A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity

We report a systematic RNA interference (RNAi) screen of 5,690 Caenorhabditis elegans genes for gene inactivations that increase lifespan. We found that genes important for mitochondrial function stand out as a principal group of genes affecting C. elegans lifespan. A classical genetic screen identified a mutation in the mitochondrial leucyl-tRNA synthetase gene (lrs-2) that impaired mitochondrial function and was associated with longer-lifespan. The long-lived worms with impaired mitochondria had lower ATP content and oxygen consumption, but differential responses to free-radical and other stresses. These data suggest that the longer lifespan of C. elegans with compromised mitochrondria cannot simply be assigned to lower free radical production and suggest a more complex coupling of metabolism and longevity.     

C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression

To verify the genome annotation and to create a resource to functionally characterize the proteome, we attempted to Gateway-clone all predicted protein-encoding open reading frames (ORFs), or the 'ORFeome,' of Caenorhabditis elegans. We successfully cloned approximately 12,000 ORFs (ORFeome 1.1), of which roughly 4,000 correspond to genes that are untouched by any cDNA or expressed-sequence tag (EST). More than 50% of predicted genes needed corrections in their intron-exon structures. Notably, approximately 11,000 C. elegans proteins can now be expressed under many conditions and characterized using various high-throughput strategies, including large-scale interactome mapping. We suggest that similar ORFeome projects will be valuable for other organisms, including humans.     

Chromosome cohesion is regulated by a clock gene paralogue TIM-1

Faithful transmission of the genome requires that a protein complex called cohesin establishes and maintains the regulated linkage between replicated chromosomes before their segregation. Here we report the unforeseen participation of Caenorhabditis elegans TIM-1, a paralogue of the Drosophila clock protein TIMELESS, in the regulation of chromosome cohesion. Our biochemical experiments defined the C. elegans cohesin complex and revealed its physical association with TIM-1. Functional relevance of the interaction was demonstrated by aberrant mitotic chromosome behaviour, embryonic lethality and defective meiotic chromosome cohesion caused by the disruption of either TIM-1 or cohesin. TIM-1 depletion prevented the assembly of non-SMC (structural maintenance of chromosome) cohesin subunits onto meiotic chromosomes; however, unexpectedly, a partial cohesin complex composed of SMC components still loaded. Further disruption of cohesin activity in meiosis by the simultaneous depletion of TIM-1 and an SMC subunit decreased homologous chromosome pairing before synapsis, revealing a new role for cohesin in metazoans. On the basis of comparisons between TIMELESS homologues in worms, flies and mice, we propose that chromosome cohesion, rather than circadian clock regulation, is the ancient and conserved function for TIMELESS-like proteins.     

Study on the Characteristics of Macromolecular Chains of Cellulose in Dilute Solutions

The characteristics of macromolecular chains of cellulose in dilute solutions are discussed. Viscometry was used for the measurement of the intrinsic viscosity [η] of four cellulose samples, which were respectively dissolved in the solvent Tri-ethylenediamine cadmium hydroxide Cadoxen (Cadoxen), Cupriethylenediamine hydroxide(Cuen), iron sodium tartrate complex(EWNN) and N,N-dimethylacetamide(DMAc)with lithium chloride(LiCI, 9% w/w). The intrinsic viscosity of the four solutions decreased in the following manner:[η]DMAc/LiCl>[η]EWNN>[η]Cuen>[η]Cadoxen. The stability of cellulose (Sample α-cellulose) dissolved in solvents Cuen and EWNN was tested by plotting the intrinsic viscosity vs. time. The values of Huggins constant K_H for the cellulose samples dissolved in solvents Cadoxen,Cuen,EWNN and DMAc/LiCl (9% w/w ) were calculated.     

Language understanding is grounded in experiential simulations: a response to Weiskopf

Several disciplines within the cognitive sciences have advanced the idea that people comprehend the actions of others, including the linguistic meanings they communicate, through embodied simulations where they imaginatively recreate the actions they observe or hear about. This claim has important consequences for theories of mind and meaning, such as that people’s use and interpretation of language emerges as a kind of bodily activity that is an essential part of ordinary cognition. Daniel Weiskopf presents several arguments against the idea that experiential simulations play a major role in immediate language use and meaning. We offer several rebuttals to Weiskopf, in which we critique his interpretation of simulation theory, present additional psycholinguistic evidence supportive of the simulation perspective, and suggest that a more traditional theory of linguistic meaning and processing has little psychological and empirical validity.     

Unraveling adaptive evolution: how a single point mutation affects the protein coregulation network

Understanding the mechanisms of evolution requires identification of the molecular basis of the multiple (pleiotropic) effects of specific adaptive mutations. We have characterized the pleiotropic effects on protein levels of an adaptive single-base pair substitution in the coding sequence of a signaling pathway gene in the bacterium Pseudomonas fluorescens SBW25. We find 52 proteomic changes, corresponding to 46 identified proteins. None of these proteins is required for the adaptive phenotype. Instead, many are found within specific metabolic pathways associated with fitness-reducing (that is, antagonistic) effects of the mutation. The affected proteins fall within a single coregulatory network. The mutation 'rewires' this network by drawing particular proteins into tighter coregulating relationships. Although these changes are specific to the mutation studied, the quantitatively altered proteins are also affected in a coordinated way in other examples of evolution to the same niche.     

Regulation of membrane trafficking and endocytosis by protein kinase C: emerging role of the pericentrion,a novel protein kinase C-dependent subset of recycling endosomes

The protein kinase C (PKC) family of isoenzymes has been shown to regulate a variety of cellular processes, including receptor desensitization and internalization, and this has sparked interest in further delineation of the roles of specific isoforms of PKC in membrane trafficking and endocytosis. Recent studies have identified a novel translocation of PKC to a juxtanuclear compartment, the pericentrion, which is distinct from the Golgi complex but epicentered on the centrosome. Sustained activation of PKC (longer than 30 min) also results in sequestration of plasma membrane lipids and proteins to the same compartment, demonstrating a global effect on endocytic trafficking. This review summarizes these studies, particularly focusing on the characterization of the pericentrion as a distinct PKC-dependent subset of recycling endosomes. We also discuss emerging insights into a role for PKC as a central hub in regulating vesicular transport pathways throughout the cell, with implications for a wide range of pathobiologic processes, e.g. diabetes and abnormal neurotransmission or receptor desensitization. Received 11 August 2006; received after revision 20 September 2006; accepted 7 November 2006     

A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation

Signalling by the Wnt family of secreted lipoproteins has essential functions in development and disease. The canonical Wnt/beta-catenin pathway requires a single-span transmembrane receptor, low-density lipoprotein (LDL)-receptor-related protein 6 (LRP6), whose phosphorylation at multiple PPPSP motifs is induced upon stimulation by Wnt and is critical for signal transduction. The kinase responsible for LRP6 phosphorylation has not been identified. Here we provide biochemical and genetic evidence for a 'dual-kinase' mechanism for LRP6 phosphorylation and activation. Glycogen synthase kinase 3 (GSK3), which is known for its inhibitory role in Wnt signalling through the promotion of beta-catenin phosphorylation and degradation, mediates the phosphorylation and activation of LRP6. We show that Wnt induces sequential phosphorylation of LRP6 by GSK3 and casein kinase 1, and this dual phosphorylation promotes the engagement of LRP6 with the scaffolding protein Axin. We show further that a membrane-associated form of GSK3, in contrast with cytosolic GSK3, stimulates Wnt signalling and Xenopus axis duplication. Our results identify two key kinases mediating Wnt co-receptor activation, reveal an unexpected and intricate logic of Wnt/beta-catenin signalling, and illustrate GSK3 as a genuine switch that dictates both on and off states of a pivotal regulatory pathway.     

Nephrocystin-5, a ciliary IQ domain protein, is mutated in Senior-Loken syndrome and interacts with RPGR and calmodulin

Nephronophthisis (NPHP) is the most frequent genetic cause of chronic renal failure in children. Identification of four genes mutated in NPHP subtypes 1-4 (refs. 4-9) has linked the pathogenesis of NPHP to ciliary functions. Ten percent of affected individuals have retinitis pigmentosa, constituting the renal-retinal Senior-Loken syndrome (SLSN). Here we identify, by positional cloning, mutations in an evolutionarily conserved gene, IQCB1 (also called NPHP5), as the most frequent cause of SLSN. IQCB1 encodes an IQ-domain protein, nephrocystin-5. All individuals with IQCB1 mutations have retinitis pigmentosa. Hence, we examined the interaction of nephrocystin-5 with RPGR (retinitis pigmentosa GTPase regulator), which is expressed in photoreceptor cilia and associated with 10-20% of retinitis pigmentosa. We show that nephrocystin-5, RPGR and calmodulin can be coimmunoprecipitated from retinal extracts, and that these proteins localize to connecting cilia of photoreceptors and to primary cilia of renal epithelial cells. Our studies emphasize the central role of ciliary dysfunction in the pathogenesis of SLSN.