Linking microarray reporters with protein functions

Background  

The analysis of microarray experiments requires accurate and up-to-date functional annotation of the microarray reporters to optimize the interpretation of the biological processes involved. Pathway visualization tools are used to connect gene expression data with existing biological pathways by using specific database identifiers that link reporters with elements in the pathways.     

Milton controls the early acquisition of mitochondria by Drosophila oocytes

Mitochondria in many species enter the young oocyte en mass from interconnected germ cells to generate the large aggregate known as the Balbiani body. Organelles and germ plasm components frequently associate with this structure. Balbiani body mitochondria are thought to populate the germ line, ensuring that their genomes will be inherited preferentially. We find that milton, a gene whose product was previously shown to associate with Kinesin and to mediate axonal transport of mitochondria, is needed to form a normal Balbiani body. In addition, germ cells mutant for some milton or Kinesin heavy chain (Khc) alleles transport mitochondria to the oocyte prematurely and excessively, without disturbing Balbiani body-associated components. Our observations show that the oocyte acquires the majority of its mitochondria by competitive bidirectional transport along microtubules mediated by the Milton adaptor. These experiments provide a molecular explanation for Balbiani body formation and, surprisingly, show that viable fertile offspring can be obtained from eggs in which the normal program of mitochondrial acquisition has been severely perturbed.     

The receptor-like tyrosine phosphatase lar is required for epithelial planar polarity and for axis determination within drosophila ovarian follicles.

The follicle cell monolayer that encircles each developing Drosophila oocyte contributes actively to egg development and patterning, and also represents a model stem cell-derived epithelium. We have identified mutations in the receptor-like transmembrane tyrosine phosphatase Lar that disorganize follicle formation, block egg chamber elongation and disrupt Oskar localization, which is an indicator of oocyte anterior-posterior polarity. Alterations in actin filament organization correlate with these defects. Actin filaments in the basal follicle cell domain normally become polarized during stage 6 around the anterior-posterior axis defined by the polar cells, but mutations in Lar frequently disrupt polar cell differentiation and actin polarization. Lar function is only needed in somatic cells, and (for Oskar localization) its action is autonomous to posterior follicle cells. Polarity signals may be laid down by these cells within the extracellular matrix (ECM), possibly in the distribution of the candidate Lar ligand Laminin A, and read out at the time Oskar is localized in a Lar-dependent manner. Lar is not required autonomously to polarize somatic cell actin during stages 6. We show that Lar acts somatically early in oogenesis, during follicle formation, and postulate that it functions in germarium intercyst cells that are required for polar cell specification and differentiation. Our studies suggest that positional information can be stored transiently in the ECM. A major function of Lar may be to transduce such signals.     

New components of the Drosophila fusome suggest it plays novel roles in signaling and transport

The fusome plays an essential role in prefollicular germ cell development within insects such as Drosophila melanogaster. Alpha-spectrin and the adducin-like protein Hu-li tai shao (Hts) are required to maintain fusome integrity, synchronize asymmetric cystocyte mitoses, form interconnected 16-cell germline cysts, and specify the initial cell as the oocyte. By screening a library of protein trap lines, we identified 14 new fusome-enriched proteins, including many associated with its characteristic vesicles. Our studies reveal that fusomes change during development and contain recycling endosomal and lysosomal compartments in females but not males. A significant number of fusome components are dispensable, because genetic disruption of tropomodulin, ferritin-1 heavy chain, or scribble, does not alter fusome structure or female fertility. In contrast, rab11 is required to maintain the germline stem cells, and to maintain the vesicle content of the spectrosome, suggesting that the fusome mediates intercellular signals that depend on the recycling endosome.     

Detection of protease-resistant cervid prion protein in water from a CWD-endemic area

Chronic wasting disease (CWD) is the only known transmissible spongiform encephalopathy affecting free-ranging wildlife. Although the exact mode of natural transmission remains unknown, substantial evidence suggests that prions can persist in the environment, implicating components thereof as potential prion reservoirs and transmission vehicles.^1–4 CWD-positive animals may contribute to environmental prion load via decomposing carcasses and biological materials including saliva, blood, urine and feces.^5–7 Sensitivity limitations of conventional assays hamper evaluation of environmental prion loads in soil and water. Here we show the ability of serial protein misfolding cyclic amplification (sPMCA) to amplify a 1.3 × 10⁻⁷ dilution of CWD-infected brain homogenate spiked into water samples, equivalent to approximately 5 × 10⁷ protease resistant cervid prion protein (PrP^CWD) monomers. We also detected PrP^CWD in one of two environmental water samples from a CWD endemic area collected at a time of increased water runoff from melting winter snow pack, as well as in water samples obtained concurrently from the flocculation stage of water processing by the municipal water treatment facility. Bioassays indicated that the PrP^CWD detected was below infectious levels. These data demonstrate detection of very low levels of PrP^CWD in the environment by sPMCA and suggest persistence and accumulation of prions in the environment that may promote CWD transmission.Key words: prions, chronic wasting disease, water, environment, serial protein misfolding cyclic amplification     

Drosophila Pxt: a cyclooxygenase-like facilitator of follicle maturation

Prostaglandins are local transient hormones that mediate a wide variety of biological events, including reproduction. The study of prostaglandin biology in a genetically tractable invertebrate model organism has been limited by the lack of clearly identified prostaglandin-mediated biological processes and prostaglandin metabolic genes, particularly analogs of cyclooxygenase (COX), the rate-limiting step in vertebrate prostaglandin synthesis. Here, we present pharmacological data that Drosophila ovarian follicle maturation requires COX-like activity and genetic evidence that this activity is supplied in vivo by the Drosophila peroxidase Pxt. pxt mutant females are sterile, and maturing follicles show defects in actin filament formation, nurse cell membrane stability and border cell migration. Maturation of pxt follicles in vitro is stimulated by prostaglandin treatment and fertility is restored in vivo to pxt mutants by expressing mammalian Cox1 protein. Our experiments suggest that prostaglandins promote Drosophila follicle maturation, in part by modulating the actin cytoskeleton, and establish Drosophila oogenesis as a model for understanding these critical biological regulators.     

A Balbiani body and the fusome mediate mitochondrial inheritance during Drosophila oogenesis

Maternally inherited mitochondria and other cytoplasmic organelles play essential roles supporting the development of early embryos and their germ cells. Using methods that resolve individual organelles, we studied the origin of oocyte and germ plasm-associated mitochondria during Drosophila oogenesis. Mitochondria partition equally on the spindle during germline stem cell and cystocyte divisions. Subsequently, a fraction of cyst mitochondria and Golgi vesicles associates with the fusome, moves through the ring canals, and enters the oocyte in a large mass that resembles the Balbiani bodies of Xenopus, humans and diverse other species. Some mRNAs, including oskar RNA, specifically associate with the oocyte fusome and a region of the Balbiani body prior to becoming localized. Balbiani body development requires an intact fusome and microtubule cytoskeleton as it is blocked by mutations in hu-li tai shao, while egalitarian mutant follicles accumulate a large mitochondrial aggregate in all 16 cyst cells. Initially, the Balbiani body supplies virtually all the mitochondria of the oocyte, including those used to form germ plasm, because the oocyte ring canals specifically block inward mitochondrial transport until the time of nurse cell dumping. Our findings reveal new similarities between oogenesis in Drosophila and vertebrates, and support our hypothesis that developing oocytes contain specific mechanisms to ensure that germ plasm is endowed with highly functional organelles.