The ArfGEF GBF-1 Is Required for ER Structure,Secretion and Endocytic Transport in C. elegans

Small GTPases of the Sar/Arf family are essential to generate transport containers that mediate communication between organelles of the secretory pathway. Guanine nucleotide exchange factor (GEFs) activate the small GTPases and help their anchorage in the membrane. Thus, GEFs in a way temporally and spatially control Sar1/Arf1 GTPase activation. We investigated the role of the ArfGEF GBF-1 in C. elegans oocytes and intestinal epithelial cells. GBF-1 localizes to the cis-Golgi and is part of the t-ER-Golgi elements. GBF-1 is required for secretion and Golgi integrity. In addition, gbf-1(RNAi) causes the ER reticular structure to become dispersed, without destroying ER exit sites (ERES) because the ERES protein SEC-16 was still localized in distinct punctae at t-ER-Golgi units. Moreover, GBF-1 plays a role in receptor-mediated endocytosis in oocytes, without affecting recycling pathways. We find that both the yolk receptor RME-2 and the recycling endosome-associated RAB-11 localize similarly in control and gbf-1(RNAi) oocytes. While RAB5-positive early endosomes appear to be less prominent and the RAB-5 levels are reduced by gbf-1(RNAi) in the intestine, RAB-7-positive late endosomes were more abundant and formed aggregates and tubular structures. Our data suggest a role for GBF-1 in ER structure and endosomal traffic.     

A thaumarchaeal provirus testifies for an ancient association of tailed viruses with archaea

Archaeal viruses, or archaeoviruses, display a wide range of virion morphotypes. Whereas the majority of those morphotypes are unique to archaeal viruses, some are more widely distributed across different cellular domains. Tailed double-stranded DNA archaeoviruses are remarkably similar to viruses of the same morphology (order Caudovirales) that infect many bacterial hosts. They have, so far, only been found in one phylum of the archaea, the Euryarchaeota, which has led to controversial hypotheses about their origin. In the present paper, we describe the identification and analysis of a putative provirus present in the genome of a mesophilic thaumarchaeon. We show that the provirus is related to tailed bacterial and euryarchaeal viruses and encodes a full complement of proteins that are required to build a tailed virion. The recently discovered wide distribution of tailed viruses in Euryarchaeota and the identification of a related provirus in Thaumarchaeota, an archaeal phylum which might have branched off before the separation of Crenarchaeota and Euryarchaeota, suggest that an association of these viruses with Archaea might be more ancient than previously anticipated.     

Cortical granule exocytosis in C. elegans is regulated by cell cycle components including separase

In many organisms, cortical granules undergo exocytosis following fertilization, releasing cargo proteins that modify the extracellular covering of the zygote. We identified cortical granules in Caenorhabditis elegans and have found that degranulation occurs in a wave that initiates in the vicinity of the meiotic spindle during anaphase I. Previous studies identified genes that confer an embryonic osmotic sensitivity phenotype, thought to result from abnormal eggshell formation. Many of these genes are components of the cell cycle machinery. When we suppressed expression of several of these genes by RNAi, we observed that cortical granule trafficking was disrupted and the eggshell did not form properly. We conclude that osmotic sensitivity phenotypes occur because of defects in trafficking of cortical granules and the subsequent formation of an impermeable eggshell. We identified separase as a key cell cycle component that is required for degranulation. Separase localized to cortically located filamentous structures in prometaphase I upon oocyte maturation. After fertilization, separase disappeared from these structures and appeared on cortical granules by anaphase I. RNAi of sep-1 inhibited degranulation in addition to causing extensive chromosomal segregation failures. Although the temperature-sensitive sep-1(e2406) allele exhibited similar inhibition of degranulation, it had minimal effects on chromosome segregation. These observations lead us to speculate that SEP-1 has two separable yet coordinated functions: to regulate cortical granule exocytosis and to mediate chromosome separation.     

Analyzing gene perturbation screens with nested effects models in R and bioconductor

Nested effects models (NEMs) are a class of probabilistic models introduced to analyze the effects of gene perturbation screens visible in high-dimensional phenotypes like microarrays or cell morphology. NEMs reverse engineer upstream/downstream relations of cellular signaling cascades. NEMs take as input a set of candidate pathway genes and phenotypic profiles of perturbing these genes. NEMs return a pathway structure explaining the observed perturbation effects. Here, we describe the package nem, an open-source software to efficiently infer NEMs from data. Our software implements several search algorithms for model fitting and is applicable to a wide range of different data types and representations. The methods we present summarize the current state-of-the-art in NEMs. AVAILABILITY: Our software is written in the R language and freely avail-able via the Bioconductor project at http://www.bioconductor.org.     

Detecting hierarchical structure in molecular characteristics of disease using transitive approximations of directed graphs

MOTIVATION: Molecular diagnostics aims at classifying diseases into clinically relevant sub-entities based on molecular characteristics. Typically, the entities are split into subgroups, which might contain several variants yielding a hierarchical model of the disease. Recent years have introduced a plethora of new molecular screening technologies to molecular diagnostics. As a result molecular profiles of patients became complex and the classification task more difficult. RESULTS: We present a novel tool for detecting hierarchical structure in binary datasets. We aim for identifying molecular characteristics, which are stochastically implying other characteristics. The final hierarchical structure is encoded in a directed transitive graph where nodes represent molecular characteristics and a directed edge from a node A to a node B denotes that almost all cases with characteristic B also display characteristic A. Naturally, these graphs need to be transitive. In the core of our modeling approach lies the problem of calculating good transitive approximations of given directed but not necessarily transitive graphs. By good transitive approximation we understand transitive graphs, which differ from the reference graph in only a small number of edges. It is known that the problem of finding optimal transitive approximation is NP-complete. Here we develop an efficient heuristic for generating good transitive approximations. We evaluate the computational efficiency of the algorithm in simulations, and demonstrate its use in the context of a large genome-wide study on mature aggressive lymphomas. AVAILABILITY: The software used in our analysis is freely available from http://compdiag.uni-regensburg.de/software/transApproxs.shtml.     

ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat

In eukaryotic cells, secretion is achieved by vesicular transport. Fusion of such vesicles with the correct target compartment relies on SNARE proteins on both vesicle (v-SNARE) and the target membranes (t-SNARE). At present it is not clear how v-SNAREs are incorporated into transport vesicles. Here, we show that binding of ADP-ribosylation factor (ARF)-GTPase-activating protein (GAP) to ER-Golgi v-SNAREs is an essential step for recruitment of Arf1p and coatomer, proteins that together form the COPI coat. ARF-GAP acts catalytically to recruit COPI components. Inclusion of v-SNAREs into COPI vesicles could be mediated by direct interaction with the coat. The mechanisms by which v-SNAREs interact with COPI and COPII coat proteins seem to be different and may play a key role in determining specificity in vesicle budding.