Laura Moffat et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "PLR-1 Controls Anteroposterior Neuronal Polarity and Downregulates Wnt Signaling"

Laura Moffat, Scott Clark, Anastasia Bakoulis

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Secreted Wnts regulate anteroposterior (AP) cell migrations, axon guidance and neuronal polarity. For example, disruption of Wnt signaling inverts ALM and PLM polarity: the anterior process adopts the length, branching pattern and synaptic properties of the wild-type posterior process and vice versa. The interneuron AVG, which is located in the retrovesicular ganglion, is polarized and has a short anterior process and a long posterior process that extends along the ventral nerve cord to the tail. plr-1 mutations caused AVG AP polarity to be reversed or symmetric, similar to the effect of Wnt mutations on ALM/PLM. In addition, plr-1 mutations altered the long-range AP cell migrations of CAN and HSN. PLR-1 has a protease-associated domain, a transmembrane domain and a RING finger domain. PLR-1 belongs to a family of transmembrane E3 ubiquitin ligases, which includes GRAIL, Goliath and plant vacuolar sorting proteins, implicated in regulating membrane trafficking. We found that PLR-1 was associated with Golgi as well as early and late endosomes, supporting the idea that PLR-1 also functions in membrane trafficking. The cytoplasmic domain, which contains the RING finger motif, was both necessary and sufficient for Golgi and endosomal localization. Both plr-1 mutations affect the RING finger domain, suggesting that ubiquitin ligase activity is required for function. plr-1-gfp was expressed in the excretory canal, tail epidermis and many neurons, including AVG, CAN and HSN. AVG, CAN and HSN expression was transient and coincided with differentiation and/or migration. Using the lin-11 promoter, we showed that PLR-1 acts autonomously to control AVG AP polarity. ALM/PLM polarity was wild-type in plr-1 mutants. Surprisingly, ectopic expression of PLR-1 in the mechanosensory neurons induced highly specific Wnt-related phenotypes, including defects in ALM/PLM polarity, PVM migration and AVM axon growth, indicating that PLR-1 can downregulate Wnt signaling in responding cells. We propose that PLR-1 regulates trafficking of a transmembrane protein, such as a receptor, needed for Wnt signaling and that plr-1 polarity and migration defects result from a misregulation of signaling.

Laura Moffat et al. (2007) International Worm Meeting "PLR-1 downregulates Wnt signaling by reducing cell surface levels of Frizzled."

Laura Moffat, Anastasia Bakoulis, Scott Clark

[International Worm Meeting, 2007]

Secreted Wnt proteins are key organizers of the nervous system along the anteroposterior (AP) body axis in vertebrates and invertebrates. In C. elegans, Wnts direct the long-range AP migrations of several neuroblasts; disruption or ectopic activation of Wnt signaling results in changes in the final positions of these cells or their descendants. Wnts also establish the intrinsic AP polarity of the mechanosensory neurons ALM and PLM. In select Wnt mutants, the polarity of ALM and PLM is reversed: the anterior process adopts the length, branching pattern and synaptic characteristics of the wild-type posterior process and vice versa. Lastly, Wnts act redundantly to guide the processes of the mechanosensory neurons AVM and PVM toward the anterior. The influence of Wnts on so many aspects of neuronal development illustrates dramatically the need for precise control over when and where Wnt signaling is activated. We identified a conserved transmembrane RING finger protein, PLR-1, that governs the response to Wnts by reducing the cell surface levels of the Wnt receptor Frizzled. Loss of PLR-1 activity in the interneuron AVG causes its AP polarity to be symmetric or reversed because signaling by the Wnts CWN-1 and CWN-2 is inappropriately activated, while ectopic expression of PLR-1 blocks Wnt signaling. Frizzleds are enriched at the plasma membrane; however, when PLR-1 and Frizzled are coexpressed, Frizzled is no longer detected at the cell surface but instead is colocalized with PLR-1 in endosomes and Golgi. We propose that PLR-1 coordinates the spatial and temporal response to Wnts during neuronal development by regulating Frizzled trafficking.

Brinda C Prasad et al. (2005) International Worm Meeting "Establishment of neuronal polarity by VPS-35 and wnt signaling."

Anastasia Bakoulis, Brinda C Prasad, Scott Clark

[International Worm Meeting, 2005]

Neurons are the apotheosis of polarized cells having axonal and dendritic extensions with different morphologies, signaling properties, cytoskeletal structures and physiological functions. Polarity becomes apparent during differentiation when neurons project processes that develop distinct characteristics. Each neuron in C. elegans exhibits an intrinsic cellular polarity and projects axons with characteristic trajectories along the dorsoventral and/or anteroposterior body axes. To understand further the cellular and molecular mechanisms that polarize a neuron and shape the initial structure of its axon, we undertook a screen for mutants with defects in the touch receptor neurons: ALM, PLM, AVM and PVM. ALM and PLM are polarized along the anteroposterior body axis with long anteriorly directed axons and short or nonexistent posteriorly directed axons. We recovered 25 mutants, which defined 18 genes, with defects in cell migration, cell fate specification and axon outgrowth, pathfinding and branching. Two mutants, lin-17(zd173) and vps-35(zd163) had neuronal polarity and cell migration defects. vps-35(zd163) caused ALM anteroposterior polarity to be reversed and PLM polarity to be symmetric, while lin-17/Fz mutations caused PLM polarity to be symmetric or reversed. vps-35 animals exhibited many phenotypes characteristic of several wnt signaling mutants, including defects in the migration of HSN, ALM and QL descendents, as well as some phenotypes not associated with wnt mutants. vps-35(zd163) contains a 148 bp deletion that likely eliminates all function. We also found that mutations in several wnt signaling genes, such as mom-3/mig-14 and lin-44/wnt, conferred defects in ALM and/or PLM polarity. Thus, we conclude that wnt signaling acts to polarize ALM and PLM and that vps-35 is needed for wnt signaling. vsp-35 encodes the C. elegans homologue of yeast and human VPS35, which is a part of a retromer complex containing VPS26, VPS29 and VPS35, that mediates endosome-to-Golgi membrane protein trafficking and transcytosis. vps-35::gfp is expressed primarily in epidermis, suggesting a possible non-cell-autonomous activity in determining ALM and PLM polarity. We are currently exploring further the role of vps-35 in wnt signaling and the regulation of ALM and PLM polarity by wnt signaling.

Eleanor Allen et al. (2004) East Coast Worm Meeting "Establishment of anteroposterior neuronal polarity"

Anastasia Bakoulis, Eleanor Allen, Scott Clark, Dave Hunt

[East Coast Worm Meeting, 2004]

Each neuron type in C. elegans projects axons with characteristic trajectories along the dorsoventral and/or anteroposterior body axes. Axons extend from the cell body at distinct orientations, revealing an intrinsic cellular polarity. Although evident in a mature neuron, when or how neuronal polarity is established is unclear. In addition, the processes that direct growth cone extensions along the anteroposterior body axis are poorly understood. To gain insight into the molecular mechanisms that determine neuronal polarity and anteroposterior axon guidance, we have begun screens for mutants with defects in the growth of the AVG axons. AVG is located in the retrovesicular ganglion and projects a very short anteriorly directed process and a long posteriorly directed process that extends to the tail. AVG pioneers the right ventral nerve bundle and is thought to provide cues that promote the proper assembly and organization of the ventral nerve cord. We isolated several mutations that alter AVG axonal outgrowth. zd101 caused the anteriorly directed AVG axon to over extend and enter the nerve ring; the posteriorly directed axon was unaffected. zd101 is an allele of klp-7 , which encodes a kinesin-related protein involved in the regulation of microtubules. We had found previously that klp-7 mutations cause neurons that normally extend only a single axon to project a second axon. Together these observations indicate that the regulation of microtubules can influence both the initiation and termination of axon extension. Several mutations were recovered that cause the apparent reversal of AVG anteroposterior polarity, as defined by the lengths of anterior and posterior axons. These mutants exhibited a range of three AVG axonal phenotypes: animals with a wildtype anterior axon and a posterior axon shorter than wild type, animals with anterior and posterior processes about equal in length and animals with a very long anterior axon and a very short or no posterior axon. When the axons were about equal in length, the anterior process terminated in the nerve ring and the posterior process stopped before the vulva. However, when the posterior axon was absent and the anterior axon was very long, the long axon typically entered the nerve ring and the looped back to the tail via the dorsal cord or lateral fascicle. These observations suggest that if the neuronal polarity of AVG is completely reversed, the long axon can be guided to the tail and that the AVG guidance cues are not restricted to the ventral nerve cord. Furthermore, these cues might only influence the direction of growth and not extension per se . We also found several genes that influence the anteroposterior polarity of other neurons, suggesting that multiple processes are involved in the establishment of anteroposterior neuronal polarity.

Robinson, Ryan et al. (2011) International Worm Meeting "PLR-1 regulates Wnt signaling by reducing cell surface levels of Frizzled and CAM-1/ROR."

Clark, Scott, Robinson, Ryan

[International Worm Meeting, 2011]

Wnts are secreted signaling proteins that regulate a variety of developmental processes by interacting with different cell surface receptors, including seven-transmembrane Frizzleds and the receptor tyrosine kinase CAM-1/ROR. During formation of the C. elegans nervous system, Wnt-Frizzled signaling controls cell migration, neuronal polarity and axon guidance along the anteroposterior body axis. We identified a transmembrane RING finger protein, PLR-1, that governs the response to Wnts by reducing the cell surface levels of Frizzled (L. Moffat, R.R., A. Bakoulis & S.C., unpublished results). Loss of PLR-1 in the neuron AVG caused its anteroposterior polarity to be symmetric or reversed because signaling by the Wnt CWN-1 was inappropriately activated; while ectopic expression of PLR-1 blocked Wnt signaling. Frizzleds are enriched at the plasma membrane; however, when PLR-1 and Frizzled were coexpressed, Frizzled was no longer detected at the cell surface but instead was colocalized with PLR-1 in endosomes and Golgi. Our results indicate that PLR-1 coordinates the spatial and temporal response to Wnts during neuronal development by regulating Frizzled trafficking. Using various fluorescently tagged transmembrane protein chimeras, we found that the extracellular domain of PLR-1 was both necessary and sufficient for interaction with the extracellular cysteine rich domain (CRD) of Frizzled and that the Frizzled CRD was both necessary and sufficient for interaction with the extracellular domain of PLR-1. These experiments and others support the model that PLR-1 and Frizzled interact via their respective extracellular/luminal domains to form a complex that is targeted to endoscopes via the activity of the PLR-1 RING finger. The CRD is a conserved feature of Frizzleds and is also present in CAM-1/ROR proteins. As such, we examined whether PLR-1 affected CAM-1/ROR trafficking. Indeed, we found that PLR-1 also reduced the cell surface levels CAM-1/ROR, showing that PLR-1 regulates the trafficking of two distinct families of Wnt receptors. We are currently investigating whether PLR-1 escorts Frizzled and CAM-1/ROR to the endosome directly from the Golgi or whether PLR-1 recruits them to the endosome from the cell surface via endocytosis.