the therapeutic potential for present and future SARS-CoV-2 variants.
AU - Dormeshkin, Dmitri
AU - Shapira, Michail
AU - Dubovik, Simon
AU - Kavaleuski, Anton
AU - Katsin, Mikalai
AU - Migas, Alexandr
AU - Meleshko, Alexander
AU - Semyonov, Sergei
ID - 12252
JF - Frontiers in Immunology
KW - Immunology
KW - Immunology and Allergy
KW - COVID-19
KW - SARS-CoV-2
KW - synthetic library
KW - RBD
KW - neutralization nanobody
KW - VHH
SN - 1664-3224
TI - Isolation of an escape-resistant SARS-CoV-2 neutralizing nanobody from a novel synthetic nanobody library
VL - 13
ER -
TY - JOUR
AB - To understand the function of neuronal circuits, it is crucial to disentangle the connectivity patterns within the network. However, most tools currently used to explore connectivity have low throughput, low selectivity, or limited accessibility. Here, we report the development of an improved packaging system for the production of the highly neurotropic RVdGenvA-CVS-N2c rabies viral vectors, yielding titers orders of magnitude higher with no background contamination, at a fraction of the production time, while preserving the efficiency of transsynaptic labeling. Along with the production pipeline, we developed suites of ‘starter’ AAV and bicistronic RVdG-CVS-N2c vectors, enabling retrograde labeling from a wide range of neuronal populations, tailored for diverse experimental requirements. We demonstrate the power and flexibility of the new system by uncovering hidden local and distal inhibitory connections in the mouse hippocampal formation and by imaging the functional properties of a cortical microcircuit across weeks. Our novel production pipeline provides a convenient approach to generate new rabies vectors, while our toolkit flexibly and efficiently expands the current capacity to label, manipulate and image the neuronal activity of interconnected neuronal circuits in vitro and in vivo.
AU - Sumser, Anton L
AU - Jösch, Maximilian A
AU - Jonas, Peter M
AU - Ben Simon, Yoav
ID - 12288
JF - eLife
KW - General Immunology and Microbiology
KW - General Biochemistry
KW - Genetics and Molecular Biology
KW - General Medicine
KW - General Neuroscience
TI - Fast, high-throughput production of improved rabies viral vectors for specific, efficient and versatile transsynaptic retrograde labeling
VL - 11
ER -
TY - JOUR
AB - To understand how potential gene manipulations affect in vitro microglia, we provide a set of short protocols to evaluate microglia identity and function. We detail steps for immunostaining to determine microglia identity. We describe three functional assays for microglia: phagocytosis, calcium response following ATP stimulation, and cytokine expression upon inflammatory stimuli. We apply these protocols to human induced-pluripotent-stem-cell (hiPSC)-derived microglia, but they can be also applied to other in vitro microglial models including primary mouse microglia.
For complete details on the use and execution of this protocol, please refer to Bartalska et al. (2022).1
AU - Hübschmann, Verena
AU - Korkut, Medina
AU - Siegert, Sandra
ID - 12117
IS - 4
JF - STAR Protocols
KW - General Immunology and Microbiology
KW - General Biochemistry
KW - Genetics and Molecular Biology
KW - General Neuroscience
SN - 2666-1667
TI - Assessing human iPSC-derived microglia identity and function by immunostaining, phagocytosis, calcium activity, and inflammation assay
VL - 3
ER -
TY - JOUR
AB - We report the complete analysis of a deterministic model of deleterious mutations and negative selection against them at two haploid loci without recombination. As long as mutation is a weaker force than selection, mutant alleles remain rare at the only stable equilibrium, and otherwise, a variety of dynamics are possible. If the mutation-free genotype is absent, generally the only stable equilibrium is the one that corresponds to fixation of the mutant allele at the locus where it is less deleterious. This result suggests that fixation of a deleterious allele that follows a click of the Muller’s ratchet is governed by natural selection, instead of random drift.
AU - Khudiakova, Kseniia
AU - Neretina, Tatiana Yu.
AU - Kondrashov, Alexey S.
ID - 9387
JF - Journal of Theoretical Biology
KW - General Biochemistry
KW - Genetics and Molecular Biology
KW - Modelling and Simulation
KW - Statistics and Probability
KW - General Immunology and Microbiology
KW - Applied Mathematics
KW - General Agricultural and Biological Sciences
KW - General Medicine
SN - 0022-5193
TI - Two linked loci under mutation-selection balance and Muller’s ratchet
VL - 524
ER -
TY - JOUR
AB - De novo protein synthesis is required for synapse modifications underlying stable memory encoding. Yet neurons are highly compartmentalized cells and how protein synthesis can be regulated at the synapse level is unknown. Here, we characterize neuronal signaling complexes formed by the postsynaptic scaffold GIT1, the mechanistic target of rapamycin (mTOR) kinase, and Raptor that couple synaptic stimuli to mTOR-dependent protein synthesis; and identify NMDA receptors containing GluN3A subunits as key negative regulators of GIT1 binding to mTOR. Disruption of GIT1/mTOR complexes by enhancing GluN3A expression or silencing GIT1 inhibits synaptic mTOR activation and restricts the mTOR-dependent translation of specific activity-regulated mRNAs. Conversely, GluN3A removal enables complex formation, potentiates mTOR-dependent protein synthesis, and facilitates the consolidation of associative and spatial memories in mice. The memory enhancement becomes evident with light or spaced training, can be achieved by selectively deleting GluN3A from excitatory neurons during adulthood, and does not compromise other aspects of cognition such as memory flexibility or extinction. Our findings provide mechanistic insight into synaptic translational control and reveal a potentially selective target for cognitive enhancement.
AU - Conde-Dusman, María J
AU - Dey, Partha N
AU - Elía-Zudaire, Óscar
AU - Garcia Rabaneda, Luis E
AU - García-Lira, Carmen
AU - Grand, Teddy
AU - Briz, Victor
AU - Velasco, Eric R
AU - Andero Galí, Raül
AU - Niñerola, Sergio
AU - Barco, Angel
AU - Paoletti, Pierre
AU - Wesseling, John F
AU - Gardoni, Fabrizio
AU - Tavalin, Steven J
AU - Perez-Otaño, Isabel
ID - 10301
JF - eLife
KW - general immunology and microbiology
KW - general biochemistry
KW - genetics and molecular biology
KW - general medicine
KW - general neuroscience
SN - 2050-084X
TI - Control of protein synthesis and memory by GluN3A-NMDA receptors through inhibition of GIT1/mTORC1 assembly
VL - 10
ER -
TY - JOUR
AU - Pranger, Christina L.
AU - Fazekas-Singer, Judit
AU - Köhler, Verena K.
AU - Pali‐Schöll, Isabella
AU - Fiocchi, Alessandro
AU - Karagiannis, Sophia N.
AU - Zenarruzabeitia, Olatz
AU - Borrego, Francisco
AU - Jensen‐Jarolim, Erika
ID - 10836
IS - 5
JF - Allergy
KW - Immunology
KW - Immunology and Allergy
SN - 0105-4538
TI - PIPE‐cloned human IgE and IgG4 antibodies: New tools for investigating cow's milk allergy and tolerance
VL - 76
ER -
TY - JOUR
AB - Vascular dysfunctions are a common feature of multiple age-related diseases. However, modeling healthy and pathological aging of the human vasculature represents an unresolved experimental challenge. Here, we generated induced vascular endothelial cells (iVECs) and smooth muscle cells (iSMCs) by direct reprogramming of healthy human fibroblasts from donors of different ages and Hutchinson-Gilford Progeria Syndrome (HGPS) patients. iVECs induced from old donors revealed upregulation of GSTM1 and PALD1, genes linked to oxidative stress, inflammation and endothelial junction stability, as vascular aging markers. A functional assay performed on PALD1 KD VECs demonstrated a recovery in vascular permeability. We found that iSMCs from HGPS donors overexpressed bone morphogenetic protein (BMP)−4, which plays a key role in both vascular calcification and endothelial barrier damage observed in HGPS. Strikingly, BMP4 concentrations are higher in serum from HGPS vs. age-matched mice. Furthermore, targeting BMP4 with blocking antibody recovered the functionality of the vascular barrier in vitro, hence representing a potential future therapeutic strategy to limit cardiovascular dysfunction in HGPS. These results show that iVECs and iSMCs retain disease-related signatures, allowing modeling of vascular aging and HGPS in vitro.
AU - Bersini, Simone
AU - Schulte, Roberta
AU - Huang, Ling
AU - Tsai, Hannah
AU - HETZER, Martin W
ID - 11055
JF - eLife
KW - General Immunology and Microbiology
KW - General Biochemistry
KW - Genetics and Molecular Biology
KW - General Medicine
KW - General Neuroscience
SN - 2050-084X
TI - Direct reprogramming of human smooth muscle and vascular endothelial cells reveals defects associated with aging and Hutchinson-Gilford progeria syndrome
VL - 9
ER -
TY - JOUR
AB - Mammalian circadian rhythms are generated by a transcription-based feedback loop in which CLOCK:BMAL1 drives transcription of its repressors (PER1/2, CRY1/2), which ultimately interact with CLOCK:BMAL1 to close the feedback loop with ~24 hr periodicity. Here we pinpoint a key difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. Both cryptochromes bind the BMAL1 transactivation domain similarly to sequester it from coactivators and repress CLOCK:BMAL1 activity. However, we find that CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We discovered a dynamic serine-rich loop adjacent to the secondary pocket in the photolyase homology region (PHR) domain that regulates differential binding of cryptochromes to the PAS domain core of CLOCK:BMAL1. Notably, binding of the co-repressor PER2 remodels the serine loop of CRY2, making it more CRY1-like and enhancing its affinity for CLOCK:BMAL1.
AU - Fribourgh, Jennifer L
AU - Srivastava, Ashutosh
AU - Sandate, Colby R
AU - Michael, Alicia Kathleen
AU - Hsu, Peter L
AU - Rakers, Christin
AU - Nguyen, Leslee T
AU - Torgrimson, Megan R
AU - Parico, Gian Carlo G
AU - Tripathi, Sarvind
AU - Zheng, Ning
AU - Lander, Gabriel C
AU - Hirota, Tsuyoshi
AU - Tama, Florence
AU - Partch, Carrie L
ID - 15153
JF - eLife
KW - General Immunology and Microbiology
KW - General Biochemistry
KW - Genetics and Molecular Biology
KW - General Medicine
KW - General Neuroscience
SN - 2050-084X
TI - Dynamics at the serine loop underlie differential affinity of cryptochromes for CLOCK:BMAL1 to control circadian timing
VL - 9
ER -
TY - JOUR
AB - Transposable elements (TEs), the movement of which can damage the genome, are epigenetically silenced in eukaryotes. Intriguingly, TEs are activated in the sperm companion cell – vegetative cell (VC) – of the flowering plant Arabidopsis thaliana. However, the extent and mechanism of this activation are unknown. Here we show that about 100 heterochromatic TEs are activated in VCs, mostly by DEMETER-catalyzed DNA demethylation. We further demonstrate that DEMETER access to some of these TEs is permitted by the natural depletion of linker histone H1 in VCs. Ectopically expressed H1 suppresses TEs in VCs by reducing DNA demethylation and via a methylation-independent mechanism. We demonstrate that H1 is required for heterochromatin condensation in plant cells and show that H1 overexpression creates heterochromatic foci in the VC progenitor cell. Taken together, our results demonstrate that the natural depletion of H1 during male gametogenesis facilitates DEMETER-directed DNA demethylation, heterochromatin relaxation, and TE activation.
AU - He, Shengbo
AU - Vickers, Martin
AU - Zhang, Jingyi
AU - Feng, Xiaoqi
ID - 12192
JF - eLife
KW - General Immunology and Microbiology
KW - General Biochemistry
KW - Genetics and Molecular Biology
KW - General Medicine
KW - General Neuroscience
SN - 2050-084X
TI - Natural depletion of histone H1 in sex cells causes DNA demethylation, heterochromatin decondensation and transposon activation
VL - 8
ER -
TY - JOUR
AB - The inner nuclear membrane (INM) is a subdomain of the endoplasmic reticulum (ER) that is gated by the nuclear pore complex. It is unknown whether proteins of the INM and ER are degraded through shared or distinct pathways in mammalian cells. We applied dynamic proteomics to profile protein half-lives and report that INM and ER residents turn over at similar rates, indicating that the INM’s unique topology is not a barrier to turnover. Using a microscopy approach, we observed that the proteasome can degrade INM proteins in situ. However, we also uncovered evidence for selective, vesicular transport-mediated turnover of a single INM protein, emerin, that is potentiated by ER stress. Emerin is rapidly cleared from the INM by a mechanism that requires emerin’s LEM domain to mediate vesicular trafficking to lysosomes. This work demonstrates that the INM can be dynamically remodeled in response to environmental inputs.
AU - Buchwalter, Abigail
AU - Schulte, Roberta
AU - Tsai, Hsiao
AU - Capitanio, Juliana
AU - HETZER, Martin W
ID - 11060
JF - eLife
KW - General Immunology and Microbiology
KW - General Biochemistry
KW - Genetics and Molecular Biology
KW - General Medicine
KW - General Neuroscience
SN - 2050-084X
TI - Selective clearance of the inner nuclear membrane protein emerin by vesicular transport during ER stress
VL - 8
ER -
TY - THES
AB - While cells of mesenchymal or epithelial origin perform their effector functions in a purely anchorage dependent manner, cells derived from the hematopoietic lineage are not committed to operate only within a specific niche. Instead, these cells are able to function autonomously of the molecular composition in a broad range of tissue compartments. By this means, cells of the hematopoietic lineage retain the capacity to disseminate into connective tissue and recirculate between organs, building the foundation for essential processes such as tissue regeneration or immune surveillance.
Cells of the immune system, specifically leukocytes, are extraordinarily good at performing this task. These cells are able to flexibly shift their mode of migration between an adhesion-mediated and an adhesion-independent manner, instantaneously accommodating for any changes in molecular composition of the external scaffold. The key component driving directed leukocyte migration is the chemokine receptor 7, which guides the cell along gradients of chemokine ligand. Therefore, the physical destination of migrating leukocytes is purely deterministic, i.e. given by global directional cues such as chemokine gradients.
Nevertheless, these cells typically reside in three-dimensional scaffolds of inhomogeneous complexity, raising the question whether cells are able to locally discriminate between multiple optional migration routes. Current literature provides evidence that leukocytes, specifically dendritic cells, do indeed probe their surrounding by virtue of multiple explorative protrusions. However, it remains enigmatic how these cells decide which one is the more favorable route to follow and what are the key players involved in performing this task. Due to the heterogeneous environment of most tissues, and the vast adaptability of migrating leukocytes, at this time it is not clear to what extent leukocytes are able to optimize their migratory strategy by adapting their level of adhesiveness. And, given the fact that leukocyte migration is characterized by branched cell shapes in combination with high migration velocities, it is reasonable to assume that these cells require fine tuned shape maintenance mechanisms that tightly coordinate protrusion and adhesion dynamics in a spatiotemporal manner.
Therefore, this study aimed to elucidate how rapidly migrating leukocytes opt for an ideal migratory path while maintaining a continuous cell shape and balancing adhesive forces to efficiently navigate through complex microenvironments.
The results of this study unraveled a role for the microtubule cytoskeleton in promoting the decision making process during path finding and for the first time point towards a microtubule-mediated function in cell shape maintenance of highly ramified cells such as dendritic cells. Furthermore, we found that migrating low-adhesive leukocytes are able to instantaneously adapt to increased tensile load by engaging adhesion receptors. This response was only occurring tangential to the substrate while adhesive properties in the vertical direction were not increased. As leukocytes are primed for rapid migration velocities, these results demonstrate that leukocyte integrins are able to confer a high level of traction forces parallel to the cell membrane along the direction of migration without wasting energy in gluing the cell to the substrate.
Thus, the data in the here presented thesis provide new insights into the pivotal role of cytoskeletal dynamics and the mechanisms of force transduction during leukocyte migration.
Thereby the here presented results help to further define fundamental principles underlying leukocyte migration and open up potential therapeutic avenues of clinical relevance.
AU - Kopf, Aglaja
ID - 6891
KW - cell biology
KW - immunology
KW - leukocyte
KW - migration
KW - microfluidics
SN - 978-3-99078-002-2
TI - The implication of cytoskeletal dynamics on leukocyte migration
ER -
TY - JOUR
AB - Eukaryotic cells are densely packed with macromolecular complexes and intertwining organelles, continually transported and reshaped. Intriguingly, organelles avoid clashing and entangling with each other in such limited space. Mitochondria form extensive networks constantly remodeled by fission and fusion. Here, we show that mitochondrial fission is triggered by mechanical forces. Mechano-stimulation of mitochondria – via encounter with motile intracellular pathogens, via external pressure applied by an atomic force microscope, or via cell migration across uneven microsurfaces – results in the recruitment of the mitochondrial fission machinery, and subsequent division. We propose that MFF, owing to affinity for narrow mitochondria, acts as a membrane-bound force sensor to recruit the fission machinery to mechanically strained sites. Thus, mitochondria adapt to the environment by sensing and responding to biomechanical cues. Our findings that mechanical triggers can be coupled to biochemical responses in membrane dynamics may explain how organelles orderly cohabit in the crowded cytoplasm.
AU - Helle, Sebastian Carsten Johannes
AU - Feng, Qian
AU - Aebersold, Mathias J
AU - Hirt, Luca
AU - Grüter, Raphael R
AU - Vahid, Afshin
AU - Sirianni, Andrea
AU - Mostowy, Serge
AU - Snedeker, Jess G
AU - Šarić, Anđela
AU - Idema, Timon
AU - Zambelli, Tomaso
AU - Kornmann, Benoît
ID - 10370
JF - eLife
KW - general immunology and microbiology
KW - general biochemistry
KW - genetics and molecular biology
KW - general medicine
KW - general neuroscience
SN - 2050-084X
TI - Mechanical force induces mitochondrial fission
VL - 6
ER -
TY - JOUR
AB - Biofilm formation is critical for the infection cycle of Vibrio cholerae. Vibrio exopolysaccharides (VPS) and the matrix proteins RbmA, Bap1 and RbmC are required for the development of biofilm architecture. We demonstrate that RbmA binds VPS directly and uses a binary structural switch within its first fibronectin type III (FnIII-1) domain to control RbmA structural dynamics and the formation of VPS-dependent higher-order structures. The structural switch in FnIII-1 regulates interactions in trans with the FnIII-2 domain, leading to open (monomeric) or closed (dimeric) interfaces. The ability of RbmA to switch between open and closed states is important for V. cholerae biofilm formation, as RbmA variants with switches that are locked in either of the two states lead to biofilms with altered architecture and structural integrity.
AU - Fong, Jiunn CN
AU - Rogers, Andrew
AU - Michael, Alicia Kathleen
AU - Parsley, Nicole C
AU - Cornell, William-Cole
AU - Lin, Yu-Cheng
AU - Singh, Praveen K
AU - Hartmann, Raimo
AU - Drescher, Knut
AU - Vinogradov, Evgeny
AU - Dietrich, Lars EP
AU - Partch, Carrie L
AU - Yildiz, Fitnat H
ID - 15154
JF - eLife
KW - General Immunology and Microbiology
KW - General Biochemistry
KW - Genetics and Molecular Biology
KW - General Medicine
KW - General Neuroscience
SN - 2050-084X
TI - Structural dynamics of RbmA governs plasticity of Vibrio cholerae biofilms
VL - 6
ER -