Biology

Transport of ER Vesicles on Actin Filaments in Neurons by Myosin V

George M. Langford et al. — Tue, 19 Oct 2010 06:45:22 PDT

Axoplasmic organelles in the giant axon of the squid have been shown to move on both actin filaments and microtubules and to switch between actin filaments and microtubules during fast axonal transport. The objectives of this investigation were to identify the specific classes of axoplasmic organelles that move on actin filaments and the myosin motors involved. We developed a procedure to isolate endoplasmic reticulum (ER) from extruded axoplasm and to reconstitute its movement in vitro. The isolated ER vesicles moved on exogenous actin filaments adsorbed to coverslips in an ATP-dependent manner without the addition of soluble factors. Therefore myosin was tightly bound and not extracted during isolation. These vesicles were identified as smooth ER by use of an antibody to an ER-resident protein, ERcalcistorin/protein disulfide isomerase (EcaSt/PDI). Furthermore, an antibody to squid myosin V was used in immunogold EM studies to show that myosin V localized to these vesicles. The antibody was generated to a squid brain myosin (p196) that was classified as myosin V based on comparisons of amino acid sequences of tryptic peptides of this myosin with those of other known members of the myosin V family. Dual labeling with the squid myosin V antibody and a kinesin heavy chain antibody showed that the two motors colocalized on the same vesicles. Finally, antibody inhibition experiments were performed with two myosin V-specific antibodies to show that myosin V motor activity is required for transport of vesicles on actin filaments in axoplasm. One antibody was made to a peptide in the globular tail domain and the other to the globular head fragment of myosin V. Both antibodies inhibited vesicle transport on actin filaments by greater than 90% compared to controls. These studies provide the first direct evidence that ER vesicles are transported on actin filaments by myosin V. These data confirm the role of actin filaments in fast axonal transport and provide support for the dual filament model of vesicle transport.

Genetics of intercellular signalling in C. elegans

Judith Austin et al. — Tue, 12 Oct 2010 09:40:51 PDT

Cell-cell interactions play a significant role in controlling cell fate during development of the nematode Caenorhabditis elegans. It has been found that two genes, glp-1 and lin-12, are required for many of these decisions. glp-1 is required for induction of mitotic proliferation in the germline by the somatic distal tip cell and for induction of the anterior pharynx early in embryogenesis. lin-12 is required for the interactions between cells of equivalent developmental potential, which allow them to take on different fates. Comparison of these two genes on a molecular level indicates that they are similar in sequence and organization, suggesting that the mechanisms of these two different sets of cell-cell interactions are similar.

Identification of Genes That Interact with glp-1, a Gene Required for Inductive Cell Interactions in Caenorhabditis elegans

Eleanor M. Maine et al. — Tue, 12 Oct 2010 09:40:51 PDT

The glp-1 gene functions in two inductive cellular interactions and in development of the embryonic hypodermis of C. elegans. We have isolated six mutations as recessive suppressors of temperature-sensitive (ts) mutations of glp-1. By mapping and complementation tests, we found that these suppressors are mutations of known dumpy (dpy) genes; dpy genes are required for development of normal body shape. Based on this result, we asked whether mutations previously isolated in screens for mutants defective in body shape could also suppress glp-1(ts). From these tests, we learned that unselected mutations of eight genes required for normal C. elegans morphogenesis, including the four already identified, suppress glp-1(ts). All of these suppressors rescue all three mutant phenotypes of glp-1(ts) (defects in embryonic induction of pharyngeal tissue, in embryonic hypodermis development, and in induction of germline proliferation). However, they do not rescue putative glp-1 null mutants and therefore do not bypass the requirement for glp-1 in development. In the light of current ideas about the molecular nature of the glp-1 and suppressor gene products, we propose an interaction between the glp-1 protein and components of the extracellular matrix and speculate that this interaction may impose spatial constraints on the decision between mitosis and meiosis in the germline.

The Drosophila Female-Specific Sex-Determination Gene, Sex-Lethal, Has Stage-, Tissue-, and Sex-Specific RNAs Suggesting Multiple Modes of Regulation

Helen K. Salz et al. — Tue, 12 Oct 2010 09:40:50 PDT

For proper sexual development of females, the Sex-lethal (Sxl) gene must be activated early in development and remain on during the rest of the life cycle. Conversely, in males, Sxl must remain functionally off through development. Here, we show that the Sxl transcription unit spans a DNA segment of greater than 20 kb and encodes at least 10 distinct, but overlapping, RNA species. These RNAs range in size from 4.4 to 1.7 kb and exhibit sex, stage, and tissue specificity. Six RNAs, three female specific and three male specific, are first detected by midembryogenesis and persist through the adult stage: Their expression reflects the on/off regulation of Sxl's activity at the level of sex-specific alternate splicing. Four Sxl RNAs are found in adult females. Two of these RNAs are dependent on the presence of a functional germ line and may be relevant to Sxl's role in adult germ-line development. All four are present in unfertilized eggs. Finally, three Sxl RNAs are found only transiently during very early embryogenesis; we suggest that the expression of these RNAs may reflect an early regulation of Sxl at the level of transcription and that these transcripts are involved in the initial selection of the Sxl activity state in response to the primary sex-determination signal, the X/A ratio.

XanthusBase: Adapting Wikipedia Principles to a Model Organism Database

Bradley I. Arshinoff et al. — Fri, 08 Oct 2010 08:27:35 PDT

xanthusBase (http://www.xanthusbase.org) is the official model organism database (MOD) for the social bacterium Myxococcus xanthus. In many respects, M.xanthus represents the pioneer model organism (MO) for studying the genetic, biochemical, and mechanistic basis of prokaryotic multicellularity, a topic that has garnered considerable attention due to the significance of biofilms in both basic and applied microbiology research. To facilitate its utility, the design of xanthusBase incorporates open-source software, leveraging the cumulative experience made available through the Generic Model Organism Database (GMOD) project, MediaWiki (http://www.mediawiki.org), and dictyBase (http://www.dictybase.org), to create a MOD that is both highly useful and easily navigable. In addition, we have incorporated a unique Wikipedia-style curation model which exploits the internet’s inherent interactivity, thus enabling M.xanthus and other myxobacterial researchers to contribute directly toward the ongoing genome annotation.

Regulation of Heterochromatin Assembly on Unpaired Chromosomes during Caenorhabditis elegans Meiosis by Components of a Small RNA-Mediated Pathway

Xingyu She et al. — Wed, 29 Sep 2010 09:58:56 PDT

Many organisms have a mechanism for down regulating the expression of non-synapsed chromosomes and chromosomal regions during meiosis. This phenomenon is thought to function in genome defense. During early meiosis in Caenorhabditis elegans, unpaired chromosomes (e.g., the male X chromosome) become enriched for a modification associated with heterochromatin and transcriptional repression, dimethylation of histone H3 on lysine 9 (H3K9me2). This enrichment requires activity of the cellular RNA-directed RNA polymerase, EGO-1. Here we use genetic mutation, RNA interference, immunofluorescence microscopy, fluorescence in situ hybridization, and molecular cloning methods to identify and analyze three additional regulators of meiotic H3K9me2 distribution: CSR-1 (a Piwi/PAZ/Argonaute protein), EKL-1 (a Tudor domain protein), and DRH-3 (a DEAH/D-box helicase). In csr-1, ekl-1, and drh-3 mutant males, we observed a reduction in H3K9me2 accumulation on the unpaired X chromosome and an increase in H3K9me2 accumulation on paired autosomes relative to controls. We observed a similar shift in H3K9me2 pattern in hermaphrodites that carry unpaired chromosomes. Based on several assays, we conclude that ectopic H3K9me2 accumulates on paired and synapsed chromosomes in these mutants. We propose alternative models for how a small RNA-mediated pathway may regulate H3K9me2 accumulation during meiosis. We also describe the germline phenotypes of csr-1, ekl-1, and drh-3 mutants. Our genetic data suggest that these factors, together with EGO-1, participate in a regulatory network to promote diverse aspects of development.

Studying gene function in Caenorhabditis elegans using RNA-mediated interference

Eleanor M. Maine — Wed, 29 Sep 2010 09:58:56 PDT

The RNA interference (RNAi) method for targeted gene silencing is widely used in Caenorhabditis elegans for large-scale functional genomic studies, analysis of limited gene sets and detailed analysis of individual gene function. The application of RNAi has identified genes that participate in various aspects of development, physiology and cell biology. In addition, RNAi has been used to identify interacting genes and to study functionally redundant genes. This review discusses the various applications of RNAi in C. elegans, focusing particularly on the analysis of developmental processes.

Intragenic Dominant Suppressors of glp-1, a Gene Essential for Cell-Signaling in Caenorhabditis elegans, Support a Role for cdcl O/SWZ6/Ankyrin Motifs in GLP-1 Function

James L. Lissemore et al. — Wed, 29 Sep 2010 08:55:30 PDT

The glp-1 gene product mediates cell-cell interactions required for cell fate specification during development in Caenorhabditis elegans. To identify genes that interact with glp-1, we screened for dominant suppressors of two temperature-sensitive glp-1 alleles and recovered 18 mutations that suppress both germline and embryonic glp-1 phenotypes. These dominant suppressors are tightly linked to glp-1 and do not bypass the requirement for a distal tip cell, which is thought to be the source of a signal that is received and transduced by the GLP-1 protein. Using single-strand conformation polymorphism (SSCP) analysis and DNA sequencing, we found that at least 17 suppressors are second-site intragenic revertants. The suppressors, like the original glp-1(ts) mutations, are all located in the cdc10/SWI6/ankyrin domain of GLP-1. cdc10/SWI6/ankyrin motifs have been shown to mediate specific protein-protein interactions in other polypeptides. We propose that the glp-1(ts) mutations disrupt contact between GLP-1 and an as yet unidentified target protein(s) and that the dominant suppressor mutations restore appropriate protein-protein interactions.

Suppressors of glp-1, a Gene Required for Cell Communication During Development in Caenorhabditis elegans, Define a Set of Interacting Genes

Eleanor M. Maine et al. — Wed, 29 Sep 2010 08:55:30 PDT

The glp-1 gene is essential for two cell interactions that control cell fate in Caenorhabditis elegans: induction of anterior pharynx in the embryo and induction of mitotic proliferation in the germ line. To identify other genes involved in these cell interactions, we have isolated suppressors of two temperature sensitive alleles of glp-1. Each of 14 recessive suppressors rescues both embryonic and germline glp-1(ts) defects. These suppressors are extragenic and define a set of six genes designated sog, for suppressor of glp-1. Suppression of glp-1 is the only obvious phenotype associated with sog mutations. Mutations in different sog genes show allele-specific intergenic noncomplementation, suggesting that the sog gene products may interact. In addition, we have analyzed a semidominant mutation that suppresses only the glp-1 germline phenotype and has a conditional feminized phenotype of its own. None of the suppressors rescues a glp-1 null mutation and therefore they do not bypass a requirement for glp-1. Distal tip cell function remains necessary for germline proliferation in suppressed animals. These suppressor mutations identify genes that may encode other components of the glp-1 mediated cell-signaling pathway or regulate glp-1 expression.

Molecular Basis of Loss-of-Function Mutations in the glp-1 Gene of Caenorhabitis elegans

Voula Kodoyianni et al. — Wed, 29 Sep 2010 08:55:29 PDT

The glp-1 gene encodes a membrane protein required for inductive cell interactions during development of the nematode Caenorhabditis elegans. Here we report the molecular characterization of 15 loss-of-function (lf) mutations of glp-1. Two nonsense mutations appear to eliminate glp-1 activity; both truncate the glp-1 protein in its extracellular domain and have a strong loss-of-function phenotype. Twelve missense mutations and one in-frame deletion map to sites within the repeated motifs of the glp-1 protein (10 epidermal growth factor [EFG]-like and 3 LNG repeats extracellularly and 6 cdc10/SW16, or ankyrin, repeats intracellularly). We find that all three types of repeated motifs are critical to glp-1 function, and two individual EFG-like repeats may have distinct functions. Intriguingly, all four missense mutations in one phenotypic class map to the N-terminal EGF-like repeats and all six missense mutations in a second phenotypic class reside in the intracellular cdc10/SW16 repeats. These two clusters of mutations may identify functional domains within the glp-1 protein.

Carboxy-Terminal Truncation Activates glp-1 Protein to Specify Vulval Fates in Caenorhabditis elegans

Susan E. Mango et al. — Wed, 29 Sep 2010 08:55:29 PDT

The glp-1 and lin-12 genes encode homologous transmembrane proteins that may act as receptors for cell interactions during development. The glp-1 product is required for induction of germ-line proliferation and for embryogenesis. By contrast, lin-12 mediates somatic cell interactions, including those between the precursor cells that form the vulval hypodermis (VPCs). Here we analyse an unusual allele of glp-1, glp-1(q35), which displays a semidominant multivulva phenotype (Muv), as well as the typical recessive, loss-of-function Glp phenotypes (sterility and embryonic lethality). We find that the effects of glp-1(q35) on VPC development mimic those of dominant lin-12 mutations, even in the absence of lin-12 activity. The glp-1(q35) gene bears a nonsense mutation predicted to eliminate the 122 C-terminal amino acids, including a ProGluSerThr (PEST) sequence thought to destabilize proteins. We suggest that the carboxy terminus bears a negative regulatory domain which normally inactivates glp-1 in the VPCs. We propose that inappropriate glp-1(q35) activity can substitute for lin-12 to determine vulval fate, perhaps by driving the VPCs to proliferate.

Analysis of the Multiple Roles of gld-I in Germline Development: Interactions With the Sex Determination Cascade and the glP-1 Signaling Pathway

Ross Francis et al. — Wed, 29 Sep 2010 08:22:39 PDT

The Caenorhabditis elegansgene gld-1 is essential for oocyte development; in gld-1 (null) hermaphrodites, a tumor forms where oogenesis would normally occur. We use genetic epistasis analysis to demonstrate that tumor formation is dependent on the sexual fate of the germline. When the germlines sex determination pathway is set in the female mode (terminal fem/fog genes inactive), gld-1 (null) germ cells exit meiotic prophase and proliferate to form a tumor, but when the pathway is set in the male mode, they develop into sperm. We conclude that the gld-1 (null) phenotype is cell-type specific and that gld-1 ( + ) acts at the end of the cascade to direct oogenesis. We also use cell ablation and epistasis analysis to examine the dependence of tumor formation on the glp-I signaling pathway. Although glp-1 activity promotes tumor growth, it is not essential for tumor formation by gld-1 (null) germ cells. These data also reveal that gld-1 ( + ) plays a nonessential (and sex nonspecific) role in regulating germ cell proliferation before their entry into meiosis. Thus gld-1 ( + ) may negatively regulate proliferation at two distinct points in germ cell development: before entry into meiotic prophase in both sexes (nonessential premeiotic gld-1 function) and during meiotic prophase when the sex determination pathway is set in the female mode (essential meiotic gld-1 function).

Grassland Root Communities: Species Distributions and How They Are Linked to Aboveground Abundance.

Douglas Frank et al. — Wed, 29 Sep 2010 06:56:38 PDT

There is little comprehensive information on the distribution of root systems among coexisting species, despite the expected importance of those distributions in determining the composition and diversity of plant communities. This gap in knowledge is particularly acute for grasslands, which possess large numbers of species with morphologically indistinguishable roots. In this study we adapted a molecular method, fluorescent fragment length polymorphism, to identify root fragments and determine species root distributions in two grasslands in Yellowstone National Park. Aboveground biomass was measured and soil cores (2 cm diam) were collected to 40 cm and 90 cm in an upland, dry grassland and a mesic, slope-bottom grassland, respectively, at peak foliar expansion. Cores were subdivided and species that occurred in each 10 cm interval were identified. The results indicated that the average number of species in 10 cm intervals (31 cm3) throughout the sampled soil profile was 3.9 and 2.8 at a dry and a mesic grassland, respectively. By contrast, average species number per 0.5 m2 determined by the presence of shoot material was 6.7 and 14.1 at dry and mesic sites, respectively. There was no relationship between soil depth and number of species per 10 cm interval in either grassland, despite the exponential decline of root biomass with soil depth at both sites. There also was no relationship between root frequency (i.e., the percentage of samples in which a species occurred) and soil depth for the vast majority of species at both sites. The preponderance of species were distributed throughout the soil profile at both sites. Assembly analyses indicated that species root occurrences were randomly assorted in all soil intervals at both sites, with the exception that F. idahoensis segregated from A. tridentata and P. spicata in 10-20 cm soil at the dry grassland. Root frequency throughout the entire sampled soil profile was positively associated with shoot biomass among species. Together these results indicated the importance of large, well proliferated root systems in establishing aboveground dominance. The findings suggest that spatial belowground segregation of species probably plays a minor role in fostering resource partitioning and species coexistence in these YNP grasslands.

Caenorhabditis elegans atx-2 Promotes Germline Proliferation and the Oocyte Fate

Eleanor M. Maine et al. — Wed, 29 Sep 2010 06:56:12 PDT

In the Caenorhabditis elegans germline, proliferation is induced by Notch-type signaling. Entry of germ cells into meiosis is triggered by activity of the GLD-1 and GLD-2 pathways, which function redundantly to promote meiosis and/or inhibit proliferation. Activation of the germline Notch-type receptor, GLP-1, ultimately inhibits the activities of the GLD-1 and GLD-2 pathways. We previously identified several ego (enhancer of glp-1) genes that promote germline proliferation and interact genetically with the GLP-1 signaling pathway. Here, we show that atx-2 is an ego gene. Our data suggest that ATX-2 is not a positive regulator of the GLP-1 signaling pathway and GLP-1 signaling is not the sole positive regulator of ATX-2 activity. Moreover, our data indicate that GLP-1 must have an additional function, which may be to repress activity of a third meiotic entry pathway that would work in parallel with the GLD-1 and GLD-2 pathways. In addition to its role in proliferation, ATX-2 acts downstream of FOG-2 to promote the female germline fate.

RNAi As a Tool for Understanding Germline Development in Caenorhabditis elegans: Uses and Cautions

Eleanor M. Maine — Wed, 29 Sep 2010 06:56:11 PDT

RNA-mediated genetic interference (RNAi) has become a very useful tool for analyzing gene function in development and other processes. RNAi can be used as a complement to traditional genetic studies or as a primary means of determining biological function. However, the efficacy of RNAi depends on a variety of factors that the researcher must take into consideration. This review focuses on germline development in the nematode, Caenorhabditis elegans, and discusses the uses and limitations of RNAi in providing new information about gene function as well as the possible endogenous role RNAi plays in germline physiology.

Oogenesis in Caenorhabditis elegans

Eleanor M. Maine — Wed, 29 Sep 2010 06:56:11 PDT

Oogenesis is the process of forming the female gamete, i.e., the ovum or egg. In Caenorhabditis elegans, gametes derive from a tissue called the germ line, which is specified early in embryonic development. Two major events occur during oogenesis: the oocyte precursor germ cell undergoes meiotic division and it accumulates substantial cytoplasm. In meiosis, two sequential rounds of cell division produce a haploid egg, with only one copy of each chromosome, from the diploid oocyte precursor cell. Simultaneously, a large volume of cytoplasm is accumulated; it contains yolk and numerous other components that are essential for early embryonic development. Meiotic progression seems to be an integral part of oogenesis, since a number of proteins are required meiotic progression and for the development of functional oocytes. For example, GLD-1, an RNA-binding protein, is required for maintenance of oocyte precursors in pachytene stage (see below); in its absence, female germ cells will enter meiosis and progress to pachytene stage, but then exit meiosis and return to mitotic proliferation. In contrast, male germ cells do not require GLD-1 for meiosis and gametogenesis.

A Conserved Mechanism for Post-Transcriptional Gene Silencing?

Eleanor M. Maine — Wed, 29 Sep 2010 06:56:10 PDT

Proteins with homology to RNA-directed RNA polymerases function in post-transcriptional gene silencing: in quelling in the fungus Neurospora crassa, RNAi in the nematode Caenorhabditis elegans, and co-suppression in the mustard plant Arabidopsis thaliana. These findings are consistent with a conserved mechanism operating in these diverse species.

EGO-1 is related to RNA-directed RNA polymerase and functions in germ-line development and RNA interference in C. elegans

Anne Smardon et al. — Wed, 29 Sep 2010 06:56:10 PDT

Background: Cell-fate determination requires that cells choose between alternative developmental pathways. For example, germ cells in the nematode worm Caenorhabditis elegans choose between mitotic and meiotic division, and between oogenesis and spermatogenesis. Germ-line mitosis depends on a somatic signal that is mediated by a Notch-type signaling pathway. The ego-1 gene was originally identified on the basis of genetic interactions with the receptor in this pathway and was also shown to be required for oogenesis. Here, we provide more insight into the role of ego-1 in germ-line development. Results: We have determined the ego-1 gene structure and the molecular basis of ego-1 alleles. Putative ego-1 null mutants had multiple, previously unreported defects in germ-line development. The ego-1 transcript was found predominantly in the germ line. The predicted EGO-1 protein was found to be related to the tomato RNA-directed RNA polymerase (RdRP) and to Neurospora crassa QDE-1, two proteins implicated in post-transcriptional gene silencing (PTGS). For a number of germ-line-expressed genes, ego-1 mutants were resistant to a form of PTGS called RNA interference. Conclusions: The ego-1 gene is the first example of a gene encoding an RdRP-related protein with an essential developmental function. The ego-1 gene is also required for a robust response to RNA interference by certain genes. Hence, a protein required for germ-line development in C. elegans may be a component of the RNA interference/PTGS machinery.

Enhancers of gZP1, a Gene Required for Cell-Signaling in Caenorhabditis elegans, Define a Set of Genes Required for Germline Development

Li Qiao et al. — Wed, 29 Sep 2010 06:56:09 PDT

The distal tip cell (DTC) regulates the proliferation or differentiation choice in the Cmorhabditis ekgans germline by an inductive mechanism. Cell signaling requires a putative receptor in the germline, encoded by the glp-1 gene, and a putative signal from the DTC, encoded by the lag-2 gene. Both glp-1 and lag-2 belong to multigene gene families whose members are essential for cell signaling during development of various tissues in insects and vertebrates as well as C. elegans. Relatively little is known about how these pathways regulate cell fate choice. To identify additional genes involved in the glp-1 signaling pathway, we carried out screens for genetic enhancers of glp-1. We recovered mutations in five new genes, named ego (enhancer of glp-1), and two previously identified genes, lag-1 and glp-4, that strongly enhance a weak glp-1 loss-of-function phenotype in the germline. Ego mutations cause multiple phenotypes consistent with the idea that gene activity is required for more than one aspect of germline and, in some cases, somatic development. Based on genetic experiments, glp-1 appears to act upstream of ego1 and ego3W. e discuss the possible functional relationships among these genes in light of their phenotypes and interactions with glp-1.

A Phylogenetic Analysis of Vertebrate and Invertebrate Notch-Related Genes

Eleanor M. Maine et al. — Wed, 29 Sep 2010 06:56:09 PDT

Members of the Notch gene family are thought to mediate inductive cell-cell interactions during develop ment of a wide variety of vertebrates and invertebrates. These genes encode transmembrane proteins that appear to act as receptors and contain three repeated sequence motifs. Two of these motifs (an epidermal growth factor like sequence and a cdc10/SW16/ankyrin sequence) have been found in a large number of unrelated proteins, while the third motif (a lin-12/Notch/glp-1 sequence) is unique to proteins of the Notch family. We present a phylogenetic analysis of 17 Notch-related genes from eight species that has implications as to the origins and relative functions of these genes in different species. Several independent gene duplications have occurred and at least one such duplication in the vertebrate lineage preceded the avian/mammalian divergence. Significantly, the overall organization of individual members of each internally repeated motif ap pears to have been conserved among species, suggesting that each repeat plays a unique role in protein function. Yet, where sequence divergence does occur among genes in vertebrate, dipteran, and nematode lineages, it may signify functional differences for specific regions in Notch-related proteins.