Two PI 3-Kinases and One PI 3-Phosphatase Together Establish the Cyclic Waves of Phagosomal PtdIns(3)P Critical for the Degradation of Apoptotic Cells

Cyclic oscillations in the level of phosphatidylinositol 3-phosphate in phagosomes, regulated by two phosphoinositide kinases and one phosphatase, are critical for phagosome maturation and degradation of apoptotic cells

An ALS-Linked Mutant SOD1 Produces a Locomotor Defect Associated with Aggregation and Synaptic Dysfunction When Expressed in Neurons of Caenorhabditis elegans

The nature of toxic effects exerted on neurons by misfolded proteins, occurring in a number of neurodegenerative diseases, is poorly understood. One approach to this problem is to measure effects when such proteins are expressed in heterologous neurons. We report on effects of an ALS-associated, misfolding-prone mutant human SOD1, G85R, when expressed in the neurons of Caenorhabditis elegans. Stable mutant transgenic animals, but not wild-type human SOD1 transgenics, exhibited a strong locomotor defect associated with the presence, specifically in mutant animals, of both soluble oligomers and insoluble aggregates of G85R protein. A whole-genome RNAi screen identified chaperones and other components whose deficiency increased aggregation and further diminished locomotion. The nature of the locomotor defect was investigated. Mutant animals were resistant to paralysis by the cholinesterase inhibitor aldicarb, while exhibiting normal sensitivity to the cholinergic agonist levamisole and normal muscle morphology. When fluorescently labeled presynaptic components were examined in the dorsal nerve cord, decreased numbers of puncta corresponding to neuromuscular junctions were observed in mutant animals and brightness was also diminished. At the EM level, mutant animals exhibited a reduced number of synaptic vesicles. Neurotoxicity in this system thus appears to be mediated by misfolded SOD1 and is exerted on synaptic vesicle biogenesis and/or trafficking

A Survey of New Temperature-Sensitive, Embryonic-Lethal Mutations in C. elegans: 24 Alleles of Thirteen Genes

To study essential maternal gene requirements in the early C. elegans embryo, we have screened for temperature-sensitive, embryonic lethal mutations in an effort to bypass essential zygotic requirements for such genes during larval and adult germline development. With conditional alleles, multiple essential requirements can be examined by shifting at different times from the permissive temperature of 15°C to the restrictive temperature of 26°C. Here we describe 24 conditional mutations that affect 13 different loci and report the identity of the gene mutations responsible for the conditional lethality in 22 of the mutants. All but four are mis-sense mutations, with two mutations affecting splice sites, another creating an in-frame deletion, and one creating a premature stop codon. Almost all of the mis-sense mutations affect residues conserved in orthologs, and thus may be useful for engineering conditional mutations in other organisms. We find that 62% of the mutants display additional phenotypes when shifted to the restrictive temperature as L1 larvae, in addition to causing embryonic lethality after L4 upshifts. Remarkably, we also found that 13 out of the 24 mutations appear to be fast-acting, making them particularly useful for careful dissection of multiple essential requirements. Our findings highlight the value of C. elegans for identifying useful temperature-sensitive mutations in essential genes, and provide new insights into the requirements for some of the affected loci

Profiling Synaptic Proteins Identifies Regulators of Insulin Secretion and Lifespan

Cells are organized into distinct compartments to perform specific tasks with spatial precision. In neurons, presynaptic specializations are biochemically complex subcellular structures dedicated to neurotransmitter secretion. Activity-dependent changes in the abundance of presynaptic proteins are thought to endow synapses with different functional states; however, relatively little is known about the rules that govern changes in the composition of presynaptic terminals. We describe a genetic strategy to systematically analyze protein localization at Caenorhabditis elegans presynaptic specializations. Nine presynaptic proteins were GFP-tagged, allowing visualization of multiple presynaptic structures. Changes in the distribution and abundance of these proteins were quantified in 25 mutants that alter different aspects of neurotransmission. Global analysis of these data identified novel relationships between particular presynaptic components and provides a new method to compare gene functions by identifying shared protein localization phenotypes. Using this strategy, we identified several genes that regulate secretion of insulin-like growth factors (IGFs) and influence lifespan in a manner dependent on insulin/IGF signaling

C. elegans Agrin Is Expressed in Pharynx, IL1 Neurons and Distal Tip Cells and Does Not Genetically Interact with Genes Involved in Synaptogenesis or Muscle Function

Agrin is a basement membrane protein crucial for development and maintenance of the neuromuscular junction in vertebrates. The C. elegans genome harbors a putative agrin gene agr-1. We have cloned the corresponding cDNA to determine the primary structure of the protein and expressed its recombinant fragments to raise specific antibodies. The domain organization of AGR-1 is very similar to the vertebrate orthologues. C. elegans agrin contains a signal sequence for secretion, seven follistatin domains, three EGF-like repeats and two laminin G domains. AGR-1 loss of function mutants did not exhibit any overt phenotypes and did not acquire resistance to the acetylcholine receptor agonist levamisole. Furthermore, crossing them with various mutants for components of the dystrophin-glycoprotein complex with impaired muscle function did not lead to an aggravation of the phenotypes. Promoter-GFP translational fusion as well as immunostaining of worms revealed expression of agrin in buccal epithelium and the protein deposition in the basal lamina of the pharynx. Furthermore, dorsal and ventral IL1 head neurons and distal tip cells of the gonad arms are sources of agrin production, but no expression was detectable in body muscles or in the motoneurons innervating them. Recombinant worm AGR-1 fragment is able to cluster vertebrate dystroglycan in cultured cells, implying a conservation of this interaction, but since neither of these proteins is expressed in muscle of C. elegans, this interaction may be required in different tissues. The connections between muscle cells and the basement membrane, as well as neuromuscular junctions, are structurally distinct between vertebrates and nematodes

To the Cloud! A Grassroots Proposal to Accelerate Brain Science Discovery

The revolution in neuroscientific data acquisition is creating an analysis challenge. We propose leveraging cloud-computing technologies to enable large-scale neurodata storing, exploring, analyzing, and modeling. This utility will empower scientists globally to generate and test theories of brain function and dysfunction

Retrolinkin, a membrane protein, plays an important role in retrograde axonal transport

Retrograde axonal transport plays an important role in the maintenance of neuronal functions, but the mechanism is poorly defined partly because the constituents of the retrograde transport system and their interactions have yet to be elucidated. Of special interest is how dynein/dynactin motor proteins interact with membrane cargoes. Here, we report that an endosomal vesicle protein, termed retrolinkin, functions as a receptor tethering vesicles to dynein/dynactin through BPAG1n4. Retrolinkin, a membrane protein highly enriched in neuronal endosomes, binds directly to BPAG1n4. Deletion of retrolinkin membrane-association domains disrupts retrograde vesicular transport, recapitulating the BPAG1 null phenotype. We propose that retrolinkin acts with BPAG1n4 to specifically regulate retrograde axonal transport. Our work lays the foundation for understanding fundamental issues of axonal transport and provides insights into the molecular mechanisms underlying human neurodegenerative disorders