The Membrane-Associated Proteins FCHo and SGIP Are Allosteric Activators of the AP2 Clathrin Adaptor Complex

The AP2 clathrin adaptor complex links protein cargo to the endocytic machinery but it is unclear how AP2 is activated on the plasma membrane. Here we demonstrate that the membrane-associated proteins FCHo and SGIP1 convert AP2 into an open, active conformation. We screened for C. elegans mutants that phenocopy the loss of AP2 subunits and found that AP2 remains inactive in fcho-1 mutants. A subsequent screen for bypass suppressors of fcho-1 nulls identified 71 compensatory mutations in all four AP2 subunits. Using a protease-sensitivity assay we show that these mutations restore the open conformation in vivo. The domain of FCHo that induces this rearrangement is not the F-BAR domain or the mu-homology domain, but rather is an uncharacterized 90 amino acid motif, found in both FCHo and SGIP proteins, that directly binds AP2. Thus, these proteins stabilize nascent endocytic pits by exposing membrane and cargo binding sites on AP2

Neuroticism Associates with Cerebral in Vivo Serotonin Transporter Binding Differently in Males and Females

Background: Neuroticism is a major risk factor for affective disorders. This personality trait has been hypothesized to associate with synaptic availability of the serotonin transporter, which critically controls serotonergic tone in the brain. However, earlier studies linking neuroticism and serotonin transporter have failed to produce converging findings. Because sex affects both the serotonergic system and the risk that neuroticism poses to the individual, sex may modify the association between neuroticism and serotonin transporter, but this question has not been investigated by previous studies. Methods: Here, we combined data from 4 different positron emission tomography imaging centers to address whether neuroticism is related to serotonin transporter binding in vivo. The data set included serotonin transporter binding potential values from the thalamus and striatum and personality scores from 91 healthy males and 56 healthy females. We specifically tested if the association between neuroticism and serotonin transporter is different in females and males. Results: We found that neuroticism and thalamic serotonin transporter binding potentials were associated in both males and females, but with opposite directionality. Higher neuroticism associated with higher serotonin transporter binding potential in males (standardized beta 0.292, P = .008), whereas in females, higher neuroticism associated with lower serotonin transporter binding potential (standardized beta -0.288, P = .014). Conclusions: The finding is in agreement with recent studies showing that the serotonergic system is involved in affective disorders differently in males and females and suggests that contribution of thalamic serotonin transporter to the risk of affective disorders depends on sex.Peer reviewe

A Genome-Wide Collection of Mos1 Transposon Insertion Mutants for the C. elegans Research Community

Methods that use homologous recombination to engineer the genome of C. elegans commonly use strains carrying specific insertions of the heterologous transposon Mos1. A large collection of known Mos1 insertion alleles would therefore be of general interest to the C. elegans research community. We describe here the optimization of a semi-automated methodology for the construction of a substantial collection of Mos1 insertion mutant strains. At peak production, more than 5,000 strains were generated per month. These strains were then subject to molecular analysis, and more than 13,300 Mos1 insertions characterized. In addition to targeting directly more than 4,700 genes, these alleles represent the potential starting point for the engineered deletion of essentially all C. elegans genes and the modification of more than 40% of them. This collection of mutants, generated under the auspices of the European NEMAGENETAG consortium, is publicly available and represents an important research resource

The Caenorhabditis elegans microtubule minus-end binding homolog PTRN-1 stabilizes synapses and neurites

Microtubule dynamics facilitate neurite growth and establish morphology, but the role of minus-end binding proteins in these processes is largely unexplored. CAMSAP homologs associate with microtubule minus-ends, and are important for the stability of epithelial cell adhesions. In this study, we report morphological defects in neurons and neuromuscular defects in mutants of the C. elegans CAMSAP, ptrn-1. Mechanosensory neurons initially extend wild-type neurites, and subsequently remodel by overextending neurites and retracting synaptic branches and presynaptic varicosities. This neuronal remodeling can be activated by mutations known to alter microtubules, and depends on a functioning DLK-1 MAP kinase pathway. We found that PTRN-1 localizes to both neurites and synapses, and our results suggest that alterations of microtubule structures caused by loss of PTRN-1 function activates a remodeling program leading to changes in neurite morphology. We propose a model whereby minus-end microtubule stabilization mediated by a functional PTRN-1 is necessary for morphological maintenance of neurons. DOI: http://dx.doi.org/10.7554/eLife.01637.00

The Center for Integrated Molecular Brain Imaging (Cimbi) database

AbstractWe here describe a multimodality neuroimaging containing data from healthy volunteers and patients, acquired within the Lundbeck Foundation Center for Integrated Molecular Brain Imaging (Cimbi) in Copenhagen, Denmark. The data is of particular relevance for neurobiological research questions related to the serotonergic transmitter system with its normative data on the serotonergic subtype receptors 5-HT1A, 5-HT1B, 5-HT2A, and 5-HT4 and the 5-HT transporter (5-HTT), but can easily serve other purposes.The Cimbi database and Cimbi biobank were formally established in 2008 with the purpose to store the wealth of Cimbi-acquired data in a highly structured and standardized manner in accordance with the regulations issued by the Danish Data Protection Agency as well as to provide a quality-controlled resource for future hypothesis-generating and hypothesis-driven studies.The Cimbi database currently comprises a total of 1100 PET and 1000 structural and functional MRI scans and it holds a multitude of additional data, such as genetic and biochemical data, and scores from 17 self-reported questionnaires and from 11 neuropsychological paper/computer tests. The database associated Cimbi biobank currently contains blood and in some instances saliva samples from about 500 healthy volunteers and 300 patients with e.g., major depression, dementia, substance abuse, obesity, and impulsive aggression. Data continue to be added to the Cimbi database and biobank

PIWI Associated siRNAs and piRNAs Specifically Require the Caenorhabditis elegans HEN1 Ortholog henn-1

Small RNAs—including piRNAs, miRNAs, and endogenous siRNAs—bind Argonaute proteins to form RNA silencing complexes that target coding genes, transposons, and aberrant RNAs. To assess the requirements for endogenous siRNA formation and activity in Caenorhabditis elegans, we developed a GFP-based sensor for the endogenous siRNA 22G siR-1, one of a set of abundant siRNAs processed from a precursor RNA mapping to the X chromosome, the X-cluster. Silencing of the sensor is also dependent on the partially complementary, unlinked 26G siR-O7 siRNA. We show that 26G siR-O7 acts in trans to initiate 22G siRNA formation from the X-cluster. The presence of several mispairs between 26G siR-O7 and the X-cluster mRNA, as well as mutagenesis of the siRNA sensor, indicates that siRNA target recognition is permissive to a degree of mispairing. From a candidate reverse genetic screen, we identified several factors required for 22G siR-1 activity, including the chromatin factors mes-4 and gfl-1, the Argonaute ergo-1, and the 3′ methyltransferase henn-1. Quantitative RT–PCR of small RNAs in a henn-1 mutant and deep sequencing of methylated small RNAs indicate that siRNAs and piRNAs that associate with PIWI clade Argonautes are methylated by HENN-1, while siRNAs and miRNAs that associate with non-PIWI clade Argonautes are not. Thus, PIWI-class Argonaute proteins are specifically adapted to associate with methylated small RNAs in C. elegans

A nuclear role for the respiratory enzyme CLK-1 in regulating mitochondrial stress responses and longevity

The coordinated regulation of mitochondrial and nuclear activities is essential for cellular respiration and its disruption leads to mitochondrial dysfunction, a hallmark of ageing. Mitochondria communicate with nuclei through retrograde signalling pathways that modulate nuclear gene expression to maintain mitochondrial homeostasis. The monooxygenase CLK-1 (human homologue COQ7) was previously reported to be mitochondrial, with a role in respiration and longevity. We have uncovered a distinct nuclear form of CLK-1 that independently regulates lifespan. Nuclear CLK-1 mediates a retrograde signalling pathway that is conserved from Caenorhabditis elegans to humans and is responsive to mitochondrial reactive oxygen species, thus acting as a barometer of oxidative metabolism. We show that, through modulation of gene expression, the pathway regulates both mitochondrial reactive oxygen species metabolism and the mitochondrial unfolded protein response. Our results demonstrate that a respiratory enzyme acts in the nucleus to control mitochondrial stress responses and longevity

Caenorhabditis elegans Cyclin D/CDK4 and Cyclin E/CDK2 Induce Distinct Cell Cycle Re-Entry Programs in Differentiated Muscle Cells

Cell proliferation and differentiation are regulated in a highly coordinated and inverse manner during development and tissue homeostasis. Terminal differentiation usually coincides with cell cycle exit and is thought to engage stable transcriptional repression of cell cycle genes. Here, we examine the robustness of the post-mitotic state, using Caenorhabditis elegans muscle cells as a model. We found that expression of a G1 Cyclin and CDK initiates cell cycle re-entry in muscle cells without interfering with the differentiated state. Cyclin D/CDK4 (CYD-1/CDK-4) expression was sufficient to induce DNA synthesis in muscle cells, in contrast to Cyclin E/CDK2 (CYE-1/CDK-2), which triggered mitotic events. Tissue-specific gene-expression profiling and single molecule FISH experiments revealed that Cyclin D and E kinases activate an extensive and overlapping set of cell cycle genes in muscle, yet failed to induce some key activators of G1/S progression. Surprisingly, CYD-1/CDK-4 also induced an additional set of genes primarily associated with growth and metabolism, which were not activated by CYE-1/CDK-2. Moreover, CYD-1/CDK-4 expression also down-regulated a large number of genes enriched for catabolic functions. These results highlight distinct functions for the two G1 Cyclin/CDK complexes and reveal a previously unknown activity of Cyclin D/CDK-4 in regulating metabolic gene expression. Furthermore, our data demonstrate that many cell cycle genes can still be transcriptionally induced in post-mitotic muscle cells, while maintenance of the post-mitotic state might depend on stable repression of a limited number of critical cell cycle regulators

Distinct Functional Constraints Partition Sequence Conservation in a cis-Regulatory Element

Different functional constraints contribute to different evolutionary rates across genomes. To understand why some sequences evolve faster than others in a single cis-regulatory locus, we investigated function and evolutionary dynamics of the promoter of the Caenorhabditis elegans unc-47 gene. We found that this promoter consists of two distinct domains. The proximal promoter is conserved and is largely sufficient to direct appropriate spatial expression. The distal promoter displays little if any conservation between several closely related nematodes. Despite this divergence, sequences from all species confer robustness of expression, arguing that this function does not require substantial sequence conservation. We showed that even unrelated sequences have the ability to promote robust expression. A prominent feature shared by all of these robustness-promoting sequences is an AT-enriched nucleotide composition consistent with nucleosome depletion. Because general sequence composition can be maintained despite sequence turnover, our results explain how different functional constraints can lead to vastly disparate rates of sequence divergence within a promoter

Intracellular Trafficking and Synaptic Function of APL-1 in Caenorhabditis elegans

Background: Alzheimer’s disease (AD) is a neurodegenerative disorder primarily characterized by the deposition of b-amyloid plaques in the brain. Plaques are composed of the amyloid-b peptide derived from cleavage of the amyloid precursor protein (APP). Mutations in APP lead to the development of Familial Alzheimer’s Disease (FAD), however, the normal function of this protein has proven elusive. The organism Caenorhabditis elegans is an attractive model as the amyloid precursor-like protein (APL-1) is the single ortholog of APP, and loss of apl-1 leads to a severe molting defect and early larval lethality. Methodology/Principal Findings: We report here that lethality and molting can be rescued by full length APL-1, C-terminal mutations as well as a C-terminal truncation, suggesting that the extracellular region of the protein is essential for viability. RNAi knock-down of apl-1 followed by drug testing on the acetylcholinesterase inhibitor aldicarb showed that loss of apl-1 leads to aldicarb hypersensitivity, indicating a defect in synaptic function. The aldicarb hypersensitivity can be rescued by full length APL-1 in a dose dependent fashion. At the cellular level, kinesins UNC-104/KIF-1A and UNC-116/kinesin-1 are positive regulators of APL-1 expression in the neurons. Knock-down of the small GTPase rab-5 also leads to a dramatic decrease in the amount of apl-1 expression in neurons, suggesting that trafficking from the plasma membrane to the early endosome is important for apl-1 function. Loss of function of a different small GTPase, UNC-108, on the contrary, leads t