Microglia are the resident macrophages of the CNS, and their functions have been extensively studied in various brain pathologies. The physiological roles of microglia in brain plasticity and ...function, however, remain unclear. To address this question, we generated CX3CR1CreER mice expressing tamoxifen-inducible Cre recombinase that allow for specific manipulation of gene function in microglia. Using CX3CR1CreER to drive diphtheria toxin receptor expression in microglia, we found that microglia could be specifically depleted from the brain upon diphtheria toxin administration. Mice depleted of microglia showed deficits in multiple learning tasks and a significant reduction in motor-learning-dependent synapse formation. Furthermore, Cre-dependent removal of brain-derived neurotrophic factor (BDNF) from microglia largely recapitulated the effects of microglia depletion. Microglial BDNF increases neuronal tropomyosin-related kinase receptor B phosphorylation, a key mediator of synaptic plasticity. Together, our findings reveal that microglia serve important physiological functions in learning and memory by promoting learning-related synapse formation through BDNF signaling.
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•CX3CR1CreER mice allow for the specific manipulation of microglial function•Microglia are important for learning and learning-dependent synaptic remodeling•Microglial BDNF is an important regulator of synaptic plasticity and function
Mice lacking microglia show defects in multiple learning tasks, with microglia-derived brain-derived neurotrophic factor being a critical factor for learning-dependent synaptic plasticity.
Mass spectrometry plays a key role in relative quantitative comparisons of proteins in order to understand their functional role in biological systems upon perturbation. In this review, we review ...studies that examine different aspects of isobaric labeling-based relative quantification for shotgun proteomic analysis. In particular, we focus on different types of isobaric reagents and their reaction chemistry (e.g., amine-, carbonyl-, and sulfhydryl-reactive). Various factors, such as ratio compression, reporter ion dynamic range, and others, cause an underestimation of changes in relative abundance of proteins across samples, undermining the ability of the isobaric labeling approach to be truly quantitative. These factors that affect quantification and the suggested combinations of experimental design and optimal data acquisition methods to increase the precision and accuracy of the measurements will be discussed. Finally, the extended application of isobaric labeling-based approach in hyperplexing strategy, targeted quantification, and phosphopeptide analysis are also examined.
Virulent biofilms are responsible for a range of infections, including oral diseases. All biofilms harbor a microbial-derived extracellular-matrix. The exopolysaccharides (EPS) formed on ...tooth-pellicle and bacterial surfaces provide binding sites for microorganisms; eventually the accumulated EPS enmeshes microbial cells. The metabolic activity of the bacteria within this matrix leads to acidification of the milieu. We explored the mechanisms through which the Streptococcus mutans-produced EPS-matrix modulates the three-dimensional (3D) architecture and the population shifts during morphogenesis of biofilms on a saliva-coated-apatitic surface using a mixed-bacterial species system. Concomitantly, we examined whether the matrix influences the development of pH-microenvironments within intact-biofilms using a novel 3D in situ pH-mapping technique. Data reveal that the production of the EPS-matrix helps to create spatial heterogeneities by forming an intricate network of exopolysaccharide-enmeshed bacterial-islets (microcolonies) through localized cell-to-matrix interactions. This complex 3D architecture creates compartmentalized acidic and EPS-rich microenvironments throughout the biofilm, which triggers the dominance of pathogenic S. mutans within a mixed-species system. The establishment of a 3D-matrix and EPS-enmeshed microcolonies were largely mediated by the S. mutans gtfB/gtfC genes, expression of which was enhanced in the presence of Actinomyces naeslundii and Streptococcus oralis. Acidic pockets were found only in the interiors of bacterial-islets that are protected by EPS, which impedes rapid neutralization by buffer (pH 7.0). As a result, regions of low pH (<5.5) were detected at specific locations along the surface of attachment. Resistance to chlorhexidine was enhanced in cells within EPS-microcolony complexes compared to those outside such structures within the biofilm. Our results illustrate the critical interaction between matrix architecture and pH heterogeneity in the 3D environment. The formation of structured acidic-microenvironments in close proximity to the apatite-surface is an essential factor associated with virulence in cariogenic-biofilms. These observations may have relevance beyond the mouth, as matrix is inherent to all biofilms.
CtIP plays an important role in homologous recombination (HR)-mediated DNA double-stranded break (DSB) repair and interacts with Nbs1 and BRCA1, which are linked to Nijmegen breakage syndrome (NBS) ...and familial breast cancer, respectively. We identified new CDK phosphorylation sites on CtIP and found that phosphorylation of these newly identified CDK sites induces association of CtIP with the N-terminus FHA and BRCT domains of Nbs1. We further showed that these CDK-dependent phosphorylation events are a prerequisite for ATM to phosphorylate CtIP upon DNA damage, which is important for end resection to activate HR by promoting recruitment of BLM and Exo1 to DSBs. Most notably, this CDK-dependent CtIP and Nbs1 interaction facilitates ATM to phosphorylate CtIP in a substrate-specific manner. These studies reveal one important mechanism to regulate cell-cycle-dependent activation of HR upon DNA damage by coupling CDK- and ATM-mediated phosphorylation of CtIP through modulating the interaction of CtIP with Nbs1, which significantly helps to understand how DSB repair is regulated in mammalian cells to maintain genome stability.
Abscisic acid (ABA) regulates abiotic stress and developmental responses including regulation of seed dormancy to prevent seeds from germinating under unfavorable environmental conditions. ABA ...HYPERSENSITIVE GERMINATION1 (AHG1) encoding a type 2C protein phosphatase (PP2C) is a central negative regulator of ABA response in germination; however, the molecular function and regulation of AHG1 remain elusive. Here we report that AHG1 interacts with DELAY OF GERMINATION1 (DOG1), which is a pivotal positive regulator in seed dormancy. DOG1 acts upstream of AHG1 and impairs the PP2C activity of AHG1 in vitro. Furthermore, DOG1 has the ability to bind heme. Binding of DOG1 to AHG1 and heme are independent processes, but both are essential for DOG1 function in vivo. Our study demonstrates that AHG1 and DOG1 constitute an important regulatory system for seed dormancy and germination by integrating multiple environmental signals, in parallel with the PYL/RCAR ABA receptor-mediated regulatory system.
The ability to perceive and respond to harmful conditions is crucial for the survival of any organism. The transcription factor DAF-16/FOXO is central to these responses, relaying distress signals ...into the expression of stress resistance and longevity promoting genes. However, its sufficiency in fulfilling this complex task has remained unclear. Using C. elegans, we show that DAF-16 does not function alone but as part of a transcriptional regulatory module, together with the transcription factor HLH-30/TFEB. Under harmful conditions, both transcription factors translocate into the nucleus, where they often form a complex, co-occupy target promoters, and co-regulate many target genes. Interestingly though, their synergy is stimulus-dependent: They rely on each other, functioning in the same pathway, to promote longevity or resistance to oxidative stress, but they elicit heat stress responses independently, and they even oppose each other during dauer formation. We propose that this module of DAF-16 and HLH-30 acts by combinatorial gene regulation to relay distress signals into the expression of specific target gene sets, ensuring optimal survival under each given threat.
In the past decade, there have been remarkable advances in proteomic technologies. Mass spectrometry has emerged as the preferred method for in-depth characterization of the protein components of ...biological systems. Using mass spectrometry, key insights into the composition, regulation and function of molecular complexes and pathways have been gained. From these studies, it is clear that mass-spectrometry-based proteomics is now a powerful 'hypothesis-generating engine' that, when combined with complementary molecular, cellular and pharmacological techniques, provides a framework for translating large data sets into an understanding of complex biological processes.
Intracellular proteins with long lifespans have recently been linked to age-dependent defects, ranging from decreased fertility to the functional decline of neurons. Why long-lived proteins exist in ...metabolically active cellular environments and how they are maintained over time remains poorly understood. Here, we provide a system-wide identification of proteins with exceptional lifespans in the rat brain. These proteins are inefficiently replenished despite being translated robustly throughout adulthood. Using nucleoporins as a paradigm for long-term protein persistence, we found that nuclear pore complexes (NPCs) are maintained over a cell’s life through slow but finite exchange of even its most stable subcomplexes. This maintenance is limited, however, as some nucleoporin levels decrease during aging, providing a rationale for the previously observed age-dependent deterioration of NPC function. Our identification of a long-lived proteome reveals cellular components that are at increased risk for damage accumulation, linking long-term protein persistence to the cellular aging process.
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•Metabolic pulse-chase labeling of rats identified long-lived proteins in rats•Long-lived proteins include nucleoporins, histone variants, and enzymes•Long-lived proteins cannot be replaced despite their robust translation•Nuclear pores are maintained over the lifespan of the organism
A system-wide identification of proteins with exceptional lifespans in the rat brain suggests that these have a higher propensity for damage. Cells utilize specific mechanisms to ensure that such proteins are maintained in a functional state through aging.
The multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the ~30 kb viral RNA genome to aid its packaging into the 80-90 nm membrane-enveloped virion. The N protein is composed of N-terminal ...RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that the N protein's central disordered domain drives phase separation with RNA, and that phosphorylation of an adjacent serine/arginine rich region modulates the physical properties of the resulting condensates. In cells, N forms condensates that recruit the stress granule protein G3BP1, highlighting a potential role for N in G3BP1 sequestration and stress granule inhibition. The SARS-CoV-2 membrane (M) protein independently induces N protein phase separation, and three-component mixtures of N + M + RNA form condensates with mutually exclusive compartments containing N + M or N + RNA, including annular structures in which the M protein coats the outside of an N + RNA condensate. These findings support a model in which phase separation of the SARS-CoV-2 N protein contributes both to suppression of the G3BP1-dependent host immune response and to packaging genomic RNA during virion assembly.
Focal adhesions undergo myosin-II-mediated maturation wherein they grow and change composition to modulate integrin signalling for cell migration, growth and differentiation. To determine how focal ...adhesion composition is affected by myosin II activity, we performed proteomic analysis of isolated focal adhesions and compared protein abundance in focal adhesions from cells with and without myosin II inhibition. We identified 905 focal adhesion proteins, 459 of which changed in abundance with myosin II inhibition, defining the myosin-II-responsive focal adhesion proteome. The abundance of 73% of the proteins in the myosin-II-responsive focal adhesion proteome was enhanced by contractility, including proteins involved in Rho-mediated focal adhesion maturation and endocytosis- and calpain-dependent focal adhesion disassembly. During myosin II inhibition, 27% of proteins in the myosin-II-responsive focal adhesion proteome, including proteins involved in Rac-mediated lamellipodial protrusion, were enriched in focal adhesions, establishing that focal adhesion protein recruitment is also negatively regulated by contractility. We focused on the Rac guanine nucleotide exchange factor β-Pix, documenting its role in the negative regulation of focal adhesion maturation and the promotion of lamellipodial protrusion and focal adhesion turnover to drive cell migration.
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