Neuronal control of maternal provisioning in response to social cues.

Mothers contribute cytoplasmic components to their progeny in a process called maternal provisioning. Provisioning is influenced by the parental environment, but the molecular pathways that transmit environmental cues between generations are not well understood. Here, we show that, in Caenorhabditis elegans, social cues modulate maternal provisioning to regulate gene silencing in offspring. Intergenerational signal transmission depends on a pheromone-sensing neuron and neuronal FMRFamide (Phe-Met-Arg-Phe)-like peptides. Parental FMRFamide-like peptide signaling dampens oxidative stress resistance and promotes the deposition of mRNAs for translational components in progeny, which, in turn, reduces gene silencing. This study identifies a previously unknown pathway for intergenerational communication that links neuronal responses to maternal provisioning. We suggest that loss of social cues in the parental environment represents an adverse environment that stimulates stress responses across generations.

Distinct functions and temporal regulation of methylated histone H3 during early embryogenesis.

During the first hours of embryogenesis, formation of higher-order heterochromatin coincides with the loss of developmental potential. Here, we examine the relationship between these two events, and we probe the processes that contribute to the timing of their onset. Mutations that disrupt histone H3 lysine 9 (H3K9) methyltransferases reveal that the methyltransferase MET-2 helps terminate developmental plasticity, through mono- and di-methylation of H3K9 (me1/me2), and promotes heterochromatin formation, through H3K9me3. Although loss of H3K9me3 perturbs formation of higher-order heterochromatin, embryos are still able to terminate plasticity, indicating that the two processes can be uncoupled. Methylated H3K9 appears gradually in developing C. elegans embryos and depends on nuclear localization of MET-2. We find that the timing of H3K9me2 and nuclear MET-2 is sensitive to rapid cell cycles, but not to zygotic genome activation or cell counting. These data reveal distinct roles for different H3K9 methylation states in the generation of heterochromatin and loss of developmental plasticity by MET-2, and identify the cell cycle as a crucial parameter of MET-2 regulation.

Regulated nuclear accumulation of a histone methyltransferase times the onset of heterochromatin formation in C. elegans embryos.

Heterochromatin formation during early embryogenesis is timed precisely, but how this process is regulated remains elusive. We report the discovery of a histone methyltransferase complex whose nuclear accumulation and activation establish the onset of heterochromatin formation in Caenorhabditis elegans embryos. We find that the inception of heterochromatin generation coincides with the accumulation of the histone H3 lysine 9 (H3K9) methyltransferase MET-2 (SETDB) into nuclear hubs. The absence of MET-2 results in delayed and disturbed heterochromatin formation, whereas accelerated nuclear localization of the methyltransferase leads to precocious H3K9 methylation. We identify two factors that bind to and function with MET-2: LIN-65, which resembles activating transcription factor 7-interacting protein (ATF7IP) and localizes MET-2 into nuclear hubs, and ARLE-14, which is orthologous to adenosine 5'-diphosphate-ribosylation factor-like 14 effector protein (ARL14EP) and promotes stable association of MET-2 with chromatin. These data reveal that nuclear accumulation of MET-2 in conjunction with LIN-65 and ARLE-14 regulates timing of heterochromatin domains during embryogenesis.

Sur7 promotes plasma membrane organization and is needed for resistance to stressful conditions and to the invasive growth and virulence of Candida albicans.

The human fungal pathogen Candida albicans causes lethal systemic infections because of its ability to grow and disseminate in a host. The C. albicans plasma membrane is essential for virulence by acting as a protective barrier and through its key roles in interfacing with the environment, secretion of virulence factors, morphogenesis, and cell wall synthesis. Difficulties in studying hydrophobic membranes have limited the understanding of how plasma membrane organization contributes to its function and to the actions of antifungal drugs. Therefore, the role of the recently discovered plasma membrane subdomains termed the membrane compartment containing Can1 (MCC) was analyzed by assessing the virulence of a sur7Δ mutant. Sur7 is an integral membrane protein component of the MCC that is needed for proper localization of actin, morphogenesis, cell wall synthesis, and responding to cell wall stress. MCC domains are stable 300-nm-sized punctate patches that associate with a complex of cytoplasmic proteins known as an eisosome. Analysis of virulence-related properties of a sur7Δ mutant revealed defects in intraphagosomal growth in macrophages that correlate with increased sensitivity to oxidation and copper. The sur7Δ mutant was also strongly defective in pathogenesis in a mouse model of systemic candidiasis. The mutant cells showed a decreased ability to initiate an infection and greatly diminished invasive growth into kidney tissues. These studies on Sur7 demonstrate that the plasma membrane MCC domains are critical for virulence and represent an important new target for the development of novel therapeutic strategies. IMPORTANCECandida albicans, the most common human fungal pathogen, causes lethal systemic infections by growing and disseminating in a host. The plasma membrane plays key roles in enabling C. albicans to grow in vivo, and it is also the target of the most commonly used antifungal drugs. However, plasma membrane organization is poorly understood because of the experimental difficulties in studying hydrophobic components. Interestingly, recent studies have identified a novel type of plasma membrane subdomain in fungi known as the membrane compartment containing Can1 (MCC). Cells lacking the MCC-localized protein Sur7 display broad defects in cellular organization and response to stress in vitro. Consistent with this, C. albicans cells lacking the SUR7 gene were more susceptible to attack by macrophages than cells with the gene and showed greatly reduced virulence in a mouse model of systemic infection. Thus, Sur7 and other MCC components represent novel targets for antifungal therapy.

Recognition of yeast by murine macrophages requires mannan but not glucan.

The first barrier against infection by Candida albicans involves fungal recognition and destruction by phagocytic cells of the innate immune system. It is well established that interactions between different phagocyte receptors and components of the fungal cell wall trigger phagocytosis and subsequent immune responses, but the fungal ligands mediating the initial stage of recognition have not been identified. Here, we describe a novel assay for fungal recognition and uptake by macrophages which monitors this early recognition step independently of other downstream events of phagocytosis. To analyze infection in live macrophages, we validated the neutrality of a codon-optimized red fluorescent protein (yEmRFP) biomarker in C. albicans; growth, hyphal formation, and virulence in infected mice and macrophages were unaffected by yEmRFP production. This permitted a new approach for studying phagocytosis by carrying out competition assays between red and green fluorescent yeast cells to measure the efficiency of yeast uptake by murine macrophages as a function of dimorphism or cell wall defects. These competition experiments demonstrate that, given a choice, macrophages display strong preferences for phagocytosis based on genus, species, and morphology. Candida glabrata and Saccharomyces cerevisiae are taken up by J774 macrophage cells more rapidly than C. albicans, and C. albicans yeast cells are favored over hyphal cells. Significantly, these preferences are mannan dependent. Mutations that affect mannan, but not those that affect glucan or chitin, reduce the uptake of yeast challenged with wild-type competitors by both J774 and primary murine macrophages. These results suggest that mannose side chains or mannosylated proteins are the ligands recognized by murine macrophages prior to fungal uptake.

Hetero-oligomeric interactions between early glycosyltransferases of the dolichol cycle.

N-Linked glycosylation begins with the formation of a dolichol-linked oligosaccharide in the endoplasmic reticulum (ER). The first two steps of this pathway lead to the formation of GlcNAc(2)-PP-dolichol, whose synthesis is sequentially catalyzed by the Alg7p GlcNAc phosphotransferase and by the dimeric Alg13p/Alg14p UDP-GlcNAc transferase on the cytosolic face of the endoplasmic reticulum. Here, we show that the Alg7p, Alg13p, and Alg14p glycosyltransferases form a functional multienzyme complex. Coimmunoprecipitation and gel filtration assays demonstrate that the Alg7p/Alg13p/Alg14p complex is a hexamer with a native molecular weight of approximately 200 kDa and an Alg7p:Alg13:Alg14p stoichiometry of 1:1:1. These results highlight and extend the striking parallels that exist between these eukaryotic UDP-GlcNAc transferases and their bacterial MraY and MurG homologs that catalyze the first two steps of the lipid-linked peptidoglycan precursor. In addition to their preferred substrate and lipid acceptors, these enzymes are similar in their structure, chemistry, temporal, and spatial organization. These similarities point to an evolutionary link between the early steps of N-linked glycosylation and those of peptidoglycan synthesis.

A new purple fluorescent color marker for genetic studies in Saccharomyces cerevisiae and Candida albicans.

The ability to visualize cellular events by linking them to color or fluorescence changes has been an invaluable tool for biology. We describe a novel plasmid-borne color marker whose expression in yeast leads to purple-colored cells that are also brightly fluorescent. This dominant marker provides a useful tool for rapidly screening plasmid maintenance using a visual or fluorescence assay in both Saccharomyces cerevisiae and Candida albicans.

Membrane topology of the Alg14 endoplasmic reticulum UDP-GlcNAc transferase subunit.

N-linked glycosylation begins in the endoplasmic reticulum with the synthesis of a highly conserved dolichol-linked oligosaccharide precursor. The UDP-GlcNAc glycosyltransferase catalyzing the second sugar addition of this precursor consists in most eukaryotes of at least two subunits, Alg14 and Alg13. Alg14 is a membrane protein that recruits the soluble Alg13 catalytic subunit from the cytosol to the face of the endoplasmic reticulum (ER) membrane where this reaction occurs. Here, we investigated the membrane topology of Saccharomyces cerevisiae Alg14 and its requirements for ER membrane association. Alg14 is predicted by most algorithms to contain one or more transmembrane spanning helices (transmembrane domains (TMDs)). We provide evidence that Alg14 contains a C-terminal cytosolic tail and an N terminus that resides within the ER lumen. However, we also demonstrate that Alg14 lacking this TMD is functional and remains peripherally associated with ER membranes, suggesting that additional domains can mediate ER association. These conclusions are based on the functional analysis of Alg13/Alg14 chimeras containing Alg13 fused at either end of Alg14 or truncated Alg14 variants lacking the predicted TMD; protease protection assays of Alg14 in intact ER membranes; and extraction of Alg14-containing ER membranes with high pH. These yeast Alg13-Alg14 chimeras recapitulate the phylogenetic diversity of Alg13-Alg14 domain arrangements that evolved in some protozoa. They encode single polypeptides containing an Alg13 domain fused to Alg14 domain in either orientation, including those lacking the Alg14 TMD. Thus, this Alg13-Alg14 UDP-GlcNAc transferase represents an unprecedented example of a bipartite glycosyltransferase that evolved by both fission and fusion.

ERS1 encodes a functional homologue of the human lysosomal cystine transporter.

Cystinosis is a lysosomal storage disease caused by an accumulation of insoluble cystine in the lumen of the lysosome. CTNS encodes the lysosomal cystine transporter, mutations in which manifest as a range of disorders and are the most common cause of inherited renal Fanconi syndrome. Cystinosin, the CTNS product, is highly conserved among mammals. Here we show that the yeast Ers1 protein and cystinosin are functional orthologues, despite sharing only limited sequence homology. Ers1 is a vacuolar protein whose loss of function results in growth sensitivity to hygromycin B. This phenotype can be complemented by the human CTNS gene but not by mutant ctns alleles that were previously identified in cystinosis patients. A genetic screen for multicopy suppressors of an ers1Delta yeast strain identified a novel gene, MEH1, which is implicated in regulating Ers1 function. Meh1 localizes to the vacuolar membrane and loss of MEH1 results in a defect in vacuolar acidification, suggesting that the vacuolar environment is critical for normal ERS1 function. This genetic system has also led us to identify Gtr1 as an Meh1 interacting protein. Like Meh1 and Ers1, Gtr1 associates with vacuolar membranes in an Meh1-dependent manner. These results demonstrate the utility of yeast as a model system for the study of CTNS and vacuolar function.