Michael E. Ailion, Ph.D. - US grants

This research project investigates how new biological species form. Laboratory-based efforts to identify genetic and molecular mechanisms of speciation in nematode worms will be aided by the isolation of new worm strains and species by members of the general public. High school students will participate directly in scientific research by collecting nematodes from rotting fruits and flowers. They will learn how research is performed and will also gain a basic understanding of genes, DNA, and evolution that is becoming increasingly more important and societally relevant. By engaging students in the practice of science, this project fulfills one of the three central dimensions in the Next Generation Science Standards recently adopted by many states across the nation and will improve scientific literacy in the general population by helping to build an informed public that understands the scientific enterprise and can better contribute to future national policy decisions that require the interpretation and evaluation of scientific data.

The process of speciation involves the evolution of reproductive barriers between populations within a species so that a single species can split. Though multiple reproductive barriers typically isolate existing species, less is known about which barriers drove the speciation process originally. The goal of the project is to identify genes that cause reproductive isolation and determine their molecular mechanisms of action. The project is focused on identifying genes that mediate incompatibility within species and will operate at three levels to: 1) determine the detailed molecular and cellular mechanism of action of a previously identified intraspecific incompatibility in the nematode C. elegans mediated by a selfish toxin/antidote gene pair, 2) identify the genes involved in a newly discovered nuclear-cytoplasmic intraspecific incompatibility in the related nematode species C. nouraguensis, and 3) systematically search for more intraspecific genetic incompatibilities within the Caenorhabditis genus in a large-scale project to isolate new strains of Caenorhabditis species using a crowdsourcing approach implemented in high school classrooms, and by systematic crosses of strains performed by undergraduate researchers to test for hybrid breakdown.

Project summary Neuronal activity can be modulated by transmitters that act through receptors coupled to heterotrimeric G proteins. Activation of these G protein signal transduction pathways leads to changes in neuronal excitability or synaptic transmission at the cellular level, and changes in behavior or memory at the organismal level. The long-term goal of our work is to identify and understand the mechanistic basis of neuromodulatory pathways. The goal of this proposal is to identify new regulatory pathways that lead to the modulation of a specific ion channel, the NALCN/NCA channel. The NALCN/NCA ion channel is a putative cation channel related to voltage-gated sodium and calcium channels, but whose precise cellular role and regulation are not well understood. However, mutations in NALCN or its associated subunits have been directly linked to human neurological diseases characterized by a range of symptoms, including abnormal movements and muscle contractions, intellectual disability, and seizures. Additionally, mutations in this channel in model organisms lead to strong neuronal phenotypes including defects in rhythmic behaviors, neuronal excitability, and synaptic function, demonstrating the physiological importance of this channel. Through a forward genetic screen in the nematode C. elegans, we found that the NCA ion channel is activated by a new signal transduction pathway acting downstream of the heterotrimeric G protein Gq. Activated Gq directly binds and stimulates the guanine nucleotide exchange activity of the Trio RhoGEF to activate the small G protein Rho. Here we will determine how this Gq-Rho pathway activates NCA by studying additional factors identified in our screen. In Aim 1, we will focus on a G protein-coupled receptor kinase. Our hypothesis is that this kinase modulates dopamine signaling that specifically regulates NCA activity through the Gq-Rho pathway. We will perform a structure- function analysis of this kinase to determine its functional domains and perform genetic and biochemical experiments to determine how this kinase interacts with dopamine receptors and members of the Rho-Nca pathway. In Aim 2, we will focus on a mitogen-activated protein kinase (MAPK) pathway that modulates Gq- Rho activation of NCA. We will identify the members of this signaling pathway and determine how they modulate output of the Gq-Rho-Nca pathway. The proposed work is significant because it will identify the signaling pathways that modulate neuronal and synaptic activity via a physiologically and medically important ion channel. The proposed work is innovative because it will close gaps in our understanding of how the NALCN/NCA ion channel is activated and identify new mechanisms of regulation of this channel.

The dense-core vesicle is an organelle that releases peptide hormones, growth factors, and biogenic amines from neurons and neuroendocrine cells in response to increases in calcium. Dense-core vesicle cargos regulate a variety of biological processes including neuronal survival and development, pain sensation, blood glucose homeostasis, and synaptic plasticity. As a consequence, numerous diseases such as mood disorders, obesity, and diabetes are caused by defects in neuropeptide and hormone secretion. Thus, it is important to determine the molecular machinery and mechanisms that orchestrate the biogenesis and release of these vesicular carriers. However, in comparison to other vesicular compartments such as synaptic vesicles, little is known about the molecular mechanisms of dense-core vesicle biogenesis, trafficking, and release. Dense-core vesicles are generated at the trans-Golgi and gain their compartmental identity in a poorly defined maturation process that occurs post-Golgi, but few molecules have been identified that function in these processes. Cargo sorting to dense-core vesicles remains a puzzle. The long-term goal of this project is to understand the molecular mechanisms by which dense-core vesicles are formed, sort cargos, and gain their compartmental identity. A first step towards this goal is to identify molecules required for dense-core vesicle biogenesis. We performed a genetic screen in the nematode C. elegans for mutants defective in dense-core vesicle function and identified a number of new proteins that act in dense-core vesicle biogenesis, including the small GTPase RAB-2 and CCCP-1, a RAB-2 effector, as well as the EARP endosomal trafficking complex. In this project, we will test how RAB-2 and its effectors interact with EARP to mediate cargo sorting to dense-core vesicles. In Aims 1 and 2, we will use genetic, biochemical and cell biological approaches in C. elegans and in the mammalian insulin-secreting cell line INS-1 832/13 to determine how and where the RAB-2 and EARP complexes are localized, whether they physically and functionally interact, and determine their precise roles in the sorting, processing, and secretion of dense-core vesicle cargos. In Aim 3, we will use a combination of in vitro biochemical approaches and in vivo functional assays to determine how the golgin-like coiled-coil protein CCCP-1 binds membranes and functions to mediate dense-core vesicle biogenesis. These studies will advance our understanding of how dense-core vesicles are generated and sort cargos, and provide general insights into mechanisms of membrane trafficking and cargo sorting controlled by multisubunit complexes that mediate trafficking between endosomal compartments and the trans-Golgi network.

Project summary The dense-core vesicle is an organelle that releases peptide hormones, growth factors, and biogenic amines from neurons and neuroendocrine cells in response to increases in calcium. Dense-core vesicle cargos regulate a variety of biological processes including neuronal survival and development, pain sensation, blood glucose homeostasis, and synaptic plasticity. As a consequence, numerous diseases such as mood disorders, obesity, and diabetes are caused by defects in neuropeptide and hormone secretion. Thus, it is important to determine the molecular machinery and mechanisms that orchestrate the biogenesis and release of these vesicular carriers. However, in comparison to other vesicular compartments such as synaptic vesicles, little is known about the molecular mechanisms of dense-core vesicle biogenesis, trafficking, and release. Dense-core vesicles are generated at the trans-Golgi and gain their compartmental identity in a poorly defined maturation process that occurs post-Golgi, but few molecules have been identified that function in these processes. Cargo sorting to dense-core vesicles remains a puzzle. The long-term goal of this project is to understand the molecular mechanisms by which dense-core vesicles are formed, sort cargos, and gain their compartmental identity. A first step towards this goal is to identify molecules required for dense-core vesicle biogenesis. We performed a genetic screen in the nematode C. elegans for mutants defective in dense-core vesicle function and identified a number of new proteins that act in dense-core vesicle biogenesis, including the small GTPase RAB-2 and CCCP-1, a RAB-2 effector, as well as the EARP endosomal trafficking complex. In this project, we will test how RAB-2 and its effectors interact with EARP to mediate cargo sorting to dense-core vesicles. In Aims 1 and 2, we will use genetic, biochemical and cell biological approaches in C. elegans and in the mammalian insulin-secreting cell line INS-1 832/13 to determine how and where the RAB-2 and EARP complexes are localized, whether they physically and functionally interact, and determine their precise roles in the sorting, processing, and secretion of dense-core vesicle cargos. In Aim 3, we will use a combination of in vitro biochemical approaches and in vivo functional assays to determine how the golgin-like coiled-coil protein CCCP-1 binds membranes and functions to mediate dense-core vesicle biogenesis. These studies will advance our understanding of how dense-core vesicles are generated and sort cargos, and provide general insights into mechanisms of membrane trafficking and cargo sorting controlled by multisubunit complexes that mediate trafficking between endosomal compartments and the trans-Golgi network.