Project description:The PROPPIN family member Atg18 is a phosphoinositide-binding protein that is composed of a seven β-propeller motif and is part of the conserved autophagy machinery. Here, we report that the Atg18 phosphorylation in the loops in the propellar structure of blade 6 and blade 7 decreases its binding affinity to phosphatidylinositol 3,5-bisphosphate in the yeast Pichia pastoris. Dephosphorylation of Atg18 was necessary for its association with the vacuolar membrane and caused septation of the vacuole. Upon or after dissociation from the vacuolar membrane, Atg18 was rephosphorylated, and the vacuoles fused and formed a single rounded structure. Vacuolar dynamics were regulated according to osmotic changes, oxidative stresses, and nutrient conditions inducing micropexophagy via modulation of Atg18 phosphorylation. This study reveals how the phosphoinositide-binding activity of the PROPPIN family protein Atg18 is regulated at the membrane association domain and highlights the importance of such phosphoregulation in coordinated intracellular reorganization.
Project description:The mitotic spindle, essential for segregating the sister chromatids into the two evolving daughter cells, is composed of highly dynamic cytoskeletal filaments, the microtubules. The dynamics of microtubules are regulated by numerous microtubule associated proteins. We identify here Developmentally regulated GTP binding protein 1 (DRG1) as a microtubule binding protein with diverse microtubule-associated functions. In vitro, DRG1 can diffuse on microtubules, promote their polymerization, drive microtubule formation into bundles, and stabilize microtubules. HeLa cells with reduced DRG1 levels show delayed progression from prophase to anaphase because spindle formation is slowed down. To perform its microtubule-associated functions, DRG1, although being a GTPase, does not require GTP hydrolysis. However, all domains are required as truncated versions show none of the mentioned activities besides microtubule binding.
Project description:KEY POINTS:Neurons in the hypothalamus of the brain which secrete the peptide kisspeptin are important regulators of reproduction, and normal reproductive development. Electrical activity, in the form of action potentials, or spikes, leads to secretion of peptides and neurotransmitters, influencing the activity of downstream neurons; in kisspeptin neurons, this activity is highly irregular, but the mechanism of this is not known. In this study, we show that irregularity depends on the presence of a particular type of potassium ion channel in the membrane, which opens transiently in response to electrical excitation. The results contribute to understanding how kisspeptin neurons generate and time their membrane potential spikes, and how reliable this process is. Improved understanding of the activity of kisspeptin neurons, and how it shapes their secretion of peptides, is expected to lead to better treatment for reproductive dysfunction and disorders of reproductive development. ABSTRACT:Kisspeptin neurons in the hypothalamus are critically involved in reproductive function, via their effect on GnRH neuron activity and consequent gonadotropin release. Kisspeptin neurons show an intrinsic irregularity of firing, but the mechanism of this remains unclear. To address this, we carried out targeted whole-cell patch-clamp recordings of kisspeptin neurons in the arcuate nucleus (Kiss1Arc ), in brain slices isolated from adult male Kiss-Cre:tdTomato mice. Cells fired irregularly in response to constant current stimuli, with a wide range of spike time variability, and prominent subthreshold voltage fluctuations. In voltage clamp, both a persistent sodium (NaP) current and a fast transient (A-type) potassium current were apparent, activating at potentials just below the threshold for spiking. These currents have also previously been described in irregular-spiking cortical interneurons, in which the A-type current, mediated by Kv4 channels, interacts with NaP current to generate complex dynamics of the membrane potential, and irregular firing. In Kiss1Arc neurons, A-type current was blocked by phrixotoxin, a specific blocker of Kv4.2/4.3 channels, and consistent expression of Kv4.2 transcripts was detected by single-cell RT-PCR. In addition, firing irregularity was correlated to the density of A-type current in the membrane. Using conductance injection, we demonstrated that adding Kv4-like potassium conductance (gKv4 ) to a cell produces a striking increase in firing irregularity, and excitability is reduced, while subtracting gKv4 has the opposite effects. Thus, we propose that Kv4 interacting dynamically with NaP is a key determinant of the irregular firing behaviour of Kiss1Arc neurons, shaping their physiological function in gonadotropin release.
Project description:Cell division cycle 42 (Cdc42) is a member of the Rho guanosine triphosphatase family and has pivotal functions in actin organization, cell migration, and proliferation. To further study the molecular mechanisms of dendritic cell (DC) regulation by Cdc42, we used Cdc42-deficient DCs. Cdc42 deficiency renders DCs phenotypically mature as they up-regulate the co-stimulatory molecule CD86 from intracellular storages to the cell surface. Cdc42 knockout DCs also accumulate high amounts of invariant chain-major histocompatibility complex (MHC) class II complexes at the cell surface, which cannot efficiently present peptide antigens (Ag's) for priming of Ag-specific CD4 T cells. Proteome analyses showed a significant reduction in lysosomal MHC class II-processing proteins, such as cathepsins, which are lost from DCs by enhanced secretion. As these effects on DCs can be mimicked by chemical actin disruption, our results propose that Cdc42 control of actin dynamics keeps DCs in an immature state, and cessation of Cdc42 activity during DC maturation facilitates secretion as well as rapid up-regulation of intracellular molecules to the cell surface.
Project description:In all organisms with circadian clocks, post-translational modifications of clock proteins control the dynamics of circadian rhythms, with phosphorylation playing a dominant role. All major clock proteins are highly phosphorylated, and many kinases have been described to be responsible. In contrast, it is largely unclear whether and to what extent their counterparts, the phosphatases, play an equally crucial role. To investigate this, we performed a systematic RNAi screen in human cells and identified protein phosphatase 4 (PPP4) with its regulatory subunit PPP4R2 as critical components of the circadian system in both mammals and Drosophila Genetic depletion of PPP4 shortens the circadian period, whereas overexpression lengthens it. PPP4 inhibits CLOCK/BMAL1 transactivation activity by binding to BMAL1 and counteracting its phosphorylation. This leads to increased CLOCK/BMAL1 DNA occupancy and decreased transcriptional activity, which counteracts the "kamikaze" properties of CLOCK/BMAL1. Through this mechanism, PPP4 contributes to the critical delay of negative feedback by retarding PER/CRY/CK1δ-mediated inhibition of CLOCK/BMAL1.
Project description:The posttranslational addition of palmitate to cysteines occurs ubiquitously in eukaryotic cells, where it functions in anchoring target proteins to membranes and in vesicular trafficking. Here we show that the Saccharomyces cerevisiae palmitoyltransferase Pfa4 enhanced heterochromatin formation at the cryptic mating-type loci HMR and HML via Rif1, a telomere regulatory protein. Acylated Rif1 was detected in extracts from wild-type but not pfa4? mutant cells. In a pfa4? mutant, Rif1-GFP dispersed away from foci positioned at the nuclear periphery into the nucleoplasm. Sir3-GFP distribution was also perturbed, indicating a change in the nuclear dynamics of heterochromatin proteins. Genetic analyses indicated that PFA4 functioned upstream of RIF1. Surprisingly, the pfa4? mutation had only mild effects on telomeric regulation, suggesting Rif1's roles at HM loci and telomeres were more complexly related than previously thought. These data supported a model in which Pfa4-dependent palmitoylation of Rif1 anchored it to the inner nuclear membrane, influencing its role in heterochromatin dynamics.
Project description:?-catenin is a key mechanosensor that forms force-dependent interactions with F-actin, thereby coupling the cadherin-catenin complex to the actin cytoskeleton at adherens junctions (AJs). However, the molecular mechanisms by which ?-catenin engages F-actin under tension remained elusive. Here we show that the ?1-helix of the ?-catenin actin-binding domain (?cat-ABD) is a mechanosensing motif that regulates tension-dependent F-actin binding and bundling. ?cat-ABD containing an ?1-helix-unfolding mutation (H1) shows enhanced binding to F-actin in vitro. Although full-length ?-catenin-H1 can generate epithelial monolayers that resist mechanical disruption, it fails to support normal AJ regulation in vivo. Structural and simulation analyses suggest that ?1-helix allosterically controls the actin-binding residue V796 dynamics. Crystal structures of ?cat-ABD-H1 homodimer suggest that ?-catenin can facilitate actin bundling while it remains bound to E-cadherin. We propose that force-dependent allosteric regulation of ?cat-ABD promotes dynamic interactions with F-actin involved in actin bundling, cadherin clustering, and AJ remodeling during tissue morphogenesis.
Project description:The ANTAR domain harnesses RNA-binding activity to promote transcription attenuation. Although several ANTAR proteins have been analyzed by high-resolution structural analyses, the residues involved in RNA-recognition and transcription attenuation have not been identified. Nor is it clear how signal-responsive domains are allosterically coupled with ANTAR domains for control of gene expression. Herein, we examined the sequence conservation of ANTAR domains to find residues that may associate with RNA. We subjected the corresponding positions of Klebsiella oxytoca NasR to site-directed alanine substitutions and measured RNA-binding activity. This revealed a functionally important patch of residues that forms amino acid pairing interactions with residues from NasR's nitrate-sensing NIT domain. We hypothesize these amino acid pairing interactions are part of an autoinhibitory mechanism that holds the structure in an "off" state in the absence of nitrate signal. Indeed, mutational disruption of these interactions resulted in constitutively active proteins, freed from autoinhibition and no longer influenced by nitrate. Moreover, sequence analyses suggested the autoinhibitory mechanism has been evolutionarily maintained by NasR proteins. These data reveal a molecular mechanism for how NasR couples its nitrate signal to RNA-binding activity, and generally show how signal-responsive domains of one-component regulatory proteins have evolved to exert control over RNA-binding ANTAR domains.
Project description:Broad-spectrum amino acid racemases (Bsrs) enable bacteria to generate non-canonical D-amino acids (NCDAAs), whose roles and impact on microbial physiology, including modulation of cell wall structure and dissolution of biofilms, are just beginning to be appreciated. Here we used a diverse array of structural, biochemical and molecular simulation studies to define and characterize how BsrV is post-translationally regulated. We discovered that contrary to Vibrio cholerae alanine racemase AlrV highly compacted active site, BsrV's is broader and can be occupied by cell wall stem peptides. We found that peptidoglycan peptides modified with NCDAAs are better stabilized by BsrV's catalytic cavity and show better inhibitory capacity than canonical muropeptides. Notably, BsrV binding and inhibition can be recapitulated by undigested peptidoglycan sacculi as it exists in the cell. Docking simulations of BsrV binding the peptidoglycan polymer generate a model where the peptide stems are perfectly accommodated and stabilized within each of the dimeŕs active sites. Taking these biochemical and structural data together, we propose that inhibition of BsrV by peptidoglycan peptides underlies a negative regulatory mechanism to avoid excessive NCDAA production. Our results collectively open the door to use "à la carte" synthetic peptides as a tool to modulate DAAs production of Bsr enzymes.
Project description:Persistent experience-driven adaptation of brain function is associated with alterations in gene expression patterns, resulting in structural and functional neuronal remodeling. How synaptic activity-in particular presynaptic performance-is coupled to gene expression in nucleus remains incompletely understood. Here, we report on a role of CtBP1, a transcriptional co-repressor enriched in presynapses and nuclei, in the activity-driven reconfiguration of gene expression in neurons. We demonstrate that presynaptic and nuclear pools of CtBP1 are interconnected and that both synaptic retention and shuttling of CtBP1 between cytoplasm and nucleus are co-regulated by neuronal activity. Finally, we show that CtBP1 is targeted and/or anchored to presynapses by direct interaction with the active zone scaffolding proteins Bassoon and Piccolo. This association is regulated by neuronal activity via modulation of cellular NAD/NADH levels and restrains the size of the CtBP1 pool available for nuclear import, thus contributing to the control of activity-dependent gene expression. Our combined results reveal a mechanism for coupling activity-induced molecular rearrangements in the presynapse with reconfiguration of neuronal gene expression.