Topalidou, Irini et al. (2011) International Worm Meeting "The Messiness of Combinatorial Control."

Topalidou, Irini, Chalfie, Martin

[International Worm Meeting, 2011]

Because of combinatorial expression, transcription factors can determine the development of more than one type of cell. For example, the transcription factor complex of MEC-3 and UNC-86 is required in embryos for the development of both the touch receptor neurons (TRNs) and the FLP neurons. Despite sharing these transcription factors, the TRNs, but not the FLP neurons, express several proteins needed for gentle touch, e.g., MEC-2, MEC-4, and MEC-18. Both the embryonic TRNs and the FLP neurons upregulate mRNAs for approximately 300 genes. Twenty three percent of the genes are upregulated in both cells. We were surprised to find that some of the commonly upregulated genes are MEC-3-regulated genes that had previously been identified as being specific to the TRNs. Although the mRNAs for these genes are detected in the FLP cells in low amounts, these mRNAs do not appear to be translated to detectable levels. These results suggest that transcriptional control is relatively inexact, but these apparent transcription errors are well tolerated and do not alter cell fate. Either a threshold amount of transcription is required for translation or these mRNAs are subjected to some sort of translational control. The FLP cells are, nonetheless, poised to make TRN-specific proteins. Expression of the transcription factor ALR-1, a downstream target of MEC-3 that ensures, but does not direct, TRN differentiation caused FLP neurons to make higher levels of the mRNAs for these genes and their proteins. This ectopic expression also prevented the FLP neurons from forming the fine branches that characterize the adult cells. Thus, alr-1 appears to change the fate of these cells. Our experiments suggest that combinatorial control, although potentially able to produce many different types of cells, can be messy. Such messiness may need to be controlled, but it also could be advantageous, allowing cells to take on additional characteristics under different conditions.

Topalidou, Irini et al. (2013) International Worm Meeting "Identifying molecules involved in dense-core vesicle biology."

Giarmarco, Michelle, Barr, Angela L., Ailion, Michael, Topalidou, Irini, Hoyt, Jill, Jarvie, Brooke

[International Worm Meeting, 2013]

Neuromodulation is a form of chemical signaling mediated by the release of neuropeptides and monoamines that activate G protein coupled receptors. The regulated release of neuromodulators is mediated by a unique organelle, the dense-core vesicle (DCV). The biogenesis, trafficking, and release of DCVs are not well understood, mainly because few proteins specific to DCV function have been identified. We previously identified at least six molecules involved in DCV function by screening for genetic suppressors of the activating Gq mutation, egl-30(tg26). Two of the new proteins, RUND-1 (a RUN domain protein) and CCCP-1 (a coiled-coil protein), act as effectors of the small GTPase RAB-2 a protein previously implicated in DCV maturation. Here we report the identification of two independent missense mutations in the same region of the coiled-coil domain of RUND-1 that give stronger phenotypes than the null mutant, implying important roles for the RUND-1 coiled-coil domain. We also characterized the function of another Gq suppressor, which encodes a conserved protein containing WD40 repeats. We show that mutants of this gene have the "unmotivated" phenotype typical of DCV mutants, characterized by little spontaneous movement on food but capable of coordinated locomotion when stimulated. The WD40 protein is required for trafficking of DCV cargos and its expression in the head cholinergic neurons partially rescues the unmotivated phenotype. We are currently investigating whether the WD40 protein interacts with the RUND-1 effector complex. To identify molecules that interact with the WD40 protein, we screened for suppressors of the WD40 unmotivated phenotype. We identified seven suppressors, at least one of which partially rescues the DCV cargo trafficking defect. By screening for additional suppressors of activated Gq to saturate the original screen, we identified three new genes that seem to be involved in DCV biology. We are currently sequencing two of our identified mutants and characterizing their DCV trafficking phenotype.

Topalidou, Irini et al. (2021) International Worm Meeting "GRK-2 signaling in sensory neurons regulates the ability of C. elegans to travel long distances"

Ailion, Michael, Topalidou, Irini

[International Worm Meeting, 2021]

G protein-coupled receptor kinases (GRKs) play key roles in terminating neuromodulator signaling by phosphorylating and deactivating G protein-coupled receptors (GPCRs). Although GRKs are critical for brain function, their particular role in regulating neuronal activity is largely unexplored. C. elegans GRK-2 is required for normal chemosensation, egg-laying, and locomotion. Our previous work suggests that GRK-2 acts in premotor interneurons to promote locomotion and swimming by negatively regulating the D2-like dopamine receptor DOP-3. Here we show that GRK-2 also regulates the ability of animals to travel long distances (exploration or roaming) in a manner independent of DOP-3. Using neuron-specific rescuing experiments we show that GRK-2 acts in multiple sensory neurons to mediate this behavior. Further analysis using mutants with defects in ciliated sensory neurons indicates that grk-2 and the cilium-structure mutants act in the same pathway to regulate exploration. We then tested candidate mutants and also used mutagenesis to identify suppressors of the exploration defect of grk-2 mutants. We identified a neuropeptide signaling pathway involving the neuropeptide FLP-1 and a neurohormonal signaling pathway involving the sulfotransferase SSU-1 that contribute to GRK-2- mediated exploration. Our studies of GRK-2 revealed neurons and molecular pathways that modulate exploration behavior.

Topalidou, Irini et al. (2019) International Worm Meeting "The GPCR kinase GRK-2 acts in premotor interneurons to positively regulate NCA channel activity."

Ailion, Michael, Bai, Jihong, Topalidou, Irini, Dong, Yongming

[International Worm Meeting, 2019]

The NALCN/NCA ion channel is a cation channel related to voltage-gated sodium and calcium channels. NALCN is a putative sodium leak channel, which regulates the resting membrane potential and excitability of invertebrate and vertebrate neurons, but its precise cellular role and regulation are unclear. The C. elegans orthologs of NALCN, NCA-1 and NCA-2, act in premotor interneurons to potentiate persistent motor circuit activity and to sustain locomotion. Through a forward genetic screen we identified the GPCR kinase GRK-2 as a modulator of NCA. To advance our genetic observations we are using an array of behavioral, electrophysiological, and imaging approaches to determine the mechanisms that connect GRK-2 to NCA activity. First, we show that grk-2 mutants have slow locomotion, fail to sustain body bends when swimming, and faint in sensitized backgrounds, suggesting that GRK-2 is a positive modulator of NCA activity for sustained locomotion. Second, we find that grk-2 mutants have severe defects in rhythmic excitatory post-synaptic currents (rEPSCs), which require NCA activity in premotor interneurons. Third, we show that expression of GRK-2 in premotor interneurons fully rescues the grk-2 locomotion defects and partially rescues the rEPSC defects, suggesting that GRK-2 acts mainly in a cell autonomous way to regulate NCA activity. Fourth, by imaging Ca2+ dynamics in the premotor interneuron AVA where NCA channels function, we find that touch-induced AVA Ca2+ responses are significantly reduced in grk-2 mutants. This study shows that GRK-2 is a positive regulator of sustained locomotion and NCA activity.

Topalidou, Irini et al. (2009) International Worm Meeting "The aristaless/Arx homolog alr-1 regulates the fate of the touch receptor neurons by acting as a transcriptional activator."

Chalfie, Martin, Topalidou, Irini

[International Worm Meeting, 2009]

The Drosophila homeobox gene aristaless (al) participates in the development of wings, legs and aristae. In humans, mutations of the al homologue (Arx) cause multiple forms of X-chromosome linked mental disorders. The C. elegans aristaless/Arx orthologue (alr-1) regulates the development of chemosensory and GABAergic neurons and is needed for the integrity of the amphids. Using cell sorting and cDNA microarrays, we identified alr-1 among the genes upregulated in embryonic touch receptor neurons (TRNs). GFP promoter and translation fusions for alr-1 are expressed in five of the six TRNs (ALM, PLM and AVM, but not PVM) throughout development. This expression depends on MEC-3. ALR-1 is functionally important in the TRNs, since alr-1 mutants are variably insensitive to gentle touch. Expression of alr-1(+) in the TRNs rescues this touch insensitivity. Using temperature-sensitive mec-8-dependent splicing, we expressed alr-1 in a temperature-dependent manner and demonstrated that alr-1 is needed for touch sensitivity throughout development. Expression of genes needed for TRN function, including mec-3, are down- regulated in alr-1 mutants in all touch neurons except PVM. We used Quantitative PCR (QPCR) to show that mRNA levels of the affected genes were ~50% of wild-type levels, a result that may explain the touch-insensitive phenotype of the alr-1 mutants. In contrast, the TRN-expression of the pan-neuronal genes unc-119 and sng-1 was unaffected in alr-1 mutants, arguing against ALR-1 acting as a general enhancer of transcription in the TRNs. These observations lead to a model in which ALR-1 positively enhances MEC-3 and, perhaps, UNC-86 activity and/or expression to regulate the fate of the TRNs. MEC-3 is needed for its own continued expression; ALR-1 may contribute to this maintained expression. Consistent with this hypothesis, ALR-1 acts as a transcriptional activator in an in vivo yeast expression system, by directly inducing expression from the mec-3 promoter and from the TRN-specific mec-18 promoter. Mutations identified in the ARX-homeobox of patients with mental retardation abolish ALR-1-dependent gene expression.

Topalidou, Irini et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "The aristaless/Arx homolog alr-1 ensures touch receptor neuron differentiation"

van Oudenaarden, Alexander, Chalfie, Martin, Topalidou, Irini

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Terminally differentiated cells must maintain their cell fate during long periods of time. This maintenance could result from autoregulatory activation of selector genes that define cell fate. For example, maintenance of the differentiation of the six touch receptor neurons (TRNs) is mediated by the autoregulatory activation of the LIM homeodomain (HD) transcription factor MEC-3 which, together with the POU HD transcription factor UNC-86, induces mec-3 and TRN-specific gene expression. Here we demonstrate that MEC-3 autoregulation is not sufficient for TRN differentiation maintenance and that an additional HD transcription factor, encoded by alr-1 , a homologue of the Drosophila aristaless (al) gene, is also required. Using phenotyp ic analysis and Single Molecule Fluorescent in Situ Hybridization, we showed that mutations in alr-1 result in variable cell function and variable mec-3 expression in the TRNs. ALR-1 prevents this variability not by increasing mec-3 expression, but by restricting expression to the higher end of the expression range, a process we call refinement. ALR-1 may also directly affect the levels of expression of mec-3 target genes. alr-1 is needed throughout development for touch sensitivity and mec-3 optimal expression. Using a yeast expression system, we showed that ALR-1 enhances MEC-3/UNC-86dependent transcription from the mec-3 promoter. Furthermore, ectopic expression of alr-1 causes the mec-3 -expressing neurons FLP and PVD to differentiate as TRNs. These results suggest that ALR-1 functions as a transcriptional activator to protect TRNs against variability and to secure TRN differentiation. Given that AL, ALR-1, and ARX are needed for the expression of other L IM HD transcription factors we propose that Aristaless proteins may also act to ensure proper differentiation in other systems.

Topalidou, Irini et al. (2009) International Worm Meeting "Determining the genetic profile of two types of C. elegans mechanosensory neurons: the touch receptor neurons and the FLP neurons."

Topalidou, Irini, Bounoutas, Alexander, Chalfie, Martin

[International Worm Meeting, 2009]

Cells acquire many of their characteristics during differentiation by expressing specific subsets of genes. The identification of this select gene pool is an important means of understanding cell fate determination. We used gfp cell sorting and cDNA microarrays to identify genes that are upregulated in the touch receptor neurons (TRNs) and FLP neurons, both of which require MEC-3 for their differentiation. We identified channel and receptor subunits, transcription factors, and calcium sensors among the 198 genes expressed at high levels in the TRNs. Using existing mutants and/or feeding RNAi, we tested the role in gentle touch sensation of most of the genes (163/189) not previously identified as needed for touch sensitivity. Eight of the genes were needed for optimal touch sensitivity; genes encoding the transcription factor ALR-1, the PON-1 homolog K11E4.3, and the GABA receptor LGC-37 were among them. We demonstrated that ALR-1 is needed in the TRNs throughout development to regulate the expression of touch neuron-specific genes. Further analysis on the role of K11E4.3 in touch sensation will be given elsewhere in this meeting (Y. Chen and M. Chalfie). Interestingly, most of the mec genes (i.e: mec-18) that were previously identified as TRN-specific were among the 203 genes upregulated in the FLP neurons. QPCR analysis for mec-18 mRNA in animals with genetically-ablated TRNs showed that mec-18 mRNA is indeed expressed in these animals, mainly at the early larvae stages, although no immuno-detectable MEC-18 is produced. Finally, a short-lived gfp driven by the mec-18 promoter (Pmec-18praja::gfp) produces fluorescence in young FLP neurons. We conclude that both post-transcriptional and transcriptional regulation play important roles in differentiating the touch receptor neurons from the FLP neurons.

Irini Topalidou et al. (2007) International Worm Meeting "An improved cell isolation method to examine gene expression in the touch receptor neurons and FLP cells."

Irini Topalidou, Martin Chalfie

[International Worm Meeting, 2007]

Previous work in our lab used cell sorting and cDNA microarrays to identify mec-3- dependent genes in embryonic touch receptor neurons (Zhang et al. Nature 418: 331-335, 2002). Those experiments required 2 X 106 cells of which 50% were GFP-positive. We have modified these methods in two ways so that we now make sufficient RNA from only 25,000 cells that are 90-95% GFP-positive. First, by characterizing the fluorescence profile of non GFP-expressing cells, we could exclude autofluorescent cells from our selection. Second, by using Ambion mRNA isolation and amplification kits, we could obtain sufficient RNA for the arrays from much less starting material. The RNA was then used on Affymetrix arrays. Using this approach we identified 201 genes differentially expressed (>4 times upregulated) in the touch receptor neurons. All 25 genes already known to be expressed in these cells were included (usually with very high scores) on our list of upregulated. Genes that were either high on the list or were interesting candidates (GABA and ACHR receptor subunits, Ca+&#43 binding proteins, potassium channel subunits, a paraoxonase homolog) were further characterized by making promoter or protein fusions with GFP. 13 of 16 new genes tested were expressed in these cells, including one, the putative receptor C49G9.1, which co-localizes with the mechanosensory channel. We have used bacterial RNAi and the RNAi-hypersensitive strains lin-35 and mec-4ts; lin-35 to demonstrate that nine of the genes, e.g., C49G9.1 and the Ca+&#43sensor C16H3.1, were needed for optimal touch sensitivity. We used a similar method to identify genes selectively expressed in the FLP neurons, a set of mec-3 expressing mechanosensory neurons in the head. Because an FLP specific promoter was not known, we isolated the cells from a line expressing P_mec-3gfp in which the touch cells were killed using a dominant mec-4 mutation (in embryos mec-3 is expressed only in touch neurons and FLP). GFP-positive cells in culture from these embryos were morphologically distinct from touch receptor neurons, suggesting that the touch neurons had been eliminated; the absence of touch neurons was confirmed using an antibody against the touch receptor-specific protein MEC-18. 398 genes were found to be differentially expressed (>4 times upregulated) in the FLP cells. Surprisingly, RNAs for several mec genes, including mec-18, were expressed in these cells. These results suggest that post-transcriptional control may prevent protein production from these genes in the FLP cells. We are currently confirming the expression pattern of genes of our candidates, one of which is the DEG/ENaC channel gene asic-1.

Cattin, Jerome et al. (2015) International Worm Meeting "Molecules involved in the biogenesis of dense-core vesicles."

Ailion, Michael, Cattin, Jerome, Topalidou, Irini

[International Worm Meeting, 2015]

The regulated release of neuromodulators is mediated by a specialized type of cellular vesicle, the dense-core vesicle (DCV). The biogenesis, maturation, and trafficking of DCVs are not well characterized, mainly because few proteins specific to DCV function are known. In a genetic screen for suppressors of constitutively active Galphaq we identified mutations in the small GTPase RAB-2, the RAB-2 effectors RUND-1 (a RUN domain protein) and CCCP-1 (a coiled-coil protein), and an uncharacterized WD40 domain-containing protein (Y87G2A.11). Here we investigate the specific function of these proteins both in C. elegans and in neuroendocrine cell lines. We show that mutants of these genes have the characteristic "unmotivated" phenotype characterized by little spontaneous movement on food but capable of coordinated locomotion once stimulated. All proteins function in the same genetic pathway, in a cell-autonomous way, and are required for trafficking of DCV but not SV (synaptic vesicle) cargos. We show that all these proteins are expressed in neuroendocrine cell lines. With the exception of Y87G2A.11, which localizes to the cytoplasm, the three other players co-localize with each other in a perinuclear region both in C. elegans and in neuroendocrine cells. Co-localization experiments in insulinoma cells (INS-1) show that these proteins partially co-localize with the trans Golgi and with proinsulin, suggesting a role in the biogenesis or early transport of secretory vesicles. We have successfully knocked out the Y87G2A.11 ortholog TSSC1 in INS-1 cells using the CRISPR technology and are now performing insulin secretion assays to investigate its role in DCV secretion. CRISPR-induced knock out of CCCP-1 and RUND-1 orthologs is underway. We are also performing in vitro binding assays of these proteins to characterize their physical interactions and yeast two hybrid and mass spec experiments to identify new interactors. Finally, we have characterized biophysical properties of CCCP-1. CCCP-1 is predicted to have long coiled-coil domains like golgins. We show that it forms oligomers and has an elongated shape, raising the possibility that it may act as a tether that links together yet unknown membranes. We are assaying its ability to bind lipids and tether membranes.

Clippinger, Amy et al. (2021) International Worm Meeting "Role of MAPK/ERK signaling in neurons"

Clippinger, Amy, Ailion, Michael, Topalidou, Irini

[International Worm Meeting, 2021]

The MAPK/ERK pathway plays a critical role in regulating neuronal activity in C. elegans. The regulation of MAP3K Raf and its targets have been well-studied in growth and development, as unregulated ERK signaling causes cancer.However, the regulation of Raf activity in neurons and how ERK signaling affects neuronal activity are not understood.Overexpression of activated LIN-45/Raf (Raf*) in neurons results in loopy locomotion. While performing a forward genetic screen for suppressors of Raf*, we identified components that (1) affect the activity of Raf and (2) are downstream targets that directly affect neuronal activity. Loss of Kinase Suppressor of Ras 1 (KSR-1) suppresses the loopy locomotion of Raf* expressing worms. KSR-1 is known to heterodimerize with Raf, and this is critical for Raf function. Our experiments demonstrate that even Raf* requires KSR-1 in neurons to promote signaling. We also identified auxiliary subunits of the voltage sensitive cation channel NCA/NALCN as suppressors of Raf*. We found Raf* and the NCA mutant nlf-1 were mutually suppressing, as nlf-1 mutants exhibit a distinct "fainting" behavior that is suppressed by Raf*. We explored by what mechanism Raf* suppresses fainting, either by enhancing impaired NCA function or via upregulation of neuronal activity via a separate pathway. As certain tumors are electrically active tissues that hijack neuronal signaling pathways to promote cancerous cell growth, these studies will relate to both neurobiology and cancer biology.