Philbrook, Alison et al. (2021) International Worm Meeting "Sensory cilia architecture shapes olfactory response dynamics"

Sengupta, Piali, Philbrook, Alison

[International Worm Meeting, 2021]

Structurally and functionally diverse sensory cilia house signal transduction molecules and play essential roles in olfaction, hearing, and photoreception. Complex cilia morphologies dictate the concentration and organization of signaling molecules within them, and are thus considered critical for precisely shaping sensory responses. However, the contribution of unique cilia morphologies to sensory signaling is poorly understood. Chemosensory neurons in C. elegans are specialized to respond to unique subsets of chemical and other stimuli and exhibit a range of morphologically distinct cilia. Cilia are built by the process of intraflagellar transport (IFT) which traffics structural and signaling components into and out of cilia. Mutations in IFT genes result in severe cilia structural defects. We and others have found that while mutations in IFT genes abolish chemical responses in a subset of chemosensory neurons, surprisingly, primary odorant responses in neurons containing complex 'wing' cilia are unaffected in these mutants. Instead, these neurons exhibit defects in sensory adaptation and desensitization. To decouple the contributions of cilia morphology and IFT to odorant responses, we generated a temperature-sensitive mutation in a C. elegans kinesin motor protein. Upon shift to the restrictive temperature, IFT is acutely blocked without immediate disruption of cilia morphology. We find that a minimal cilium length is required for odorant detection in rod-like simple cilia. However, in wing cilia, their morphological complexity shapes desensitization but not adaptation of the sensory response. Instead, regulated trafficking of G protein-coupled receptors (GPCRs) may drive adaptation to odorants. Our results suggest that signaling-dependent removal of GPCRs is mediated by phosphorylation via the G-protein coupled receptor kinase GRK-2, followed by BBSome-mediated ciliary exit of GPCRs. We also identify a role for arrestin-mediated endocytosis of GPCRs at the cilia base in shaping sensory response dynamics. Together, our work demonstrates how specialized cilia morphologies contribute to the unique responses of individual chemosensory neurons in C. elegans, and highlights the importance of ciliary structural diversity in shaping sensory behaviors.

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.

He, Siwei et al. (2015) International Worm Meeting "Transcriptional control of postsynaptic remodeling through regulated expression of an immunoglobulin superfamily protein."

He, Siwei, Francis, Michael, Miller, David, Taub, Daniel, Gabel, Christopher, McWhirter, Rebecca, Philbrook, Alison

[International Worm Meeting, 2015]

Neural circuits are extensively remodeled during development; however, the mechanisms underlying this process and the timing of rewiring remain largely unknown. Here we describe a transcriptionally regulated immunoglobulin super family protein, OIG-1, that fine-tunes synaptic plasticity in remodeling GABAergic motor neurons. DD class GABAergic motor neurons reverse polarity in the first larval (L1) stage. In newly born animals, DDs receive cholinergic inputs in the dorsal nerve cord but these are switched to the ventral side by the end of the L1 stage. The relocation of the DD postsynaptic apparatus can be monitored with the acetylcholine receptor (AChR) subunit, ACR-12::GFP. OIG-1 is highly expressed in early DD neurons where it antagonizes the relocation of ACR-12::GFP from the dorsal side. During the L1/L2 transition, OIG-1 is down-regulated by the transcription factor, IRX-1/Iroquois, in DD neurons to coincide with the translocation of postsynaptic ACR-12 to the ventral side. In VD class GABAergic motor neurons, which normally do not remodel, the transcription factor, UNC-55/COUP-TF turns off IRX-1, thus maintaining high levels of OIG-1 to block the removal of dorsally-located ACR-12 receptors. OIG-1 is secreted from GABAergic motor neurons but its anti-plasticity function is cell-autonomous and does not require secretion. Instead, we propose that secretion offers a rapid mechanism for clearing functionally active, intracellular OIG-1 thereby unleashing the postsynaptic remodeling program. Our study provides a novel mechanism by which synaptic remodeling is set in motion, through the temporal and spatial regulation of an Ig domain protein. .

Kazatskaya, Anna et al. (2020) MicroPubl Biol

de Bono, Mario, Amin-Wetzel, Niko, Sengupta, Piali, Philbrook, Alison, Kazatskaya, Anna, Yuan, Lisa

[MicroPubl Biol, 2020]

A subset of sensory neurons in C. elegans contains compartmentalized sensory structures termed cilia at their distal dendritic ends (Ward et al. 1975; Perkins et al. 1986; Doroquez et al. 2014). Cilia present on different sensory neuron types are specialized both in morphology and function, and are generated and maintained via shared and cell-specific molecules and mechanisms (Perkins et al. 1986; Evans et al. 2006; Mukhopadhyay et al. 2007; Mukhopadhyay et al. 2008; Morsci and Barr 2011; Doroquez et al. 2014; Silva et al. 2017). The bilaterally symmetric pair of URX oxygen-sensing neurons in the C. elegans head (Figure 1A) is thought to be non-ciliated (Ward et al. 1975; Doroquez et al. 2014) but nevertheless exhibits intriguing morphological similarities with ciliated sensory neurons. URX dendrites extend to the nose where they terminate in large bulb-like complex structures (Ward et al. 1975; Doroquez et al. 2014; Cebul et al. 2020) (Figure 1A). These structures concentrate oxygen-sensing signaling molecules (Gross et al. 2014; Mclachlan et al. 2018) suggesting that similar to cilia, these structures are specialized for sensory functions. Microtubule growth events similar to those observed in ciliated sensory neurons were also reported at the distal dendritic regions of URX, implying the presence of a microtubule organizer such as a remodeled basal body (Harterink et al. 2018). Moreover, a subset of ciliary genes is expressed in URX (Kunitomo et al. 2005; Harterink et al. 2018; Mclachlan et al. 2018). We tested the hypothesis that URX dendrites contain cilia at their distal ends.

Cuentas-Condori A et al. (2019) Elife "C. elegans neurons have functional dendritic spines."

He S, Mulcahy B, Miller DM, Zhen M, Palumbos S, Cuentas-Condori A

[Elife, 2019]

Dendritic spines are specialized postsynaptic structures that transduce presynaptic signals, are regulated by neural activity and correlated with learning and memory. Most studies of spine function have focused on the mammalian nervous system. However, spine-like protrusions have been reported in <i>C. elegans</i> (Philbrook et al. 2018), suggesting that the experimental advantages of smaller model organisms could be exploited to study the biology of dendritic spines. Here, we used super-resolution microscopy, electron microscopy, live-cell imaging and genetics to show that <i>C. elegans</i> motor neurons have functional dendritic spines that: (1) are structurally defined by a dynamic actin cytoskeleton; (2) appose presynaptic dense projections; (3) localize ER and ribosomes; (4) display calcium transients triggered by presynaptic activity and propagated by internal Ca⁺⁺ stores; (5) respond to activity-dependent signals that regulate spine density. These studies provide a solid foundation for a new experimental paradigm that exploits the power of <i>C. elegans</i> genetics and live-cell imaging for fundamental studies of dendritic spine morphogenesis and function.

Mariana Simaes et al. (2006) European Worm Meeting "Phenotypic Analysis and Mapping of a New C. elegans Mab Mutant, mab-29"

Mariana Simaes, Alison Woollard

[European Worm Meeting, 2006]

Mariana Simes and Alison Woollard. Mab (Male ABnormal) mutants have defects in male tail copulatory structures and therefore have a reduced ability to mate. Nonetheless, these mutations can be isolated without difficulties accompanying lethality since the normal population consists of hermaphrodites. In a recent F1 screen for new Mab mutants, one such mutant was isolated, mab-29. Male tails have swollen bursa/ rays and reduced fan, suggesting a defect in morphogenesis. Extensive phenotypic analysis of the male tail using conventional microscopy and specific tissue markers shows abnormal attachment of the rays and phasmid to the hypodermal sheath, resulting in the striking appearance of the lumpy and amorphous rays and reduced fan.. By using recombination mapping, mab-29 has been mapped to an interval on Linkage Group X. We are using a combination of three-factor crosses and SNP snip-mapping approaches to narrow the genomic region in which mab-29 is found. Snip mapping is being carried out using snips in the interval of about three map units, between lin-2 and unc-9.. Our mapping strategy and progress towards defining the mab-29 map position will be presented. Further analysis depends on the nature of this gene.

Toby Braun et al. (2006) European Worm Meeting "Searching for rnt-1 Interacting Genes in C. elegans"

Toby Braun, Alison Woollard

[European Worm Meeting, 2006]

Toby Braun and Alison Woollard. RNT-1 is the only C. elegans member of the Runx family of transcriptional regulators, which are postulated to act both as oncogenes and tumour suppressors in mammalian cells. Work in our lab has shown that rnt-1 is a rate-limiting regulator of seam cell proliferation during C. elegans development (see abstract by Nimmo et al).. Apart from this control of seam cell proliferation, rnt-1 shows a partial embryonic lethality in RNAi experiments. This suggests that rnt-1 may act together with other, as yet unknown factors, to regulate some essential aspect of embryonic development.. To uncover such redundant factors, we are currently performing two synthetic lethal screens in a rnt-1 mutant background, using a genome wide RNAi feeding library and a random mutagenesis screen. Our approach allows us to easily discriminate between factors that are lethal during development in their own right and factors that are only lethal in conjunction with the absence of rnt-1 (synthetic lethality).. We are convinced that this genome-wide screen will elucidate further factors that are involved in the control of C. elegans development.

Caroline Bowen et al. (2006) European Worm Meeting "RNA Degradation in the Nematode Worm C.elegans"

Caroline Bowen, Alison Woollard

[European Worm Meeting, 2006]

Caroline Bowen and Alison Woollard RNA turnover is necessary to maintain basal levels of proteins, through mRNA regulation, and to remove aberrant mRNA transcripts. Here we examine processes of RNA degradation in C.elegans. This approach is based on a functional investigation of the C.elegans components of the RNA degradation pathways, identified from homology to other organisms.. The 5-3 RNA degradation pathway in C.elegans has been previously identified (Newbury and Woollard 2004). Here we investigate the 3-5 pathway in C.elegans, starting with the identification of its core and accessory components. 3-5 exoribonucleic acid decay, first identified in S.cerevisae, is brought about by a complex of exoribonucleases, helicases and associated factors, known as the exosome, (Mitchell et al 1997). The exosome was hailed as a proteosome for RNA, due to the degradation properties of the heteromeric complex.. We have used RNAi and an available mutant to show that disruption of the exosome complex, through the targeted destruction of individual components, results in worms with severe developmental problems including slowed growth rates. A full length GFP tagged exosome component, has shown the component to be largely confined to the nucleus of all cells, including the germline. Phenotypes associated with depletion of the exosome components include slow growth and developmental problems. These are attributed to effects on rRNA processing. When exosome components are silenced, there is a failure of the degradation of specific cleaved rRNA precursors, which in turn effects ribosome function and subsequently protein synthesis. We will present our findings on the nature of these rRNA processing defects, which will help to clarify the molecular basis of the phenotypes we observe.

Pete Appleford et al. (2006) European Worm Meeting "cis-acting Factors and Downstream Targets of the T-Box Transcription Factor mab-9"

Alison Woollard, Pete Appleford

[European Worm Meeting, 2006]

Pete Appleford and Alison Woollard. The C. elegans T-box gene mab-9 was originally isolated in a screen for worms with mating defects. Loss of correct MAB-9 expression results in a defect in cell-fate specification of the hindgut cells B & F. In male worms, this leads to loss of the B-cell lineage which forms the larger part of the male-specific tail structures. mab-9 mutants also have axon guidance defects which result in poor backward movement.. We are interested in identifying cis-acting elements important for the regulation of MAB-9 expression. Deletion of sections of the endogenous 5 sequence from a promoter-only mab-9 expression construct has revealed regions required for VNC, hindgut and hypodermal components of the wild-type expression pattern. Cloning of the sequence required for VNC expression into a pes-10 minimal promoter vector confirms that this part of the promoter is sufficient to drive expression in these nerve cells. Further dissection of the VNC promoter is in progress.. Sequence comparison of the promoters of C. elegans mab-9 and its nearest C. briggsae homologue identified several areas of conserved sequence which could contain important regulatory elements. Deletion of 2 of these from a mab-9::gfp rescuing construct resulted in complete loss of tail hypodermal expression. Furthermore, insertion of these elements into the pes-10 minimal promoter vector proved sufficient to drive expression in posterior hypodermis. Worms rescued with a mab-9::gfp rescuing construct lacking these 2 regions displayed neomorphic tail defects which suggest a previously unknown role of mab-9 in hypodermal morphogenesis.. Downstream targets of mab-9 were identified by a microarray-based approach. We used RNA from synchronized L1/L2 populations of him-8 and mab-9; him-8 worms to probe Affymetrix chips. Targets which were downregulated/upregulated in a mab-9 mutant were confirmed by semi-quantitative RT-PCR. Promoter-only gfp fusions are being made to selected targets to compare expression levels in him-8 and mab-9; him-8 worms..

Monsalve, G.C. et al. (2009) International Worm Meeting "Timing the Molting Cycle."

Monsalve, G.C., Davie, J., Frand, A.R.

[International Worm Meeting, 2009]

Nematodes develop through periodic molts but molecular mechanisms that set the pace of the molting cycle are not well understood. Molting involves detachment of the old exoskeleton from the underlying hypodermis and synthesis of the new exoskeleton, an extracellular matrix composed largely of collagens. Epidermal stem cells divide in sync with the first three molts but terminally differentiate at the final molt. A period of behavioral quiescence (lethargus) accompanies remodeling of the exoskeleton. Animals subsequently use specialized movements to escape the old skeleton (ecdyse). Improper coordination of these cell biological and behavioral events can be fatal. We aim to identify genes and molecules that regulate the timing of the molting cycle. The best-characterized biological timers control circadian rhythms in the physiology and behavior of humans and other animals. In theory, worm counterparts of the core circadian clock proteins might also regulate the ultradian rhythm of the molting cycle. We are therefore investigating the role of clock gene homologs in the reiterative molecular and behavioral events characteristic of molting. As a complementary approach, we conducted a forward genetic screen for mutants that molt prematurely, using expression of the mlt-10p::gfp-pest reporter to indicate synthesis of a new cuticle. In a pilot screen of approximately 18,000 genomes, we indentified one candidate that expressed the mlt-10 reporter too early and then partly shed the L1 exoskeleton. This particular animal perished, possibly due to the exposure of an incomplete L2 skeleton to the environment. In ongoing work, a clonal screen will allow the recovery of lethal mutations from siblings of affected animals. To further investigate the physiologic regulation of the molting cycle, we have cloned and characterized new mutations that cause supernumerary molts after puberty. These alleles emerged from a genetic screen conducted by Alison Frand and Sascha Russel in the laboratory of Gary Ruvkun. By studying the regulation of the molting cycle, we expect to uncover novel but conserved mechanisms for the endocrine control of development and behavior.