Favello A et al. (1995) Methods Cell Biol "Genomic DNA sequencing methods."

Wilson RK, Hillier L, Favello A

[Methods Cell Biol, 1995]

Sequence analysis of cosmids from C. elegans and other organisms currently is best done using the random or "shotgun" strategy (Wilson et al., 1994). After shearing by sonication, DNA is used to prepare M13 subclone libraries which provide good coverage and high-quality sequence data. The subclones are assembled and the data edited using software tools developed especially for C. elegans genomic sequencing. These same tools facilitate much of the subsequent work to complete both strands of the sequence and resolve any remaining ambiguities. Analysis of the finished sequence is then accomplished using several additional computer tools including Genefinder and ACeDB. Taken together, these methods and tools provide a powerful means for genome analysis in the nematode.

Cockcroft S et al. (2016) Biochem Soc Trans "RdgB reciprocally transfers PA and PI at ER-PM contact sites to maintain PI(4,5)P2 homoeostasis during phospholipase C signalling in Drosophila photoreceptors."

Cockcroft S, Raghu P, Garner K, Gomez-Espinoza E, Yadav S

[Biochem Soc Trans, 2016]

Phosphatidylinositol (PI) is the precursor lipid for the synthesis of PI 4,5-bisphosphate [PI(4,5)P2] at the plasma membrane (PM) and is sequentially phosphorylated by the lipid kinases, PI 4-kinase and phosphatidylinositol 4-phosphate (PI4P)-5-kinase. Receptor-mediated hydrolysis of PI(4,5)P2 takes place at the PM but PI resynthesis occurs at the endoplasmic reticulum (ER). Thus PI(4,5)P2 resynthesis requires the reciprocal transport of two key intermediates, phosphatidic acid (PA) and PI between the ER and the PM. PI transfer proteins (PITPs), defined by the presence of the PITP domain, can facilitate lipid transfer between membranes; the PITP domain comprises a hydrophobic cavity with dual specificity but accommodates a single phospholipid molecule. The class II PITP, retinal degeneration typeB (RdgB) is a multi-domain protein and its PITP domain can bind and transfer PI and PA. In Drosophila photoreceptors, a well-defined G-protein-coupled phospholipase C (PLC) signalling pathway, phototransduction defects resulting from loss of RdgB can be rescued by expression of the PITP domain provided it is competent for both PI and PA transfer. We propose that RdgB proteins maintain PI(4,5)P2 homoeostasis after PLC activation by facilitating the reciprocal transport of PA and PI at ER-PM membrane contact sites.

Randazzo PA et al. (2000) Sci STKE "Molecular aspects of the cellular activities of ADP-ribosylation factors."

Hsu VW, Randazzo PA, Nie Z, Miura K

[Sci STKE, 2000]

Adenosine diphosphate-ribosylation factor (Arf) proteins are members of the Arf arm of the Ras superfamily of guanosine triphosphate (GTP)-binding proteins. Arfs are named for their activity as cofactors for cholera toxin-catalyzed adenosine diphosphate-ribosylation of the heterotrimeric G protein Gs. Physiologically, Arfs regulate membrane traffic and the actin cytoskeleton. Arfs function both constitutively within the secretory pathway and as targets of signal transduction in the cell periphery. In each case, the controlled binding and hydrolysis of GTP is critical to Arf function. The activities of some guanine nucleotide exchange factors (GEFs) and guanosine triphosphatase (GTPase)-activating proteins (GAPs) are stimulated by phosphoinositides, including phosphatidylinositol 3,4,5-trisphosphate (PIP3) and phosphatidylinositol 4,5-bisphosphate (PIP2), and phosphatidic acid (PA), likely providing both a means to respond to regulatory signals and a mechanism to coordinate GTP binding and hydrolysis. Arfs affect membrane traffic in part by recruiting coat proteins, including COPI and clathrin adaptor complexes, to membranes. However, Arf function likely involves many additional biochemical activities. Arf activates phospholipase D and phosphatidylinositol 4-phosphate 5-kinase with the consequent production of PA and PIP2, respectively. In addition to mediating Arf's effects on membrane traffic and the actin cytoskeleton, PA and PIP2 are involved in the regulation of Arf. Arf also works with Rho family proteins to affect the actin cytoskeleton. Several Arf-binding proteins suspected to be effectors have been identified in two-hybrid screens. Arf-dependent biochemical activities, actin cytoskeleton changes, and membrane trafficking may be integrally related. Understanding Arf's role in complex cellular functions such as protein secretion or cell movement will involve a description of the temporal and spatial coordination of these multiple Arf-dependent events.

Merida I et al. (2008) Biochem J "Diacylglycerol kinases: at the hub of cell signalling."

Merida I, Avila-Flores A, Merino E

[Biochem J, 2008]

DGKs (diacylglycerol kinases) are members of a unique and conserved family of intracellular lipid kinases that phosphorylate DAG (diacylglycerol), catalysing its conversion into PA (phosphatidic acid). This reaction leads to attenuation of DAG levels in the cell membrane, regulating a host of intracellular signalling proteins that have evolved the ability to bind this lipid. The product of the DGK reaction, PA, is also linked to the regulation of diverse functions, including cell growth, membrane trafficking, differentiation and migration. In multicellular eukaryotes, DGKs provide a link between lipid metabolism and signalling. Genetic experiments in Caenorhabditis elegans, Drosophila melanogaster and mice have started to unveil the role of members of this protein family as modulators of receptor-dependent responses in processes such as synaptic transmission and photoreceptor transduction, as well as acquired and innate immune responses. Recent discoveries provide new insights into the complex mechanisms controlling DGK activation and their participation in receptor-regulated processes. After more than 50 years of intense research, the DGK pathway emerges as a key player in the regulation of cell responses, offering new possibilities of therapeutic intervention in human pathologies, including cancer, heart disease, diabetes, brain afflictions and immune dysfunctions.

Reed E et al. (2018) J Neurochem "Animal models of Wilson disease."

Lutsenko S, Bandmann O, Reed E

[J Neurochem, 2018]

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism manifesting with hepatic, neurological and psychiatric symptoms. The limitations of the currently available therapy for WD (particularly in the management of neuropsychiatric disease), together with our limited understanding of key aspects of this illness (e.g. neurological vs hepatic presentation) justify the ongoing need to study WD in suitable animal models. Four animal models of WD have been established: the Long-Evans Cinnamon rat, the toxic-milk mouse, the Atp7b knockout mouse and the Labrador retriever. The existing models of WD all show good similarity to human hepatic WD and have been helpful in developing an improved understanding of the human disease. As mammals, the mouse, rat and canine models also benefit from high homology to the human genome. However, important differences exist between these mammalian models and human disease, particularly the absence of a convincing neurological phenotype. This review will first provide an overview of our current knowledge of the orthologous genes encoding ATP7B and the closely related ATP7A protein in C. elegans, Drosophila and zebrafish (Danio rerio) and then summarise key characteristics of rodent and larger mammalian models of ATP7B-deficiency. This article is protected by copyright. All rights reserved.

Cerpa W et al. (2005) Mol Aspects Med "Is there a role for copper in neurodegenerative diseases?"

Cerpa W, Inestrosa NC, Varela-Nallar L, Minniti AN, Reyes AE

[Mol Aspects Med, 2005]

Copper is an essential metal in living organisms; thus, the maintenance of adequate copper levels is of vital importance and is highly regulated. Dysfunction of copper metabolism leading to its excess or deficiency results in severe ailments. Two examples of illnesses related to alterations in copper metabolism are Menkes and Wilson diseases. Several proteins are involved in the maintenance of copper homeostasis, including copper transporters and metal chaperones. In the last several years, the beta-amyloid-precursor protein (beta-APP) and the prion protein (PrP(C)), which are related to the neurodegenerative disorders Alzheimer and prion diseases respectively, have been associated with copper metabolism. Both proteins bind copper through copper-binding domains that also have been shown to reduce copper in vitro. Moreover, this ability to reduce copper is associated with a neuroprotective effect exerted by the copper-binding domain of both proteins against copper in vivo. In addition to a functional link between copper and beta-APP or PrP(C), evidence suggests that copper has a role in Alzheimer and prion diseases. Here, we review the evidence that supports both, the role of beta-APP and PrP(C), in copper metabolism and the putative role of copper in neurodegenerative diseases.

Emmons SW (1987) Cell "Mechanisms of C. elegans development."

Emmons SW

[Cell, 1987]

What are the respective roles in multicellular development of mechansims acting at the level of the cell and mechanisms acting at the level of the cell group? It's an old question, and one that is central to the problem of developmental biology. Even early in this century it had long been debated "whether the character of growth and morphogenesis is a cause or a result of the corresponding activities on the part of the component cells individually considered" (E.B. Wilson, The Cell in Development and Heredity, Macmillan, 1925, p. 1029). The question is now being reexamined in the nematode Caenorhabditis elegans, an organism whose embryonic and postembryonic development are easily observed. Initial studies emphasized the reproducibility and, thus, the apparent cell-autonomy of development in the animal. Little flexibility in cell division patterns or differentiation was found in blastomere isolation experiments or after microsurgery with a laser beam. More recent results, however, demonstrate that cellular interactions are more important. These new results, combined with new molecular techniques that make it possible to isolate genes defined by mutations and to reintroduce cloned genes into the germ line, open the way to a molecular analysis of developmental mechanisms that are likely to be widespread in the animal kingdom.

Wang ZW (2010) J Neurosci Res "Origin of quantal size variation and high-frequency miniature postsynaptic currents at the Caenorhabditis elegans neuromuscular junction."

Wang ZW

[J Neurosci Res, 2010]

The neuromuscular junction (NMJ) of Caenorhabditis elegans has proved to be a very useful model synapse for investigating molecular mechanisms of synaptic transmission. Intriguingly, miniature postsynaptic currents (minis) at this synapse occur at an unusually high frequency (50-90 Hz in wild-type worms) and show large variation in quantal size (from <10 pA to >200 pA). It is important to understand the cellular and molecular bases for these properties of minis in order to interpret electrophysiological data from this synapse properly. Existing data suggest that several factors may contribute to the high frequency and quantal size variation, including 1) the establishment of multiple NMJs with each body-wall muscle cell, 2) diversity of postsynaptic receptors (two acetylcholine receptors and one GABA receptor), 3) association of one presynaptic site with several body-wall muscle cells, 4) effects of Ca(2+) at the presynaptic site, and 5) a possibly elevated (less negative) resting membrane potential in motoneurons. Neither the frequency nor the quantal size of minis is affected by electrical coupling of body-wall muscle cells. Furthermore, quantal size variation is not due to synchronized multivesicular release. Analyses of the C. elegans NMJ may lead to a better understanding of the mechanisms controlling the frequency and quantal size of minis of other synapses as well.