Desbois, Muriel et al. (2013) International Worm Meeting "A non-trapping method for live imaging of specific neuronal connections."

Desbois, Muriel, Buelow, Hannes

[International Worm Meeting, 2013]

The nervous system is a complex network that senses and processes information and is essential for the survival of both vertebrates and invertebrates. Information within the network is transmitted through specialized cell-cell contacts, including synaptic connections and GAP junctions. Importantly, the network is not static and is believed to change during development, learning but also during pathological or normal age-related decline. Previous strategies to label specific synapses in living animals 'trap' the synapses by fixing the connections, thus precluding dynamic studies. To circumvent this problem, we are adapting a technique called BLINC (Biotin Labeling of INtercellular Contacts) for live imaging of specific synapses in C. elegans. BLINC is based on the biotinylation of an acceptor peptide by the E. Coli biotin ligase BirA, both are fused to two interacting proteins. In analogy to work in cell culture, we fused the BirA ligase to neurexin and expressed the fusion in a set of neurons. We then fused the acceptor peptide to neuroligin and expressed this construct in another group of neurons, known to form connections with the former set of neurons. During the formation of synapses/connections between the two groups of neurons, the neuroligin and the neurexin form a complex, leading the ligase to transfer a biotin to the acceptor peptide. This "biotin mark" is specifically detected by a monovalent streptavidin fused to a fluorescent protein which is transgenically secreted from the coelomocytes. In preliminary findings we observe specific staining in living animals consistent with known connections between both sets of neurons. We will report on our progress, but expect that this new technique will allow to visualize formation and dynamics of specific neuronal connections in vivo under different experimental conditions.

Desbois, Muriel et al. (2021) International Worm Meeting "Ubiquitin ligase activity inhibits CDK-5 to promote axon termination"

opperman, karla, Desbois, Muriel, Grill, Brock, Amezquita, Jonathan

[International Worm Meeting, 2021]

Cdk5 is an atypical cyclin-dependent kinase that is involved in neurodegenerative disease, nervous system development and neuronal plasticity. It also has important non-neuronal roles in the DNA damage response, immune response, angiogenesis and cancer. Cdk5 is positively regulated primarily by its activators, P35 and P39. How Cdk5 is negatively regulated remains relatively poorly understood, in particular in post-mitotic neurons and in the nervous system in vivo. Here, we identify the E3 ubiquitin ligase RPM-1 as a negative regulator of CDK-5 in C. elegans. This discovery originated from in vivo affinity purification proteomics with an RPM-1 biochemical 'trap' that enriched ubiquitination substrates, which included CDK-5. A combination of both biochemical and genetic findings demonstrated that RPM-1 ubiquitin ligase activity restricts CDK-5. Co-immunoprecipitation using endogenous proteins tagged by CRISPR/Cas9 engineering confirmed that CDK-5 binds to RPM-1. CDK-5 also binds to FSN-1, the F-box protein that is the substrate recognition module of the RPM-1 ubiquitin ligase complex. These results suggest that CDK-5 is an RPM-1 ubiquitination substrate. Genetic results indicated that RPM-1 inhibits CDK-5 to promote axon termination in both ALM mechanosensory neurons and SAB motor neurons. Outcomes from CRISPR gene-editing and cell-specific rescue experiments showed that RPM-1 restricts the kinase activity of CDK-5 cell-autonomously in neurons. Thus, we have identified RPM-1 as a ubiquitin ligase that inhibits CDK-5 in vivo in the nervous system. We further demonstrate that CDK-5 kinase activity needs to be restrained for proper axon termination. Understanding how Cdk5 is inhibited could have important implications given its role in neurodegenerative disease and emerging links to neurodevelopmental disorders, including intellectual disability.

Desbois, Muriel et al. (2015) International Worm Meeting "Directional trans-synaptic labeling of specific neuronal connections in live animals."

Bulow, H.nnes E., Desbois, Muriel, Cook, Steven J., Emmons, Scott W.

[International Worm Meeting, 2015]

Understanding animal behavior and development requires visualization and analysis of their synaptic connectivity, but existing methods are laborious or, may not depend on trans-synaptic interactions. Here we describe a transgenic approach for in vivo labeling of specific connections in Caenorhabditis elegans, which we term iBLINC. The method is based on BLINC (Biotin Labeling of INtercellular Contacts) and involves trans-synaptic enzymatic transfer of biotin by the Escherichia coli biotin ligase BirA onto an acceptor peptide. A BirA fusion with the presynaptic cell adhesion molecule NRX-1/neurexin is expressed presynaptically, whereas a fusion between the acceptor peptide and the postsynaptic protein NLG-1/neuroligin is expressed postsynaptically. The biotinylated acceptor peptide::NLG-1/neuroligin fusion is detected by a monomeric streptavidin::fluorescent protein fusion transgenically secreted into the extracellular space. Physical contact between neurons is not sufficient to create a fluorescent signal suggesting that synapse formation is required. The labeling approach captures the directionality of synaptic connections, and quantitative analyses of synapse patterns display excellent concordance with electron micrograph reconstructions. Experiments using photoconvertible fluorescent proteins suggest that the method can be utilized for studies of protein dynamics at the synapse. Applying this technique, we find connectivity patterns of defined connections to vary across a population of wild type animals. In aging animals, specific segments of synaptic connections are more susceptible to decline than others, consistent with dedicated mechanisms of synaptic maintenance. Taken together, we have developed an enzyme-based, trans-synaptic labeling method that allows high-resolution analyses of synaptic connectivity as well as protein dynamics at specific synapses of live animals.

Yim, Hyunsoo et al. (2017) International Worm Meeting "Possible synaptic remodeling of IL2 neurons for behavioral plasticity in C.elegans."

Lee, Junho, Yim, Hyunsoo, Son, Sangwon

[International Worm Meeting, 2017]

Many organisms undergo remodeling of neural circuits among developmental stages, and it is fundamental for them to adopt stage-specific behaviors. Nictation, a dauer-specific dispersal behavior of the C.elegans, is regulated by IL2 neurons which were known as cholinergic sensory neurons. As dauer is an alternative developmental stage and the larvae show quite different behavioral pattern, it is strongly expected that the neural circuit of the dauer stage may have undergone synaptic remodeling. In this study we examined the possibility that IL2 neurons make a novel synapse with body wall muscle. We tried to confirm the neuromuscular junctions between IL2 and BWM by the co-localization of pre- and pos-synaptic markers, and by iBLINC [1]. Further studies include the investigation of signaling pathways responsible for the IL2 NMJ remodeling in dauer larvae by candidate screening, and the necessity of IL2 NMJ in the nictation behavior. Our study will contribute to the understanding of the developmental plasticity of the synapse remodeling and the behavior. [1] Desbois, M. et al. (2015) Directional Trans-Synaptic Labeling of Specific Neuronal Connections in Live Animals. Genetics 200(3), 697-705.