Cook, Steven J. et al. (2021) MicroPubl Biol

Vidal, Berta, Hobert, Oliver, Cook, Steven J.

[MicroPubl Biol, 2021]

Single neuron-specific drivers are important tools for visualizing neuron anatomy, manipulating neuron activity and gene rescue experiments. We report here that genomic regions upstream of the C. elegans bHLH-PAS gene hlh-34 can be used to drive gene expression exclusively in the AVH interneuron pair and not, as previously reported, the AVJ interneuron pair.

Cook, Steven J. et al. (2017) International Worm Meeting "Investigating the structure and sexual dimorphisms of the C. elegans connectome."

Bulow, Hannes E., Brittin, Christopher A., Nguyen, Ken C.Q., Hall, David H., Tang, Leo T.-H., Jarrell, Travis A., Cook, Steven J., Yakovlev, Maksim, Emmons, Scott W., Wang, Yi, Hobert, Oliver, Bayer, Emily A.

[International Worm Meeting, 2017]

We present the first whole-animal maps of synaptic connectivity, including anatomical connection strength, of both adult sexes of a species. Our results are based on analyses of legacy and new serial section electron micrographs (EMs) of the C. elegans nervous system and the tissues it innervates. In a graph representation of connectivity, the pathways of information flow can be arranged hierarchically, revealing a largely feed forward structure of shallow (1-5 synapses) depth. Our reconstruction has revealed that muscles and other end-organs are more extensively cross-connected than previously reported. Sensory information converges and diverges widely throughout the fully-connected neural network. The sexes differ not only by the addition of sex-specific neurons and muscles, but also at numerous points in the connectivity of shared neurons. Differences between the hermaphrodite and male reconstructions could be either inter-individual differences or differences due to genetic sex. To distinguish between these possibilities, we examined a subset of 7 synaptic connections that were respectively stronger in the male reconstruction, 4 that were stronger in the hermaphrodite reconstruction, and 4 that were similar, using in vivo trans-synaptic labeling. In each instance, the difference seen in the reconstructions was confirmed in multiple animals. Extrapolating these results to the number of statistically significant differences in the reconstructions, we conclude that there is an unexpectedly large number of sexually dimorphic connections. These connections were mainly located in the nerve ring, and embedded within the connectome at least one synapse away from any sex-specific neuron. Our results showed that AVA receives sex-specific input from ADL, ASH, and AVF in the hermaphrodite, while RIB receives sex-specific input from IL1, IL2, and RIA in the male. AIM, which has been reported to change its neurotransmitter from glutamate in the hermaphrodite to acetylcholine in the male, makes a strong male-specific connection to AIB. These hubs of sex-specific connectivity also maintained the majority of their sex-shared output. Our results suggest that the genetic sex of the nervous system allows for diverse synaptic patterns in a relatively small nervous system.

McGhee JD (1987) Biochemistry "Purification and characterization of a carboxylesterase from the intestine of the nematode Caenorhabditis elegans."

McGhee JD

[Biochemistry, 1987]

The major intestinal esterase from the nematode Caenorhabditis elegans has been purified to essential homogeneity. Starting from whole worms, the overall purification is 9000-fold with a 10% recovery of activity. The esterase is a single polypeptide chain of Mr 60,000 and is stoichiometrically inhibited by organophosphates. Substrate preferences and inhibition patterns classify the enzyme as a carboxylesterase (EC 3.1.1.1), but the physiological function is unknown. The sequence of 13 amino acid residues at the esterase N- terminus has been determined. This partial sequence shows a surprisingly high degree of similarity to the N-terminal sequence of two carboxylesterases recently isolated from Drosophila mojavensis [Pen, J., van Beeumen, J., & Beintema, J. J. (1986) Biochem. J. 238, 691-699].

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.

Philbrook, Alison et al. (2015) International Worm Meeting "Investigation of spine-like synaptic structures on GABAergic DD neurons."

Philbrook, Alison, Hall, David H., Francis, Michael M., Cook, Steven J.

[International Worm Meeting, 2015]

Synapses are dynamic structures that undergo selective strengthening or weakening to refine their patterns of connectivity. Even after the nervous system has developed, dendritic spines grow and retract, and the placement of synapses can be reengineered to form new neural circuits. In order to investigate molecular mechanisms by which particular synapses are either remodeled or stabilized, we are studying C. elegans GABAergic circuits. We previously identified an ionotropic acetylcholine receptor (ACR-12) that is expressed at synapses onto GABAergic neurons and regulates their activity. In order to investigate mechanisms underlying receptor clustering and synapse dynamics, we sought to establish a system in which we could examine the subcellular localization of ACR-12 receptors in individual neurons in vivo. We generated a transgenic strain expressing GFP-tagged ACR-12 receptors in the GABA DD motor neurons and focused our efforts on a single neuron, DD1. The DD1 cell body and processes are spatially separated from the other DD neurons, enabling in vivo visualization of synapse dynamics on the single DD1 neuronal process. We find that ACR-12 clusters are restricted to the dorsal side in first larval stage (L1) animals. In contrast, punctate ACR-12-GFP fluorescence is localized within a defined spatial domain of the ventral DD1 process in adult animals. These results are consistent with previous studies suggesting that synapses onto the DD neurons undergo developmental remodeling at the end of the L1 stage. In adults, ACR-12 clusters are concentrated at the tips of spine-like dendritic protrusions and are apposed by presynaptic vesicle markers, consistent with a synaptic localization. Spine-like structures are also apparent in volumetric reconstructions of the ventral DD1 dendrite from electron micrographs. Finally, genetic manipulations that reduce cholinergic transmission decrease spine number, suggesting mechanisms for their activity-dependent regulation. Together, our findings raise the interesting possibility that these spine-like structures in DD1 represent an evolutionary precursor to mammalian dendritic spines. We are now working to investigate molecular requirements for the development and maintenance of these synapses and will present our findings.

Murakami S et al. (1999) Curr Biol "Life extension and stress resistance in Caenorhabditis elegans modulated by the tkr-1 gene."

Johnson TE, Murakami S

[Curr Biol, 1999]

In this Brief Communication, which appeared in the 14 September 1998 issue of Current Biology, the UV dose was reported erroneously. The dose reported was 20 J/m2 but the actual dose used was 0.4 J/cm2. Also, the gene formally referred to as tkr-1 has since been renamed old-1 (overexpression longevity determination).

Tang, Leo T.-H. et al. (2017) International Worm Meeting "A lateralized circuit of sensory neurons in C. elegans."

Bulow, H.nnes E., Emmons, Scott W., Cook, Steven J., Tang, Leo T.-H.

[International Worm Meeting, 2017]

Lateral specialization of the central nervous system is a well-established feature across species, yet the underlying mechanism on how such functional asymmetry arise is largely unknown. Roughly two thirds of C. elegans neurons are present as L-R homologous pairs. By evaluating new data from reconstruction of serial section electron micrographs (wormwiring.org), we found that most L-R neurons display laterally similar connectivity and connection strength. However, we found that two amphid neurons known to be asymmetric in gene expression and function, ASE and AWC, displayed lateralized connectivity (15 ASEL->AWC synapses vs. 2 ASER->AWC synapses). To corroborate and evaluate this chemical synapse pattern in a large sample of live animals, we generated a reporter strain labelling ASE->AWC connections using in vivo Biotin Labelling of Intercellular Contact (iBLINC). We observe that roughly 85% of animals contain more ASEL->AWC synaptic puncta than ASER->AWC. Time-course imaging revealed this connection is present throughout larval development and symmetric at early larval stages (median, left: 3, right: 2) but becomes lateralized by a larger increase of ASEL synaptic puncta from late larval to adult stages (median 5) as compared to the increase in that of ASER (median 2.5). We next evaluated the impact of ASEL/ASER cell-fate on connectivity in lsy-6 mutant (two ASERs) and lsy-6 misexpression (two ASELs) backgrounds. While we found modest changes in the number of synaptic puncta, a presynaptic cell-fate change was insufficient to symmetrize this connection. We are currently investigating the impact of other cell-fate determinants, laterally expressed genes, and environmental cues on this connection. In conclusion we have added to the rich literature of ASE and AWC asymmetries by describing a lateralized synaptic connection and in part its genetic determinants. We hope that our studies will yield insight into how genes and/or the environment determine the development and plasticity of functional lateralized circuits.

Ramos CG et al. (2014) J Bacteriol "Retraction for Ramos et al., MtvR is a global small noncoding regulatory RNA in Burkholderia cenocepacia."

Feliciano JR, da Costa PJ, Ramos CG, Grilo AM, Leitao JH

[J Bacteriol, 2014]

Volume 195, no. 16, p. 35143523, 2013. A number of problems related to images published in this paper have been brought to our attention. Figure 1D contains duplicated images in lanes S and LE, and Fig. 4D and 6B contain images previously published in articles in this journal and in Microbiology and Microbial Pathogenesis, i.e., the following: C. G. Ramos, S. A. Sousa, A. M. Grilo, J. R. Feliciano, and J. H. Leitao, J. Bacteriol. 193:15151526, 2011. doi:10.1128/JB.01374-11. S. A. Sousa, C. G. Ramos, L. M. Moreira, and J. H. Leitao, Microbiology 156:896908, 2010. doi:10.1099/mic.0.035139-0. C. G. Ramos, S. A. Sousa, A. M. Grilo, L. Eberl, and J. H. Leitao, Microb. Pathog. 48:168177, 2010. doi: 10.1016/j.micpath.2010.02.006. Therefore, we retract the paper. We deeply regret this situation and apologize for any inconvenience to the editors and readers of Journal of Bacteriology, Microbial Pathogenesis, and Microbiology.

Nillegoda NB et al. (2015) Nature "Crucial HSP70 co-chaperone complex unlocks metazoan protein disaggregation."

Berynskyy M, Morimoto RI, Bukau B, Stengel F, Kirstein J, Szlachcic A, Arnsburg K, Stank A, Scior A, Nillegoda NB, Gao X, Guilbride DL, Aebersold R, Wade RC, Mayer MP

[Nature, 2015]

Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states. Healthy metazoan cells effectively eliminate intracellular protein aggregates, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control.

Jarrell, Travis A. et al. (2013) International Worm Meeting "The connectome of the anterior nervous system of the C. elegans adult male."

Nguyen, Kenneth, Bloniarz, Adam E., Brittin, Christopher A., Cook, Steven J., Hall, David H., Emmons, Scott W., Xu, Meng, Jarrell, Travis A., Wang, Yi

[International Worm Meeting, 2013]

The innate behavioral repertoires of the two sexes of a species are guided by differing reproductive priorities. C. elegans male copulation is controlled by a neural network in the tail in which a majority of the neurons and muscles are specific to the male. But known differences in olfactory preferences and exploratory tendencies emanate from behaviors controlled by circuits in the head, where the complement of neurons is nearly identical in the two sexes. We determined connectivity in the anterior nervous system of the adult male from a 1,500 section-long thin section EM series extending from near the tip of the nose, through the nerve ring, and part way into the retrovesicular ganglion. This region contains the bulk of the synapses, excluding ventral cord nmj's. To make a comparison to the hermaphrodite, we re-reconstructed legacy Cambridge micrographs using our software, which allows us to score synaptic weights (see abstract by Cook et al). While our analysis is at an early stage, we can already see the essential result: in the adjacency matrices that display the connectivity, it is difficult to spot differences that appear greater than would be expected given the inherent variability of neuronal wiring. Known circuits in navigation and other responses are conserved. Thus behavioral differences likely emerge from differing circuit properties rather than differing connectivity. There are two possible exceptions: AIM synapses onto AIB and RIA synapses onto RIB in the male only. One set of male-specific synapses expected involves the male-specific head CEM sensory neurons, and the tail EF interneurons, which receive extensive input from the copulatory circuits in the tail and extend processes through the ventral nerve cord into the nerve ring. Both of these neuron classes have as their strongest targets the AVB command interneurons for forward locomotion. This suggests one of their functions may be to inhibit forward locomotion when a hermaphrodite is sensed or during copulation. They make additional connections to be further explored.