Regulators of yeast endocytosis identified by systematic quantitative analysis

Endocytosis of receptors at the plasma membrane is controlled by a complex mechanism that includes clathrin, adaptors, and actin regulators. Many of these proteins are conserved in yeast yet lack observable mutant phenotypes, which suggests that yeast endocytosis may be subject to different regulatory mechanisms. Here, we have systematically defined genes required for internalization using a quantitative genome-wide screen that monitors localization of the yeast vesicle-associated membrane protein (VAMP)/synaptobrevin homologue Snc1. Genetic interaction mapping was used to place these genes into functional modules containing known and novel endocytic regulators, and cargo selectivity was evaluated by an array-based comparative analysis. We demonstrate that clathrin and the yeast AP180 clathrin adaptor proteins have a cargo-specific role in Snc1 internalization. We additionally identify low dye binding 17 (LDB17) as a novel conserved component of the endocytic machinery. Ldb17 is recruited to cortical actin patches before actin polymerization and regulates normal coat dynamics and actin assembly. Our findings highlight the conserved machinery and reveal novel mechanisms that underlie endocytic internalization.     

The Drosophila homolog of NTF-2, the nuclear transport factor-2, is essential for immune response

Nuclear transport factor-2 (NTF-2) functions in yeast and mammalian cell culture in targeting proteins into the nucleus. The Drosophila homolog, DNTF-2, is an essential component of the nuclear import machinery, since ntf mutants are lethal. Interestingly, hypomorphic alleles show specific phenotypes. Some are viable, but the number of omatidia in the eye is severely reduced. The immune response in the Drosophila larval fat body is also affected; the three NF-κB/Rel proteins Dorsal, Dif and Relish do not target to the nucleus after infection, and, consequently, the expression of the anti-microbial peptide genes drosomycin, attacin and drosocin is severely impaired. Hence, in spite of its general requirement in many developmental processes, DNTF-2 has a higher specific requirement in the development of the eye and in the immune response. We also found that DNTF-2 interacts directly with Mbo/DNup88, which does not contain phenylalanine-glycine-rich repeats, but has been shown to function in the import of Rel proteins.     

The nuclear transport machinery as a regulator of Drosophila development

The developmental processes that give rise to the animal body plan are exceedingly complex. Model systems such as Drosophila melanogaster have yielded profound insight into roles of conserved genes and genetic pathways in development. Drosophila development begins with the formation of sperm and eggs, and proceeds through several morphologically distinct stages including development of the early embryo, larval instars, formation of pupae, and differentiation of adult tissues. The nuclear transport of proteins and RNAs represents a critical step in the regulation of gene expression during every stage of development and tissue differentiation. Studies of the nuclear transport machinery in Drosophila refute the notion that nuclear transport is strictly a housekeeping process without specific regulatory roles in development. Rather, they support the idea that the basal nuclear transport machinery has adapted during the evolution of the metazoan body plan to play important regulatory roles in key developmental events.     

The RNAi machinery controls distinct responses to environmental signals in the basal fungus Mucor circinelloides

Background

RNA interference (RNAi) is a conserved mechanism of genome defence that can also have a role in the regulation of endogenous functions through endogenous small RNAs (esRNAs). In fungi, knowledge of the functions regulated by esRNAs has been hampered by lack of clear phenotypes in most mutants affected in the RNAi machinery. Mutants of Mucor circinelloides affected in RNAi genes show defects in physiological and developmental processes, thus making Mucor an outstanding fungal model for studying endogenous functions regulated by RNAi. Some classes of Mucor esRNAs map to exons (ex-siRNAs) and regulate expression of the genes from which they derive. To have a broad picture of genes regulated by the silencing machinery during vegetative growth, we have sequenced and compared the mRNA profiles of mutants in the main RNAi genes by using RNA-seq. In addition, we have achieved a more complete phenotypic characterization of silencing mutants.

Results

Deletion of any main RNAi gene provoked a deep impact in mRNA accumulation at exponential and stationary growth. Genes showing increased mRNA levels, as expected for direct ex-siRNAs targets, but also genes with decreased expression were detected, suggesting that, most probably, the initial ex-siRNA targets regulate the expression of other genes, which can be up- or down-regulated. Expression of 50% of the genes was dependent on more than one RNAi gene in agreement with the existence of several classes of ex-siRNAs produced by different combinations of RNAi proteins. These combinations of proteins have also been involved in the regulation of different cellular processes. Besides genes regulated by the canonical RNAi pathway, this analysis identified processes, such as growth at low pH and sexual interaction that are regulated by a dicer-independent non-canonical RNAi pathway.

Conclusion

This work shows that the RNAi pathways play a relevant role in the regulation of a significant number of endogenous genes in M. circinelloides during exponential and stationary growth phases and opens up an important avenue for in-depth study of genes involved in the regulation of physiological and developmental processes in this fungal model.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1443-2) contains supplementary material, which is available to authorized users.     

Unique and Shared Functions of Nuclear Lamina LEM Domain Proteins in Drosophila

The nuclear lamina is an extensive protein network that contributes to nuclear structure and function. LEM domain (LAP2, emerin, MAN1 domain, LEM-D) proteins are components of the nuclear lamina, identified by a shared ∼45-amino-acid motif that binds Barrier-to-autointegration factor (BAF), a chromatin-interacting protein. Drosophila melanogaster has three nuclear lamina LEM-D proteins, named Otefin (Ote), Bocksbeutel (Bocks), and dMAN1. Although these LEM-D proteins are globally expressed, loss of either Ote or dMAN1 causes tissue-specific defects in adult flies that differ from each other. The reason for such distinct tissue-restricted defects is unknown. Here, we generated null alleles of bocks, finding that loss of Bocks causes no overt adult phenotypes. Next, we defined phenotypes associated with lem-d double mutants. Although the absence of individual LEM-D proteins does not affect viability, loss of any two proteins causes lethality. Mutant phenotypes displayed by lem-d double mutants differ from baf mutants, suggesting that BAF function is retained in animals with a single nuclear lamina LEM-D protein. Interestingly, lem-d double mutants displayed distinct developmental and cellular mutant phenotypes, suggesting that Drosophila LEM-D proteins have developmental functions that are differentially shared with other LEM-D family members. This conclusion is supported by studies showing that ectopically produced LEM-D proteins have distinct capacities to rescue the tissue-specific phenotypes found in single lem-d mutants. Our findings predict that cell-specific mutant phenotypes caused by loss of LEM-D proteins reflect both the constellation of LEM-D proteins within the nuclear lamina and the capacity of functional compensation of the remaining LEM-D proteins.     

The CURLY LEAF Interacting Protein BLISTER Controls Expression of Polycomb-Group Target Genes and Cellular Differentiation of Arabidopsis thaliana

Polycomb-group (Pc-G) proteins are important regulators of many developmental processes in plants and animals and repress gene expression by imparting histone H3 lysine 27 trimethylation (H3K27me3). Here, we present the identification of the novel, plant-specific Arabidopsis thaliana protein BLISTER (BLI), which interacts with the Pc-G histone methyltransferase CURLY LEAF (CLF). We map the interaction of BLI with CLF to a predicted coiled-coil domain in BLI that shares similarity with STRUCTURAL MAINTENANCE OF CHROMOSOMES proteins. BLI colocalizes with CLF in the nucleus, shows an overlapping expression pattern with CLF throughout plant development that is strongest in dividing cells, and represses a subset of Pc-G target genes. Loss of BLI results in a pleiotropic developmental mutant phenotype, indicating that BLI prevents premature differentiation. Furthermore, bli mutants exhibit severe epidermal defects, including loss of cell adhesion, outgrowth of cells, and increased cotyledon cell size. As these phenotypes have not been observed in Pc-G mutants, we propose that BLI has functions related to Pc-G proteins but can also act independently in Arabidopsis development.     

STAR, a gene family involved in signal transduction and activation of RNA

A new subfamily of KH-domain-containing RNA-binding proteins is encoded by genes that are conserved from yeast to humans. Mutations with interesting developmental phenotypes have been identified in Caemorbabditis elegans, Drosophila and mouse. It is hypothesized that these bifunctional proteins provide a rich source of interesting molecular information about development and define a new cellular pathway that links signal transduction directly to RNA metabolism.     

Looking at Cerebellar Malformations through Text-Mined Interactomes of Mice and Humans

We have generated and made publicly available two very large networks of molecular interactions: 49,493 mouse-specific and 52,518 human-specific interactions. These networks were generated through automated analysis of 368,331 full-text research articles and 8,039,972 article abstracts from the PubMed database, using the GeneWays system. Our networks cover a wide spectrum of molecular interactions, such as bind, phosphorylate, glycosylate, and activate; 207 of these interaction types occur more than 1,000 times in our unfiltered, multi-species data set. Because mouse and human genes are linked through an orthological relationship, human and mouse networks are amenable to straightforward, joint computational analysis. Using our newly generated networks and known associations between mouse genes and cerebellar malformation phenotypes, we predicted a number of new associations between genes and five cerebellar phenotypes (small cerebellum, absent cerebellum, cerebellar degeneration, abnormal foliation, and abnormal vermis). Using a battery of statistical tests, we showed that genes that are associated with cerebellar phenotypes tend to form compact network clusters. Further, we observed that cerebellar malformation phenotypes tend to be associated with highly connected genes. This tendency was stronger for developmental phenotypes and weaker for cerebellar degeneration.     

Proteomic and gene expression differences between post-diapause and subitaneous offspring phenotypes in the cyclic parthenogen <Emphasis Type="Italic">Daphnia pulex</Emphasis>

In temperate waters, post-diapause and subitaneous offspring in Daphnia vary in life history traits and metabolic rates, and presumably are adapted to the seasonal environments in which they dwell. These offspring types result from different developmental programs of the same genetic background, representing the phenomenon of phenotypic plasticity. We aimed to explore the molecular mechanism of this phenotypic plasticity in Daphnia pulex from an intermittent population by applying a high-throughput proteomic analysis and expression analysis of several genes. The study revealed 176 proteins that were differentially expressed among offspring phenotypes. Post-diapause and subitaneous females clearly differed in their upregulated protein profiles and gene expression levels. There were more upregulated proteins with oxidoreductase and binding activity in post-diapause offspring, whereas more upregulated proteins with transporter and transferase activity were seen in subitaneous offspring. In contrast to subitaneous phenotype, over 1.5-fold more of the proteins that were upregulated in post-diapause phenotype are involved in metabolism and biosynthesis. Expression levels of several selected genes linked to cellular metabolism were also higher in post-diapause females. The greatest difference, 5-fold upregulation in post-diapause compared to subitaneous offspring, was recorded for the target of rapamycin-like (TOR) protein. Expression of ribosomal proteins in this offspring phenotype was also increased. These upregulations suggest that the TOR signaling pathway is involved and may be responsible for the regulation of the developmental program underlying post-diapause and subitaneous offspring phenotypes in Daphnia. Gene regulatory patterns observed in post-diapause and subitaneous offspring were in agreement with the expectations based on previously observed organismal traits of these Daphnia offspring types.     

Evolution of developmental traits

The evolution of plant development can be studied in many different ways, each of which provides new insights into how plants have been modified over evolutionary time. DNA sequencing shows that most developmental genes are under purifying selection and that obvious adaptive change in proteins is rare. This may indicate that most change occurs in cis-regulatory sequences, that tests for detecting selection lack power, or both. Gene duplications are common and often correlate with divergence of function, as predicted by theory. Studies of gene expression illuminate similarities among structures in disparate plant groups and indicate that the same genes have been deployed repeatedly for similar developmental ends. Comparative functional studies remain uncommon, but promise to illuminate how changing proteins lead to changes in development. Precise characterization of phenotypes by studies of developmental morphology is beginning to occur in some taxonomic groups. The genetic variation necessary for morphological change must originate as allelic polymorphism within populations; such polymorphism has been identified in grasses and in sunflowers, although it is often cryptic.     

Assessment of the Requirements for Magnesium Transporters in Bacillus subtilis

Magnesium is the most abundant divalent metal in cells and is required for many structural and enzymatic functions. For bacteria, at least three families of proteins function as magnesium transporters. In recent years, it has been shown that a subset of these transport proteins is regulated by magnesium-responsive genetic control elements. In this study, we investigated the cellular requirements for magnesium homeostasis in the model microorganism Bacillus subtilis. Putative magnesium transporter genes were mutationally disrupted, singly and in combination, in order to assess their general importance. Mutation of only one of these genes resulted in strong dependency on supplemental extracellular magnesium. Notably, this transporter gene, mgtE, is known to be under magnesium-responsive genetic regulatory control. This suggests that the identification of magnesium-responsive genetic mechanisms may generally denote primary transport proteins for bacteria. To investigate whether B. subtilis encodes yet additional classes of transport mechanisms, suppressor strains that permitted the growth of a transporter-defective mutant were identified. Several of these strains were sequenced to determine the genetic basis of the suppressor phenotypes. None of these mutations occurred in transport protein homologues; instead, they affected housekeeping functions, such as signal recognition particle components and ATP synthase machinery. From these aggregate data, we speculate that the mgtE protein provides the primary route of magnesium import in B. subtilis and that the other putative transport proteins are likely to be utilized for more-specialized growth conditions.     

Conjugation genes are common throughout the genus Rickettsia and are transmitted horizontally

Rickettsia are endosymbionts of arthropods, some of which are vectored to vertebrates where they cause disease. Recently, it has been found that some Rickettsia strains harbour conjugative plasmids and others encode some conjugative machinery within the bacterial genome. We investigated the distribution of these conjugation genes in a phylogenetically diverse collection of Rickettsia isolated from arthropods. We found that these genes are common throughout the genus and, in stark contrast to other genes in the genome, conjugation genes are frequently horizontally transmitted between strains. There is no evidence to suggest that these genes are preferentially transferred between phylogenetically related strains, which is surprising given that closely related strains infect similar host species. In addition to detecting patterns of horizontal transmission between diverse Rickettsia species, these findings have implications for the evolution of pathogenicity, the evolution of Rickettsia genomes and the genetic manipulation of intracellular bacteria.