Topalidou I et al. (2017) G3 (Bethesda) "Caenorhabditis elegans HIF-1 Is Broadly Required for Survival in Hydrogen Sulfide."

Topalidou I, Miller DL

[G3 (Bethesda), 2017]

Hydrogen sulfide is common in the environment, and is also endogenously produced by animal cells. Although hydrogen sulfide is often toxic, exposure to low levels of hydrogen sulfide improves outcome in a variety of mammalian models of ischemia-reperfusion injury. In Caenorhabditis elegans, the initial transcriptional response to hydrogen sulfide depends on the hif-1 transcription factor, and hif-1 mutant animals die when exposed to hydrogen sulfide. In this study, we use rescue experiments to identify tissues in which hif-1 is required to survive exposure to hydrogen sulfide. We find that expression of hif-1 from the unc-14 promoter is sufficient to survive hydrogen sulfide. Although unc-14 is generally considered to be a pan-neuronal promoter, we show that it is active in many non-neuronal cells as well. Using other promoters, we show that pan-neuronal expression of hif-1 is not sufficient to survive exposure to hydrogen sulfide. Our data suggest that hif 1 is required in many different tissues to direct the essential response to hydrogen sulfide.

Topalidou I et al. (2017) PLoS Genet "Dopamine negatively modulates the NCA ion channels in C. elegans."

Cooper K, Topalidou I, Ailion M, Pereira L

[PLoS Genet, 2017]

The NALCN/NCA ion channel is a cation channel related to voltage-gated sodium and calcium channels. NALCN has been reported to be a sodium leak channel with a conserved role in establishing neuronal resting membrane potential, but its precise cellular role and regulation are unclear. The Caenorhabditis elegans orthologs of NALCN, NCA-1 and NCA-2, act in premotor interneurons to regulate motor circuit activity that sustains locomotion. Recently we found that NCA-1 and NCA-2 are activated by a signal transduction pathway acting downstream of the heterotrimeric G protein Gq and the small GTPase Rho. Through a forward genetic screen, here we identify the GPCR kinase GRK-2 as a new player affecting signaling through the Gq-Rho-NCA pathway. Using structure-function analysis, we find that the GPCR phosphorylation and membrane association domains of GRK-2 are required for its function. Genetic epistasis experiments suggest that GRK-2 acts on the D2-like dopamine receptor DOP-3 to inhibit Go signaling and positively modulate NCA-1 and NCA-2 activity. Through cell-specific rescuing experiments, we find that GRK-2 and DOP-3 act in premotor interneurons to modulate NCA channel function. Finally, we demonstrate that dopamine, through DOP-3, negatively regulates NCA activity. Thus, this study identifies a pathway by which dopamine modulates the activity of the NCA channels.

Topalidou I et al. (2011) Proc Natl Acad Sci U S A "Shared gene expression in distinct neurons expressing common selector genes."

Topalidou I, Chalfie M

[Proc Natl Acad Sci U S A, 2011]

Expression of the mec-3/unc-86 selector gene complex induces the differentiation of the touch receptor neurons (TRNs) of Caenorhabditis elegans. These genes are also expressed in another set of embryonically derived mechanosensory neurons, the FLP neurons, but these cells do not share obvious TRN traits or proteins. We have identified ~300 genes in each cell type that are up-regulated at least threefold using DNA microarrays. Twenty-three percent of these genes are up-regulated in both cells. Surprisingly, some of the common genes had previously been identified as TRN-specific. Although the FLP neurons contain low amounts of the mRNAs for these TRN genes, they do not have detectable proteins. These results suggest that transcription control is relatively inexact but that these apparent errors of transcription are tolerated and do not alter cell fate. Previous studies showed that loss of the EGL-44 and EGL-46 transcription factors cause the FLP neurons to acquire TRN-like traits. Here, we show that similar changes occur (e.g., the expression of both the TRN mRNAs and proteins) when the FLP neurons ectopically express the auxiliary transcription factor ALR-1 (Aristaless related), which ensures, but does not direct, TRN differentiation. Thus, the FLP neurons can acquire a TRN-like fate but use multiple levels of regulation to ensure they do not. Our data indicate that expression of common master regulators in different cell types can result in inappropriate expression of effector genes. This misexpression makes these cells vulnerable to influences that could cause them to acquire alternative fates.

Topalidou I et al. (2017) Genetics "The NCA-1 and NCA-2 Ion Channels Function Downstream of Gq and Rho To Regulate Locomotion in Caenorhabditis elegans."

Topalidou I, Watanabe S, Cooper K, Jorgensen EM, Ailion M, Chen PA

[Genetics, 2017]

The heterotrimeric G protein Gq positively regulates neuronal activity and synaptic transmission. Previously, the Rho guanine nucleotide exchange factor Trio was identified as a direct effector of Gq that acts in parallel to the canonical Gq effector phospholipase C. Here we examine how Trio and Rho act to stimulate neuronal activity downstream of Gq in the nematode Caenorhabditis elegans Through two forward genetic screens, we identify the cation channels NCA-1 and NCA-2, orthologs of mammalian NALCN, as downstream targets of the Gq/Rho pathway. By performing genetic epistasis analysis using dominant activating mutations and recessive loss-of-function mutations in the members of this pathway, we show that NCA-1 and NCA-2 act downstream of Gq in a linear pathway. Through cell-specific rescue experiments, we show that function of these channels in head acetylcholine neurons is sufficient for normal locomotion in C. elegans Our results suggest that NCA-1 and NCA-2 are physiologically relevant targets of neuronal Gq-Rho signaling in C. elegans.

Topalidou I et al. (2012) Curr Biol "Genetically separable functions of the MEC-17 tubulin acetyltransferase affect microtubule organization."

Nguyen KC, Heppenstall P, Politi KA, Somhegyi H, Topalidou I, Chalfie M, Hall DH, Keller C, Kalebic N

[Curr Biol, 2012]

BACKGROUND: Microtubules (MTs) are formed from the lateral association of 11-16 protofilament chains of tubulin dimers, with most cells containing 13-protofilament (13-p) MTs. How these different MTs are formed is unknown, although the number of protofilaments may depend on the nature of the - and -tubulins. RESULTS: Here we show that the enzymatic activity of the Caenorhabiditis elegans -tubulin acetyltransferase (-TAT) MEC-17 allows the production of 15-p MTs in the touch receptor neurons (TRNs) MTs. Without MEC-17, MTs with between 11 and 15 protofilaments are seen. Loss of this enzymatic activity also changes the number and organization of the TRN MTs and affects TRN axonal morphology. In contrast, enzymatically inactive MEC-17 is sufficient for touch sensitivity and proper process outgrowth without correcting the MT defects. Thus, in addition to demonstrating that MEC-17 is required for MT structure and organization, our results suggest that the large number of 15-p MTs, normally found in the TRNs, is not essential for mechanosensation. CONCLUSION: These experiments reveal a specific role for -TAT in the formation of MTs and in the production of higher order MTs arrays. In addition, our results indicate that the -TAT protein has functions that require acetyltransferase activity (such as the determination of protofilament number) and others that do not (presence of internal MT structures).

Topalidou I et al. (2016) PLoS Genet "The EARP Complex and Its Interactor EIPR-1 Are Required for Cargo Sorting to Dense-Core Vesicles."

Pappas AL, Ailion M, MacCoss MJ, Cooper K, Merrihew GE, Cattin-Ortola J, Topalidou I

[PLoS Genet, 2016]

The dense-core vesicle is a secretory organelle that mediates the regulated release of peptide hormones, growth factors, and biogenic amines. Dense-core vesicles originate from the trans-Golgi of neurons and neuroendocrine cells, but it is unclear how this specialized organelle is formed and acquires its specific cargos. To identify proteins that act in dense-core vesicle biogenesis, we performed a forward genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We previously reported the identification of two conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core vesicle cargo-sorting. Here we identify several additional conserved factors important for dense-core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated recycling protein (EARP) complex. By assaying behavior and the trafficking of dense-core vesicle cargos, we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants in the RAB-2 pathway. Genetic epistasis data indicate that RAB-2, EIPR-1 and EARP function in a common pathway. In addition, using a proteomic approach in rat insulinoma cells, we show that EIPR-1 physically interacts with the EARP complex. Our data suggest that EIPR-1 is a new interactor of the EARP complex and that dense-core vesicle cargo sorting depends on the EARP-dependent trafficking of cargo through an endosomal sorting compartment.

Topalidou I et al. (2011) Proc Natl Acad Sci U S A "Caenorhabditis elegans aristaless/Arx gene alr-1 restricts variable gene expression."

van Oudenaarden A, Topalidou I, Chalfie M

[Proc Natl Acad Sci U S A, 2011]

Variable expressivity of mutant phenotypes in genetically identical individuals is a phenomenon widely reported but poorly understood. For example, mutations in the gene encoding the transcription factor ALR-1 in Caenorhabditis elegans result in variable touch receptor neuron (TRN) function. Using single-molecule in situ hybridization, we demonstrate that this phenotypic variability reflects enhanced variability in the expression of the selector gene mec-3, which is needed, together with unc-86, for the differentiation of the TRNs. In a yeast expression system, ALR-1 enhances MEC-3/UNC-86-dependent transcription from the mec-3 promoter, showing that ALR-1 can enhance bulk mec-3 expression. We show that, due to stochastic fluctuations, autoregulation of mec-3 is not sufficient for TRN differentiation; ALR-1 provides a second positive feedback loop that increases mec-3 expression, by restricting variability, and thus ensures TRN differentiation. Our results link fluctuations in gene expression to phenotypic variability, which is seen in many mutant strains, and provide an explicit demonstration of how variable gene expression can be curtailed in developing cells to ensure their differentiation. Because ALR-1 and similar proteins (Drosophila Aristaless and human ARX) are needed for the expression of other transcription factors, we propose that proteins in this family may act to ensure differentiation more generally.

Topalidou I et al. (2019) Mol Biol Cell "EIPR1 controls dense-core vesicle cargo retention and EARP complex localization in insulin-secreting cells."

Hummer B, Topalidou I, Ailion M, Asensio CS, Cattin-Ortola J

[Mol Biol Cell, 2019]

Dense-core vesicles (DCVs) are secretory vesicles found in neurons and endocrine cells. DCVs package and release cargos including neuropeptides, biogenic amines, and peptide hormones. We recently identified the endosome-associated recycling protein (EARP) complex and the EARP-interacting protein EIPR-1 as proteins important for controlling levels of DCV cargos in <i>C. elegans</i> neurons. Here we determine the role of mammalian EIPR1 in insulinoma cells. We find that in <i>Eipr1</i> KO cells, there is reduced insulin secretion, and mature DCV cargos such as insulin and carboxypeptidase E (CPE) accumulate near the trans-Golgi network and are not retained in mature DCVs in the cell periphery. In addition, we find that EIPR1 is required for the stability of the EARP complex subunits and for the localization of EARP and its association with membranes, but EIPR1 does not affect localization or function of the related Golgi-associated retrograde protein (GARP) complex. EARP is localized to two distinct compartments related to its function: an endosomal compartment and a DCV biogenesis-related compartment. We propose that EIPR1 functions with EARP to control both endocytic recycling and DCV maturation.

Topalidou I et al. (2008) European Worm Meeting "THE C. ELEGANS ARISTALESS/ARX HOMOLOG ALR-1 DEFINES THE FUNCTION OF THE TOUCH RECEPTOR NEURONS BY ACTING AS A POSITIVE REGULATOR OF TRANSCRIPTION"

Topalidou I, Chalfie M

[European Worm Meeting, 2008]

The highly conserved homeodomain protein Aristaless (Al) was initially. described in Drosophila where it affects the development of legs, wings and. terminal appendages (aristae). The human Aristaless-related homeobox gene. (ARX) is involved in the development of human brain. Mutations in ARX have. been recently found in patients with multiple forms of X-chromosome-linked. mental retardation. The C. elegans homolog, alr-1, is needed for the. regulation of chemosensory and GABAergic motor neuron development. The in. vivo role of Al, ARX, or alr-1 in the observed developmental and behavioral. phenotypes is unclear. Based on genetic data and reporter assays ARX is. thought to act as a bi-functional transcriptional regulator. Using cell. sorting and cDNA miscroarrays we identified alr-1 among genes. differentially expressed in the touch receptor neurons in C. elegans. We. used a GFP-promoter fusion to verify that alr-1 is expressed in these cells. and to show that expression depends on the LIM-homeodomain protein and. touch receptor neuron-cell fate regulator MEC-3. ALR-1 is needed in the. touch receptor neurons, since alr-1 mutants (including animals deleted for. the gene) had impaired touch insensitivity, which could be rescued by touch. neuron expression of an alr-1(+) transgene. Using GFP expression or. antibodies we showed that several genes needed for touch neuron function. are down- regulated in alr-1 mutants. Using Quantitative PCR (QPCR) we. found that mRNA levels were ~50% of wild-type levels, a result that may. explain the largely touch-insensitive phenotype of these animals. The above. observations lead to a model in which ALR-1 functions positively in the. touch neurons to define the absolute levels of expression of touch neuron-. related genes. To test this hypothesis we used a yeast expression system to. demonstrate that heterologous expression of ALR-1 is sufficient to. upregulate the expression from touch neurons-specific promoters. These data. indicate that ALR-1 can act as a transcriptional activator in vivo. We are. currently exploring the putative co-operation between MEC-3 and ALR-1 for. the activation of touch neurons-related genes. Finally, we are. investigating the effect of mutations identified in the ARX genes of. patients with mental retardation on the binding and activating capacity of. ALR-1.

O'Hagan R et al. (2021) Bio Protoc "Fixation and Immunostaining of Endogenous Proteins or Post-translational Modificationsin Caenorhabditis elegans."

O'Hagan R, Topalidou I

[Bio Protoc, 2021]

Although the advent of genetically-encoded fluorescent markers, such as the green fluorescent protein (GFP; Chalfie <i>et al.</i>, 1994 ), has enabled convenient visualization of gene expression <i>in vivo</i>, this method is generally not effective for detecting post-translational modifications because they are not translated from DNA sequences. Genetically-encoded, fluorescently-tagged transgene products can also be misleading for observing expression patterns because transgenes may lack endogenous regulatory DNA elements needed for precise regulation of expression that could result in over or under expression. Fluorescently-tagged proteins created by CRISPR genome editing are less prone to defective expression patterns because the loci retain endogenous DNA elements that regulate their transcription (Nance and Frokjaer-Jensen, 2019). However, even CRISPR alleles encoding heritable fluorescently-tagged protein markers can result in defects in function or localization of the gene product if the fluorescent tag obstructs or otherwise interferes with important protein interaction domains or affects the protein structure. Indirect immunofluorescence is a method for detecting endogenous gene expression or post-translational modifications without the need for transgenesis or genome editing. Here, we present a reliable protocol in which <i>C. elegans</i> nematodes are fixed, preserved, and permeabilized for staining with a primary antibody to bind proteins or post-translational modifications, which are then labeled with a secondary antibody conjugated to a fluorescent dye. Use of this method may be limited by the availability of (or ability to generate) a primary antibody that binds the epitope of interest in fixed animals. Thousands of animals are simultaneously subjected to a series of chemical treatments and washes in a single centrifuge tube, allowing large numbers of identically-treated stained animals to be examined. We have successfully used this protocol (O' Hagan <i>et al.</i>, 2011 and 2017; Power <i>et al.</i>, 2020 ) to preserve and detect post-translational modifications of tubulin in <i>C. elegans</i> ciliated sensory neurons and to detect non-modified endogenous protein (Topalidou and Chalfie, 2011).