B Grant et al. (1999) International C. elegans Meeting "Receptor-Mediated Endocytosis in C. elegans"

L Pedraza, DH Hall, B Grant, Y Zhang, D Hirsh

[International C. elegans Meeting, 1999]

Endocytosis, the vesicle-mediated internalization of membrane or membrane bound macromolecules, mediates many key aspects of cellular function and development. These functions include uptake of nutrients, synaptic recycling, membrane turnover, and regulation of growth-factor receptor signaling. Although the basic kinetics and morphology of endocytic processes have been well described, many questions remain about the identity and function of the proteins which mediate endocytosis. We have identified C. elegans mutants ( r eceptor- m ediated e ndocytosis genes) in which endocytosis of a YP170(yolk protein)::GFP fusion by oocytes is defective. We describe here two of these genes: rme-2 , which encodes a putative receptor for yolk proteins, and rme-1 , which encodes a novel endosome associated protein, which may mediate endocytic trafficking in all cells. rme-2 mutants produce slightly small oocytes which appear morphologically normal but are refractory to the uptake of YP170::GFP and endogenous yolk. We found that rme-2 mapped near a predicted member of the LDL-receptor superfamily T11F8.3, and that each of our 3 rme-2 alleles was associated with specific lesions within the T11F8.3 gene. Furthermore, T11F8.3(RNAi) closely phenocopied the rme-2 mutant phenotype. Affinity purified antisera against the predicted intracellular and extracellular domains of RME-2 specifically stained the oocyte plasma membrane. Under certain circumstances RME-2 and YP170::GFP can co-localize to the same endocytic vesicles within oocytes. Furthermore, muscle cells forced to ectopically express RME-2 bound YP170::GFP, unlike wild-type muscle. These findings indicate that RME-2 is the C. elegans yolk receptor. We have also analyzed the rme-1 gene, which encodes a member of a new class of EH domain protein conserved through evolution. rme-1 mutants display reduced yolk uptake by oocytes, and accumulate large vacuolar structures in intestine and hypodermis. We find that RME-1 is a widely expressed cytoplamic protein associated with endosomal membranes. Experiments are underway to analyze rme-1 function in the endosomes of the intestine, and to study the rme-1 phenotype by electron microscopy.

Swords, S. et al. (2019) International Worm Meeting "Using BioID as a tool to explore the endosomal interactome of RME-8 and SNX-1 in C. elegans."

Swords, S., Grant, B.

[International Worm Meeting, 2019]

BioID is a technique that uses fusion to a promiscuous biotin ligase to covalently label proteins in the immediate vicinity of a protein of interest. When biotin is supplied in excess, the promiscuous biotin ligase will biotinylate proteins interacting or proximal to the protein of interest to which it is fused. By exploiting the strength of the streptavidin-biotin interaction, affinity purification can be used to pull down all biotinylated proteins in the sample and mass spectrometry (MS) analysis can be performed to identify interactors of the protein of interest. This method has been used in cell culture lines extensively, but has not been implemented to define a protein's interaction space in an intact multicellular organism. I aim to optimize this system so that BioID can be used to identify novel interactors of C. elegans RME-8, a protein involved in cargo trafficking, along with other proteins that our lab is interested in such as RME-1 and SNX-1. C. elegans lines were created that expressed C. elegans optimized BioID2 fused to GFP and our proteins of interest. After adapting a cell-culture oriented protocol from Gingras & Hesketh to C. elegans, western blotting showed that BioID2::GFP::SNX-1 biotinylated endogenous RME-8, a known interactor. However, initial mass spectometry indicated that the rate of biotinylation was not sufficient to outcompete the noise generated by biotinylated proteins that are targets of C. elegans' endogenous biotin ligase protein, BPL-1. Unfortunately bpl-1 mutant animals are virtually sterile, and thus cannot be used in these experiments. Our new experiments are focused on a new version of the promiscuous biotin ligase called turboID, with much higher (>10x) enzymatic activity than BioID2.

Rodriguez, W. et al. (2019) International Worm Meeting "Syndapin interacting proteins in recycling endosome function."

Rodriguez, W., Grant, B.

[International Worm Meeting, 2019]

Membrane transport and transmembrane protein trafficking is essential for the maintenance of cell and tissue architecture. Endocytic recycling, or the selective return of internalized macromolecules to the cell surface from endosomes, is a key process for cell and tissue function, but is poorly understood compared to other aspects of the endocytic pathway. We seek a better understanding of the underlying molecular mechanisms that drive endocytic recycling. To achieve this, much of our work focuses on in vivo studies in the model organism C. elegans, using advanced genetics and cell biology to characterize the function of proteins involved in endocytic recycling. Many of our studies focus on endocytic recycling in a system that our laboratory first pioneered, the C. elegans intestine. This system allows the analysis of trafficking mechanisms in the context of a living epithelial tube, with native polarity, and in its normal physiological context within the intact animal. Importantly, we have identified a key recycling regulator, the F-BAR protein SDPN-1/Syndapin/PACSIN, required for endocytic recycling, with apparent effects on separation of recycling endosomes and early endosomes. Our objective is to determine the mechanism by which SDPN-1 interacting proteins control endocytic recycling. We are taking advantage of previous work of the C. elegans SH3-ome project, following up on specific binding partners of the SDPN-1 SH3 domain to define the SDPN-1 mechanism of action.

Malini Jagdeo et al. (2001) International C. elegans Meeting "NOVEL HELIX-LOOP-HELIX PROTEINS IN CAENORHABDITIS ELEGANS."

Malini Jagdeo, Tracee McMiller, Casonya M Johnson

[International C. elegans Meeting, 2001]

Helix-loop-helix (HLH) proteins are transcription factors that are involved in cellular specification, differentiation, and differential gene expression in development. Normal pancreatic functioning, brain and eye morphogenesis, and skeletal muscle development are just some aspects of human development that are regulated by HLH proteins. The nematode, Caenorhabditis elegans, is an ideal model to study myogenic related HLH proteins since it has a simple genetic system and is known to produce numerous transcriptional regulators. After identifying six DNA sequences that are likely to encode myogenic HLH genes in C. elegans, we used RT-PCR, RNA interference, and promoter fusions to determine which of the genes are expressed, and the timing and localization of their expression. (Research supported by NSF grant # MCB9986640 and by MBRS/RISE grant # R25GM58094)

Petr Liby et al. (2006) European Worm Meeting "Inhibition of bir-1, the Homologue of Human Survivin, Induces Changes of Expression of Developmentally Active Collagen Genes in L1 Larval Stage"

Marta Kostrouchova, Eva Brozova, Marketa Kostrouchova, Michal Pohludka, Jaroslav Vohanka, Petr Liby, Zdenek Kostrouch

[European Worm Meeting, 2006]

Petr Liby1, Marketa Kostrouchova1, Michal Pohludka1, Jaroslav Vohanka2, Eva Brozova2, Marta Kostrouchova2 and Zdenek Kostrouch1. BIR-1 is a member of the Inhibitors of Apoptosis protein family that is involved in the regulation of microtubule organization, condensation of chromosomes and cell division in C. elegans as well as in vertebrates. In C. elegans, bir-1 is organized in an operon together with SKIP, a transcription and splicing cofactor. bir-1 inhibition induces developmental defects including dpy and egl phenotypes. Here we studied the effect of bir-1 inhibition by RNAi in L1 larval stage using whole genome microarrays (Affymetrix). Microarrays detected changes in expression of several developmentally active collagen genes in bir-1 inhibited larvae. We further characterized the effect of bir-1 inhibition on selected collagen gene expression and induction of dpy phenotype. Acknowledgement: We thank Drs. A. Fire for GFP construct, vectors and host used in RNAi and M.W. Krause for support and advice. We thank the NIDDK Microarrays facility for performing the microarrays analysis. The work was supported by the grant 303/03/0333 from the Czech Science Foundation, by the grant NC-7554 from the Ministry of Health of the Czech Republic and by the grant 0021620806 from the Ministry of Education, Youth and Sports of the Czech Republic.

Pant, S. et al. (2009) International Worm Meeting "AMPH-1/Amphiphysin/BIN1: A Novel Regulator of Endocytic Recycling."

Patel, K., Caplan, S., Carr, C., Grant, B., Pant, S., Sharma, M.

[International Worm Meeting, 2009]

Receptor-mediated endocytosis is crucial for the internalization of receptor-bound ligands of many types. Cargo-laden internalized vesicles fuse with early endosomes. Endosome maturation allows sorting with degradation in the late endosomes and lysosomes or recycling to the plasma membrane to participate in further rounds of endocytosis. Endocytic recycling maintains cellular homeostasis of membrane and lipid components and in polarized cells allows accurate sorting to maintain distinct apical and basolateral domains. The Grant Lab utilizes Caenorhabditis elegans as a model system to uncover the molecular players involved in endocytic recycling. We have previously established that the RME-1 protein is found on the endocytic recycling compartment and mediates endocytic recycling in several C. elegans tissues. RME-1 is a conserved protein homologous to four mammalian proteins EHD1-EHD4. The protein is an ATPase with an ATP binding N-terminal P-loop that is critical for membrane association and homo-oligomerization. The recent EHD2 crystal structure reveals that, while being ATPases, EHD/RME-1 family proteins bear structural similarity to the clathrin-coated pit GTPase Dynamin. The C-termini of RME-1 family proteins contain a single Eps15-homology (EH) domain. Classically, EH domains function as protein interaction motifs in endocytosis proteins, binding target proteins through Asparagine-Proline-Phenylalanine (NPF) motifs. Mammalian EHD proteins bind to Syndapin, Rabenosyn5, EHBP1, and Numb in this manner.Utilizing an RNAi based approach in a GFP-RME-1 expressing worm strain, we screened for proteins causing alteration in recycling endosome morphology. We focused on predicted worm proteins bearing multiple NPF motifs. Through this screen we identified C. elegansAMPH-1/Amphiphysin as a novel regulator of endocytic recycling. Utilizing genetic analysis we establish a novel role for AMPH-1 in the regulation of recycling endosome morphology and recycling of transmembrane cargo. We demonstrate that endogenous RME-1 colocalizes with and associates with endogenous AMPH-1, and this association has a role in recycling. We show that in vitro recombinant purified RME-1 and AMPH-1 bind and tubulate phosphatidylserine liposomes independently, but their properties are distinct when in complex. We propose that the AMPH-1/RME-1 complex functions at the recycling endosome, promoting tubulation and/or scission of cargo carriers destined for the plasma membrane. We show phylogenetic conservation of the role of Amphiphysin family proteins in endosome function using siRNA analysis in HeLa cells.

Fluet A et al. (1998) West Coast Worm Meeting "AGGREGATION AND TOXICITY OF HUMAN b-AMYLOID PEPTIDE VARIANTS EXPRESSED IN C. ELEGANS"

Fluet A, Johnson CJ, Fay DS, Link CD

[West Coast Worm Meeting, 1998]

We have engineered transgenic C. elegans that express the human b-amyloid peptide, which appears to play a central role in the pathology of Alzheimer's disease. As described previously, these worms, which express the peptide under the control of the unc-54 promoter, are unhealthy, have larval vacuoles, and exhibit a progressive paralysis. To better understand the molecular mechanism of b peptide toxicity, we would like to determine which, if any, of these phenotypes are specific to the expression of b peptide, and which are non-specific effects resulting from overexpression of a foreign peptide. To address this question we have sought to develop non-toxic b peptide variants, which should 'unmask' the non-specific phenotypes. We have specifically attempted to design b peptide variants that are incapable of aggregating into amyloid fibers, which have previously been reported to be directly correlated with b peptide toxicity. Single amino acid changes in the b(1-42) peptide have provided us with two interesting variants that both fail to produce amyloid aggregates: L17P and M35C. Intriguingly, multiple L17P transgenic lines all show the progressive paralysis observed in strains expressing wild-type b peptide. All M35C lines, however, are significantly healthier than the strains expressing wild-type b peptide. While the L17P variant may allow us to differentiate between b peptide's ability to form aggregates and its toxicity, the M35C variant is currently being used to differentiate between specific and non-specific toxicity resulting from b peptide expression. We have also isolated a second non-aggregating version of the b peptide, the single-chain b dimer. Like the M35C variant, this version of the b peptide does not aggregate and animals that express it are healthier than those expressing the wild-type b peptide. Quantitative immunoblot analyses on these strains demonstrate that the differences we see in aggregation of the variants are due to qualitative differences in the peptides and not quantitative differences in their levels of expression. These results also suggest that the progressive paralysis phenotype is specifically due to b peptide toxicity. We are currently using the non- or less-toxic b peptide variants in conjunction with the wild-type b peptide to look for molecular correlates of b peptide toxicity. We have preliminary results from immunoblots of Hsp16 expression that suggest that this heat shock protein is upregulated in response to b amyloid expression, and that this upregulation is significantly reduced in lines expressing the non-aggregating b dimer.

Garriga, G. et al. (2019) International Worm Meeting "Quantitative study of dopaminergic phenotypes via computer-aided video analysis."

Nelms, B., Garriga, G.

[International Worm Meeting, 2019]

Dopamine is an important neurotransmitter that regulates a myriad of brain functions ranging from motor control to memory and learning. Dysregulation of dopamine is associated with multiple disorders including Parkinson's disease (PD), attention deficit hyperactivity disorder (ADHD), addiction, and schizophrenia, among others. In order to better understand dopamine function at the molecular level, we are investigating the roles and regulation of dopaminergic genes using Caenorhabditis elegans (C. elegans) as a model system. We are conducting swimming induced paralysis (SWIP) assays using a computer-aided video analysis system, allowing us to collect more nuanced data to uncover new phenotypes and discover roles for genes involved in dopaminergic signaling. We have selected eight genes to focus on in this study. Each of these eight genes is highly enriched in dopamine neurons, based on RNASeq data from our laboratory, and mutant strains for these genes show accelerated swimming-induced paralysis when compared to the wild-type strain, N2, a phenotype characteristic of hyperdopaminergic signaling. Based on our computer-aided video analysis, we propose to gain more insight into specific rates and patterns of paralysis of these mutant strains in an effort to identify novel genes critical for dopaminergic functioning. Funding support from NSF CREST Grant #HRD1547757, NIH BD2K R25 Grant #1R25MD010396-01, and DOE Title VII Grant MD-HBCU #P382G090004.

Wen C-h et al. (1995) International C. elegans Meeting "sel-9 AND sel-10: GENETICS, MAPPING AND CLONING"

Hubbard EJA, Wen C-h, Greenwald IS

[International C. elegans Meeting, 1995]

The lin-12 gene plays a central role in several different cell fate decisions requiring cell interactions and may encode a receptor for intercellular signals. lin-12(n676n930) is a hypomorphic allele of lin-12 at 25C. Five sel genes, including sel-9 and sel-10, were isolated in a screen for suppressors of lin-12(n676n930), and therefore may contribute to cell fate decisions mediated by lin-12 (see also abstract by Grant and Greenwald).

Marketa Kostrouchova et al. (2006) European Worm Meeting "Valproic Acid Affects Gene Expression and Development in Caenorhabditis elegans"

Zdenek Kostrouch, Marta Kostrouchova, Marketa Kostrouchova, Petr Liby

[European Worm Meeting, 2006]

Marketa Kostrouchova1, Petr Liby1, Marta Kostrouchova2 and Zdenek Kostrouch1. Valproic acid (VPA, 2-propylpentanoic acid), a drug widely used in the treatment of epilepsy and bipolar disorder, has been shown to posses anti-cancer activity. VPA suppresses tumor growth, induces tumor cell differentiation and reduces the formation of metastases. Multiple mechanisms were shown to be part of VPAs mode of action, including inhibition of histone deacetylase activity, inhibition of glycogen synthase 3 and interaction with the regulation of nuclear hormone receptors. We studied the effect of VPA on the development and gene expression in C. elegans. VPA delivered to worms by microinjections to the ovarial syncytium or by incubating worms in various concentrations of VPA (1 to 10 mM) caused a dose dependent delay of larval development and lethality in two developmental stages: in late embryogenesis and in L1 larval stage. Surprisingly, most animals were able to overcome the effect of VPA, suggesting a compensatory or balancing mechanism. Microarray experiments show that genes affected by VPA can be divided to at least three different categories suggesting multiple mechanisms to be involved in VPA mode of action. Acknowledgement: We thank Dr. M.W. Krause for support and advice. We thank the NIDDK Microarrays facility for performing the microarrays analysis. The work was supported by the grant 303/03/0333 from the Czech Science Foundation, by the grant NC-7554 from the Ministry of Health of the Czech Republic and by the grant 0021620806 from the Ministry of Education, Youth and Sports of the Czech Republic.