Zimmer M (2009) Chem Soc Rev "GFP: from jellyfish to the Nobel prize and beyond."

Zimmer M

[Chem Soc Rev, 2009]

On December 10, 2008 Osamu Shimomura, Martin Chalfie and Roger Tsien were awarded the Nobel Prize in Chemistry for "the discovery and development of the green fluorescent protein, GFP". The path taken by this jellyfish protein to become one of the most useful tools in modern science and medicine is described. Osamu Shimomura painstakingly isolated GFP from hundreds of thousands of jellyfish, characterized the chromophore and elucidated the mechanism of Aequorean bioluminescence. Martin Chalfie expressed the protein in E. coli and C. elegans, and Roger Tsien developed a palette of fluorescent proteins that could be used in a myriad of applications.

Tokuoka SM et al. (2008) Mol Biol Cell "The mood stabilizer valproate inhibits both inositol- and diacylglycerol-signaling pathways in Caenorhabditis elegans."

Tokuoka SM, Saiardi A, Nurrish SJ

[Mol Biol Cell, 2008]

Monitoring Editor: Tom U. Martin The anti-epileptic valproate (VPA) is widely used in the treatment of bipolar disorder, although the mechanism of its action in the disorder is unclear. We show here that VPA inhibits both inositol phosphate and diacylglycerol (DAG) signaling in C.elegans. VPA disrupts two behaviors regulated by the inositol 1,4,5-Tris phosphate (IP3): defecation and ovulation. VPA also inhibits two activities regulated by DAG signaling: acetylcholine (ACh) release and egg-laying. The effects of VPA on DAG signaling are relieved by phorbol ester, a DAG analog, suggesting that VPA acts to inhibit DAG production. VPA reduces levels of DAG and IP1, but PIP2 is slightly increased, suggesting that Phospholipase C mediated hydrolysis of PIP2 to form DAG and IP3 is defective in the presence of VPA.

Gower NJ et al. (2005) Mol Biol Cell "Inositol 1,4,5-trisphosphate signaling regulates mating behavior in Caenorhabditis elegans males."

Gower NJ, Walker DS, Baylis HA

[Mol Biol Cell, 2005]

Monitoring Editor: Martin Chalfie Complex behavior requires the coordinated action of the nervous system and nonneuronal targets. Male mating in Caenorhabditis elegans consists of a series of defined behavioral steps that lead to the physiological outcomes required for successful impregnation. We demonstrate that signaling mediated by inositol 1,4,5-trisphosphate (IP3) is required at several points during mating. Disruption of IP3 receptor (itr-1) function results in dramatic loss of male fertility, due to defects in turning behavior (during vulva location), spicule insertion and sperm transfer. To elucidate the signaling pathways responsible, we knocked down the six C. elegans genes encoding phospholipase C (PLC) family members. egl-8, which encodes PLC-beta, functions in spicule insertion and sperm transfer. itr-1 and egl-8 are widely expressed in the male reproductive system. An itr-1 gain-of-function mutation rescues infertility caused by egl-8 RNA interference, indicating that egl-8 and itr-1 function together as central components of the signaling events controlling sperm transfer.

Sela N et al. (2008) Biol Direct "Transduplication resulted in the incorporation of two protein-coding sequences into the turmoil-1 transposable element of C. elegans."

Ast G, Makalowski W, Sela N, Pupko T, Stern A

[Biol Direct, 2008]

ABSTRACT: Transposable elements may acquire unrelated gene fragments into their sequences in a process called transduplication. Transduplication of protein-coding genes is common in plants, but is unknown of in animals. Here, we report that the Turmoil-1 transposable element in C. elegans has incorporated two protein-coding sequences into its inverted terminal repeat (ITR) sequences. The ITRs of Turmoil-1 contain a conserved RNA recognition motif (RRM) that originated from the rsp-2 gene and a fragment from the protein-coding region of the cpg-3 gene. We further report that an open reading frame specific to C. elegans may have been created as a result of a Turmoil-1 insertion. Mutations at the 5'' splice site of this open reading frame may have reactivated the transduplicated RRM motif. Reviewers: This article was reviewed by Dan Graur and William Martin. For the full reviews, please go to the Reviewers'' Reports section.

Qian H et al. (2008) FASEB J "Levamisole resistance resolved at the single-channel level in Caenorhabditis elegans."

Powell-Coffman JA, Robertson AP, Qian H, Martin RJ

[FASEB J, 2008]

Sydney Brenner promoted Caenorhabditis elegans as a model organism, and subsequent investigations pursued resistance to the nicotinic anthelmintic drug levamisole in C. elegans at a genetic level. These studies have advanced our understanding of genes associated with neuromuscular transmission and resistance to the antinematodal drug. In lev-8 and lev-1 mutant C. elegans, levamisole resistance is associated with reductions in levamisole-activated whole muscle cell currents. Although lev-8 and lev-1 are known to code for nicotinic acetylcholine receptor (nAChR) subunits, an explanation for why these currents get smaller is not available. In wild-type adults, nAChRs aggregate at neuromuscular junctions and are not accessible for single-channel recording. Here we describe a use of LEV-10 knockouts, in which aggregation is lost, to make in situ recordings of nAChR channel currents. Our observations provide an explanation for levamisole resistance produced by LEV-8 and LEV-1 mutants at the single-channel level.-Qian, H., Robertson, A. P., Powell-Coffman, J. A., and Martin, R. J. Levamisole resistance resolved at the single-channel level in Caenorhabditis elegans.

Squirrell JM et al. (2006) Mol Biol Cell "CAR-1, a protein that localizes with the mRNA decapping component DCAP-1, is required for cytokinesis and ER organization in Caenorhabditis elegans embryos."

Squirrell JM, Anderson P, Eggers ZT, Grimson A, Lyons GE, White JG, Luedke N, Saari B

[Mol Biol Cell, 2006]

Monitoring Editor: Martin Chalfie The division of one cell into two requires the coordination of multiple components. We describe a gene, car-1, whose product may provide a link between disparate cellular processes. Inhibition of car-1 expression in C. elegans embryos causes late cytokinesis failures: cleavage furrows ingress but subsequently regress and the spindle midzone fails to form, even though midzone components are present. The localized accumulation of membrane that normally develops at the apex of the cleavage furrow during the final phase of cytokinesis does not occur and organization of the endoplasmic reticulum is aberrant, indicative of a disruption in membrane trafficking. The car-1 gene has homologues in a number of species, including proteins that associate with RNA binding proteins. CAR-1 localizes to P granules (germ-line specific ribonucleoprotein particles) and discrete, developmentally regulated cytoplasmic foci. These foci also contain DCAP-1, a protein involved in decapping mRNAs. Thus, CAR-1, a protein likely to be associated with RNA metabolism, plays an essential role in the late stage of cytokinesis, suggesting a novel link between RNA, membrane trafficking and cytokinesis in the C. elegans embryo.

Hu J et al. (2006) Mol Biol Cell "Casein kinase II and calcineurin modulate TRPP function and ciliary localization."

Knobel KM, Barr MM, Hu J, Bae YK

[Mol Biol Cell, 2006]

Monitoring Editor: Martin Chalfie Cilia serve as sensory devices in a diversity of organisms and their defects contribute to many human diseases. In primary cilia of kidney cells, the transient receptor potential polycystin (TRPP) channels polycystin-1 (PC-1) and polycystin-2 (PC-2) act as a mechanosensitive channel, with defects resulting in autosomal dominant polycystic kidney disease. In sensory cilia of Caenorhabditis elegans male-specific neurons, the TRPPs LOV-1 and PKD-2 are required for mating behavior. The mechanisms regulating TRPP ciliary localization and function are largely unknown. We identified the regulatory subunit of the serine-theonine casein kinase II (CK2) as a binding partner of LOV-1 and human PC-1. CK2 and the calcineurin phosphatase TAX-6 modulate male mating behavior and PKD-2 ciliary localization. The phospho-defective mutant PKD-2(S534A) localizes to cilia while a phospho-mimetic PKD-2(S534D) mutant is largely absent from cilia. Calcineurin is required for PKD-2 ciliary localization, but is not essential for ciliary gene expression, ciliogenesis, or localization of cilium structural components. This unanticipated function of calcineurin may be important for regulating ciliary protein localization. A dynamic phosphorylation-dephosphorylation cycle may represent a mechanism for modulating TRPP activity, cellular sensation, and ciliary protein localization.

Glodowski DR et al. (2007) Mol Biol Cell "RAB-10 regulates glutamate receptor recycling in a cholesterol-dependent endocytosis pathway."

Rongo C, Grant BD, Glodowski DR, Chen CC, Schaefer H

[Mol Biol Cell, 2007]

Monitoring Editor: Tom U. Martin Regulated endocytosis of AMPA-type glutamate receptors (AMPARs) is critical for synaptic plasticity. However, the specific combination of clathrin-dependent and independent mechanisms that mediate AMPAR trafficking in vivo has not been fully characterized. Here we examine the trafficking of the AMPAR subunit GLR-1 in C. elegans. GLR-1 is localized on synaptic membranes, where it regulates reversals of locomotion in a simple behavioral circuit. Animals lacking RAB-10, a small GTPase required for endocytic recycling of intestinal cargo, are similar in phenotype to animals lacking LIN-10, a PDZ-domain containing protein: GLR-1 accumulates in large accretions and animals display a decreased frequency of reversals. Mutations in unc-11 (AP180) or itsn-1 (Intersectin 1), which reduce clathrin-dependent endocytosis, suppress the lin-10 but not rab-10 mutant phenotype, suggesting that LIN-10 functions after clathrin-mediated endocytosis. By contrast, cholesterol depletion, which impairs lipid raft formation and clathrin-independent endocytosis, suppresses the rab-10 but not the lin-10 phenotype, suggesting that RAB-10 functions after clathrin-independent endocytosis. Animals lacking both genes display additive GLR-1 trafficking defects. We propose that RAB-10 and LIN-10 recycle AMPARs from intracellular endosomal compartments to synapses along distinct pathways, each with distinct sensitivities to cholesterol and the clathrin-mediated endocytosis machinery.

Schmidt DJ et al. (2005) Mol Biol Cell "Functional analysis of cytoplasmic dynein heavy chain in Caenorhabditis elegans with fast-acting temperature-sensitive mutations."

Rose DJ, Schmidt DJ, Saxton MW, Strome S

[Mol Biol Cell, 2005]

Monitoring Editor: Martin Chalfie Cytoplasmic dynein, a minus-end-directed microtubule motor, has been implicated in many cellular and developmental processes. Identification of specific cellular processes that rely directly on dynein would be facilitated by a means to induce specific and rapid inhibition of its function. We have identified conditional variants of C. elegans dynein heavy chain (DHC-1) that lose function within a minute of a modest temperature upshift. Mutant embryos generated at elevated temperature show defects in centrosome separation, pronuclear migration, rotation of the centrosome/nucleus complex, bipolar spindle assembly, anaphase chromosome segregation, and cytokinesis. Our analyses of mutant embryos generated at permissive temperature and then upshifted quickly just before events of interest indicate that DHC-1 is required specifically for rotation of the centrosome/nucleus complex, for chromosome congression to a well-ordered metaphase plate, and for timely initiation of anaphase. Our results do not support the view that DHC-1 is required for anaphase B separation of spindle poles and chromosomes. A P-loop mutation identified in two independent dominant temperature-sensitive alleles of dhc-1, when engineered into the DHC1 gene of S. cerevisiae, conferred a dominant temperature-sensitive dynein loss-of-function phenotype. This suggests that temperature-sensitive mutations can be created for time-resolved function analyses of dyneins and perhaps other P-loop proteins in a variety of model systems.

Ratemi, Zakaria et al. (2019) MicroPubl Biol

Kiss, Robert S, Ratemi, Zakaria, Rocheleau, Christian E

[MicroPubl Biol, 2019]

The excessive storage of neutral lipids in lipid droplets (LDs) is a consequence of excess dietary nutrient uptake and the primary cause of major metabolic disorders, including obesity, diabetes, and atherosclerosis (Eckel et al., 2005). Several studies investigating proteins associated with the monolayer surface of LDs have identified the Rab18 GTPase as a component with a role in the regulation of fat storage and lipid mobilization (Martin et al., 2005; Ozeki et al., 2005). Rab18, a small GTPase protein localized on the surface of LDs, plays a key role in several LD-related processes, including lipogenesis, lipolysis, and lipophagy (Pulido et al., 2011; Schulze et al., 2017). Although Rab18 has been previously associated with multiple roles in lipid metabolism, including mediating the apposition of LDs to the ER, its fundamental function remains disputed (Ozeki et al., 2005). To better understand the function of Rab18 in fat control at the organismal level, we proceeded to investigate the effect of rab-18 knockout on overall lipid abundance in C. elegans.To determine if RAB-18 regulates fat storage in C. elegans, we first investigated the fat content of rab-18(ok2020) mutants compared to wild type (N2) worms by performing fixed Nile Red (NR) and Oil Red O (ORO) staining experiments under fed conditions. Each NR and ORO experiment was performed with 5 different samples of N2 and rab-18(ok2020) worms and 10 worms were analyzed from each sample. Three separate NR and ORO experiments were performed for a total of 150 images per strain. rab-18 mutants displayed a significant reduction in NR fluorescence intensity (Figure 1A, B) and ORO staining intensity (Figure 1A, C) compared to the control N2 worms. This reduction suggests a significant decrease in lipid content in rab-18(ok2020) mutants compared to N2 worms. The less pronounced reduction in the fat content of rab-18(ok2020) mutants observed in ORO experiments is attributed to the lower sensitivity of ORO in quantifying changes of lipid abundance compared to NR (Escorcia et al., 2018).The fixed NR staining experiment of wild type (N2) and rab-18(ok2020) mutant worms was also performed under short-term (6 hours) fasting conditions to determine whether the previously observed decrease in fat content of mutant worms under fed conditions was amplified or reduced under short-term fasting conditions. Moreover, fixed NR staining under fasting conditions serves as an internal control to ensure the accuracy of the staining method as the reduction in LD fat content through lipolysis upon short-term fasting is widely acknowledged in the literature (Srinivasan et al., 2015). Three separate NR staining experiments were performed and 10-15 images were captured per strain for each experiment. Representative images are shown in Figure 1D and quantified in Figure 1E. A significant reduction in NR fluorescence intensity was observed in rab-18(ok2020) mutants as compared to wild type (N2) under fasted conditions, suggesting a significant decrease in lipid content. Moreover, we demonstrate a decrease in fat content under short-term (6 hours) starving conditions in both wild type and rab-18(ok2020) mutant worms, which is in line with findings that short-term fasting depletes body fat stores due to the increased expression of ATGL (atgl-1) lipase mediating lipolysis (Srinivasan et al., 2015). Thus, RAB-18 promotes lipid storage under both fed and fasted conditions and is not required for lipolysis during short-term fasting.