Irini Topalidou

The EARP Complex and Its Interactor EIPR-1 Are Required for Cargo Sorting to Dense-Core Vesicles.

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.

The NCA-1 and NCA-2 Ion Channels Function Downstream of Gq and Rho To Regulate Locomotion in Caenorhabditis elegans

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.

The dense‐core vesicle maturation protein CCCP‐1 binds RAB‐2 and membranes through its C‐terminal domain

Dense‐core vesicles (DCVs) are secretory organelles that store and release modulatory neurotransmitters from neurons and endocrine cells. Recently, the conserved coiled‐coil protein CCCP‐1 was identified as a component of the DCV biogenesis pathway in the nematode Caenorhabditis elegans. CCCP‐1 binds the small GTPase RAB‐2 and colocalizes with it at the trans‐Golgi. Here, we report a structure‐function analysis of CCCP‐1 to identify domains of the protein important for its localization, binding to RAB‐2, and function in DCV biogenesis. We find that the CCCP‐1 C‐terminal domain (CC3) has multiple activities. CC3 is necessary and sufficient for CCCP‐1 localization and for binding to RAB‐2, and is required for the function of CCCP‐1 in DCV biogenesis. In addition, CCCP‐1 binds membranes directly through its CC3 domain, indicating that CC3 may comprise a previously uncharacterized lipid‐binding motif. We conclude that CCCP‐1 is a coiled‐coil protein that binds an activated Rab and localizes to the Golgi via its C‐terminus, properties similar to members of the golgin family of proteins. CCCP‐1 also shares biophysical features with golgins; it has an elongated shape and forms oligomers.

Dopamine negatively modulates the NCA ion channels in C. elegans

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.

Caenorhabditis elegans HIF-1 Is Broadly Required for Survival in Hydrogen Sulfide

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 outcomes 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 nonneuronal 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.

The SEK-1 p38 MAP Kinase Pathway Modulates Gq Signaling in Caenorhabditis elegans

Gq is a heterotrimeric G protein that is widely expressed in neurons and regulates neuronal activity. To identify pathways regulating neuronal Gq signaling, we performed a forward genetic screen in Caenorhabditis elegans for suppressors of activated Gq. One of the suppressors is an allele of sek-1, which encodes a mitogen-activated protein kinase kinase (MAPKK) in the p38 MAPK pathway. Here, we show that sek-1 mutants have a slow locomotion rate and that sek-1 acts in acetylcholine neurons to modulate both locomotion rate and Gq signaling. Furthermore, we find that sek-1 acts in mature neurons to modulate locomotion. Using genetic and behavioral approaches, we demonstrate that other components of the p38 MAPK pathway also play a positive role in modulating locomotion and Gq signaling. Finally, we find that mutants in the SEK-1 p38 MAPK pathway partially suppress an activated mutant of the sodium leak channel, NCA-1/NALCN, a downstream target of Gq signaling. Our results suggest that the SEK-1 p38 pathway may modulate the output of Gq signaling through NCA-1(unc-77).

EIPR1 controls dense-core vesicle cargo sorting and EARP complex localization in insulinoma cells

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 C. elegans neurons. Here we determine the role of mammalian EIPR1 in insulinoma cells. We find that in Eipr1 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.

Modulation of Gq-Rho Signaling by the ERK MAPK Pathway Controls Locomotion in Caenorhabditis elegans

The heterotrimeric G protein Gq regulates neuronal activity through distinct downstream effector pathways. In addition to the canonical Gq effector phospholipase Cβ, the small GTPase Rho was recently identified as a conserved effector of Gq. To identify additional molecules important for Gq signaling in neurons, we performed a forward genetic screen in the nematode Caenorhabditis elegans for suppressors of the hyperactivity and exaggerated waveform of an activated Gq mutant. We isolated two mutations affecting the MAP kinase scaffold protein KSR-1 and found that KSR-1 modulates locomotion downstream of, or in parallel to, the Gq-Rho pathway. Through epistasis experiments, we found that the core ERK MAPK cascade is required for Gq-Rho regulation of locomotion, but that the canonical ERK activator LET-60/Ras may not be required. Through neuron-specific rescue experiments, we found that the ERK pathway functions in head acetylcholine neurons to control Gq-dependent locomotion. Additionally, expression of activated LIN-45/Raf in head acetylcholine neurons is sufficient to cause an exaggerated waveform phenotype and hypersensitivity to the acetylcholinesterase inhibitor aldicarb, similar to an activated Gq mutant. Taken together, our results suggest that the ERK MAPK pathway modulates the output of Gq-Rho signaling to control locomotion behavior in C. elegans.

The ERK MAPK pathway modulates Gq-dependent locomotion in Caenorhabditis elegans

The heterotrimeric G protein Gq regulates neuronal activity through distinct downstream effector pathways. In addition to the canonical Gq effector phospholipase Cβ, the small GTPase Rho was recently identified as a conserved effector of Gq. To identify additional molecules important for Gq signaling in neurons, we performed a forward genetic screen in the nematode Caenorhabditis elegans for suppressors of the hyperactivity and exaggerated waveform of an activated Gq mutant. We isolated two mutations affecting the MAP kinase scaffold protein KSR-1 and found that KSR-1 modulates locomotion downstream of or in parallel to the Gq-Rho pathway. Through epistasis experiments, we found that the core ERK MAPK cascade is required for Gq-Rho regulation of locomotion, but that the canonical ERK activator LET-60/Ras may not be required. Through neuron-specific rescue experiments, we found that the ERK pathway functions in head acetylcholine neurons to control Gq-dependent locomotion. Additionally, expression of activated LIN-45/Raf in head acetylcholine neurons is sufficient to cause an exaggerated waveform phenotype and hypersensitivity to the acetylcholinesterase inhibitor aldicarb, similar to an activated Gq mutant. Taken together, our results suggest that the ERK MAPK pathway modulates the output of Gq-Rho signaling to control locomotion behavior in C. elegans.

C. elegans HIF-1 is broadly required for survival in hydrogen sulfide

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.