50 research outputs found
Des changements dynamiques de la synapse révélés par une nouvelle méthode trans-synaptique pour visualiser les connections neuronales in vivo chez C. elegans
The nervous system is a complex network that senses and processes information and is essential for the survival of both vertebrates and invertebrates as it is involved in behavior responses. Information within the network is transmitted through specialized cell-cell contacts, including synaptic connections. Importantly, the network is not static and is believed to change during development and learning, as well as during pathological or normal age-related decline. Studying the nervous system in vivo requires the use of animal models such as Caenorhabditis elegans. Understanding of behavior and development requires visualization and analysis of synaptic connectivity. However, existing methods are laborious and may not depend on trans-synaptic interactions, or otherwise ‘trap’ the synapses by fixing the connections, thus precluding dynamic studies. In order to study synaptic modifications, we developed a new transgenic approach for in vivo labeling of specific connections in C. elegans, called iBLINC (in vivo Biotin Labeling of INtercellular Contacts). iBLINC involves the biotinylation of an acceptor peptide (AP) by the Escherichia coli biotin ligase BirA. Both are fused to two interacting post- and pre-synaptic proteins, respectively. The biotinylated acceptor peptide fusion is detected by a monomer streptavidin fused to a fluorescent protein that is transgenically expressed and secreted into the extracellular space. The method is directional, bright and dynamic. Moreover it correlates well with electron micrograph reconstruction. Using this new technique to observe synapses, which are part of the thermosensory circuitry of C. elegans, during aging, we could conclude that the connection pattern varies with age and within a population. Changes of the number and size of the synapses were observed during aging. Some segments of the synaptic region seem to be more affected than others by the aging process. Those results were corroborated by using a GABAergic pre-synaptic marker which allowed us to visualize a decline of the vesicle number with aging. In summary, in this thesis I explained a new in vivo trans-synaptic method to visualize synapses in C. elegans. Then I demonstrated that a natural decline in the number of synapses as well as the number of vesicles occurs during aging.Le système nerveux est un réseau complexe qui détecte et analyse les informations. Ces informations sont transmises entre cellules grâce à des connections synaptiques et des jonctions communicantes. Ce réseau n’est pas statique et évolue au cours du développement, de l’apprentissage mais aussi durant le processus de vieillissement – naturels ou pathologiques. Comprendre le système nerveux et son fonctionnement requiert une analyse des connections synaptiques in vivo chez un animal model tel que Caenorhabditis elegans. Cependant les techniques actuellement disponibles pour C. elegans sont laborieuses, ne dépendent pas forcément d’une interaction trans-synaptique ou fixent la synapse. Par conséquent, ces approches ne permettent pas de réaliser des études de populations et dynamiques des modifications synaptiques. Dans ce manuscrit, je décris tout d’abord une nouvelle technique pour visualiser les synapses in vivo chez le vers C. elegans. Cette technique appelé iBLINC (in vivo Biotin Labeling of INtercellular Contacts) qui consiste en la biotinylation d’un peptide par une ligase d’Escherichia coli, BirA. Ces deux molécules sont fusionnées à des protéines trans-membranaires qui forment un complexe à la synapse. La biotinylation est détectée grâce à une streptavidin monomérique taguée avec un fluorophore qui est secrétée dans l’espace extracellulaire. J’ai démontré que cette technique est directionnelle et dynamique. En utilisant iBLINC pour visualiser des synapses faisant partie du circuit sensoriel de C. elegans, une évolution du nombre et de la taille des synapses a pu être observée avec l’âge. Il semblerait que ce changement soit dépendant du segment de la zone synaptique observée. Ces résultats ont été corroborés par l’observation d’une diminution du nombre de vésicules pendant le vieillissement grâce à un marqueur pré-synaptique des synapses GABAergique de la jonction neuromusculaire. Pour conclure, ce manuscrit décrit une nouvelle technique permettant d’observer les synapses chez le vers vivant et démontre une évolution naturelle du nombre de synapses et du nombre de vésicules pré-synaptiques avec l’âge
Optimized protocol for in vivo affinity purification proteomics and biochemistry using C. elegans
We present an optimized protocol for in vivo affinity purification proteomics and biochemistry using the model organism C. elegans. We describe steps for target tagging, large-scale culture, affinity purification using a cryomill, mass spectrometry and validation of candidate binding proteins. Our approach has proven successful for identifying protein-protein interactions and signaling networks with verified functional relevance. Our protocol is also suitable for biochemical evaluation of protein-protein interactions in vivo.For complete details on the use and execution of this protocol, please refer to Crawley et al.,1 Giles et al.,2 and Desbois et al.
Supporting information for Ubiquitin ligase activity inhibits Cdk5 to control axon termination. S1 Table [Dataset]
Transgenic and CRISPR Strains.Peer reviewe
Supporting information for Ubiquitin ligase activity inhibits Cdk5 to control axon termination. S3 Table [Dataset]
CRISPR Targeting Sequences and Repair Templates.Peer reviewe
Supporting information for Ubiquitin ligase activity inhibits Cdk5 to control axon termination. S3 Fig [Dataset]
Quantitation of axon termination defects in ALM neurons for indicated genotypes. Reduced frequency of axon termination defects occurs in cdk-5; rpm-1 double mutants, but not pct-1; rpm-1 double mutants. Suppression of termination defects is not increased in cdk-5; pct-1; rpm-1 triple mutants compared to cdk-5; rpm-1 double mutants. Results suggest that CDK-5 and PCT-1 do not function redundantly during axon termination. Means (bars) are shown for 6 or more counts (black dots, 20 or more worms/count) for each genotype. Error bars indicate SEM. Significance assessed using Student’s t-test. * p<0.05, ** p<0.01.Peer reviewe
Supporting information for Ubiquitin ligase activity inhibits Cdk5 to control axon termination. S4 Table [Dataset]
Primers used for genotyping and cloning.Peer reviewe
Ubiquitin ligase activity inhibits Cdk5 to control axon termination
The Cdk5 kinase plays prominent roles in nervous system development, plasticity, behavior and disease. It also has important, non-neuronal functions in cancer, the immune system and insulin secretion. At present, we do not fully understand negative regulatory mechanisms that restrict Cdk5. Here, we use Caenorhabditis elegans to show that CDK-5 is inhibited by the RPM-1/FSN-1 ubiquitin ligase complex. This atypical RING ubiquitin ligase is conserved from C. elegans through mammals. Our finding originated from unbiased, in vivo affinity purification proteomics, which identified CDK-5 as a putative RPM-1 substrate. CRISPR-based, native biochemistry showed that CDK-5 interacts with the RPM-1/FSN-1 ubiquitin ligase complex. A CRISPR engineered RPM-1 substrate ‘trap’ enriched CDK-5 binding, which was mediated by the FSN-1 substrate recognition module. To test the functional genetic relationship between the RPM-1/FSN-1 ubiquitin ligase complex and CDK-5, we evaluated axon termination in mechanosensory neurons and motor neurons. Our results indicate that RPM-1/FSN-1 ubiquitin ligase activity restricts CDK-5 to control axon termination. Collectively, these proteomic, biochemical and genetic results increase our understanding of mechanisms that restrain Cdk5 in the nervous system.B.G. was supported by the National Institutes of Health Grant R01 NS072129. O.C. was supported by the Severo Ochoa Postdoctoral Program and a Marie Sklodowska-Curie Actions Individual Fellowship.Peer reviewe
Supporting information for Ubiquitin ligase activity inhibits Cdk5 to control axon termination. S5 Fig [Dataset]
RPM-1 and CDK-5 function in parallel to regulate synapse formation in ALM mechanosensory neurons. A) Schematic shows primary axon and collateral synaptic branch of ALM mechanosensory neurons. Highlighted in green on collateral branch are presynaptic sites labeled by RAB-3::GFP. B) Shown are representative confocal images of presynaptic RAB-3::GFP puncta for indicated genotypes. For wildtype, images are shown for GFP::RAB-3 (upper left, green), RFP expressed in mechanosensory neurons (PmecRFP; upper middle, magenta) and merged image (upper right). C) Quantitation of GFP::RAB-3 puncta in ALM neurons for indicated genotypes. Note rpm-1 mutants have reduced numbers of RAB-3 puncta, and these defects are enhanced in cdk-5; rpm-1 double mutants. Means (bars) are shown for each genotype (black dots indicate individual worms). Error bars indicate SEM. Significance assessed using Student’s t-test with Bonferroni correction. ns, non-significant; * p<0.05. Scale bar 10 μm.Peer reviewe
Supporting information for Ubiquitin ligase activity inhibits Cdk5 to control axon termination. S2 Fig [Dataset]
Quantitation indicates ALM axon termination defects are reduced in cdka-1; rpm-1 double mutants compared to rpm-1 single mutants. Means (bars) are shown for 4 or more counts (black dots, 20 or more worms/count) for each genotype. Error bars indicate SEM. Significance assessed using Student’s t-test. * p<0.05.Peer reviewe
Supporting information for Ubiquitin ligase activity inhibits Cdk5 to control axon termination. S1 Fig [Dataset]
A) LC MS-MS spectra for two CDK-5 peptides identified in GS::RPM-1 LD samples. B) Highlighted in CDK-5 sequence are four unique CDK-5 peptides (red) identified in GS::RPM-1 LD substrate ‘trap’ samples. C) LC MS-MS spectrum of one CDKA-1 peptide identified in GS::RPM-1 LD samples. D) Highlighted in CDKA-1 sequence are two unique peptides (red) identified in GS::RPM-1 LD samples.Peer reviewe