A genome-wide resource for the analysis of protein localisation in Drosophila
- Max Planck Institute of Cell Biology and Genetics, Germany
- Max Planck Institute of Biochemistry, Germany
- Tata Institute of Fundamental Research, India
- Centre for Cellular and Molecular Platforms, National Centre for Biological Sciences, India
- Heidelberg Institute of Theoretical Studies, Germany
- University of California, Los Angeles, United States
- Trinity College Dublin, Ireland
Abstract
The Drosophila genome contains >13,000 protein coding genes, the majority of which remain poorly investigated. Important reasons include the lack of antibodies or reporter constructs to visualise these proteins. Here we present a genome-wide fosmid library of 10,000 GFP-tagged clones, comprising tagged genes and most of their regulatory information. For 880 tagged proteins we created transgenic lines and for a total of 207 lines we assessed protein expression and localisation in ovaries, embryos, pupae or adults by stainings and live imaging approaches. Importantly, we visualised many proteins at endogenous expression levels and found a large fraction of them localising to subcellular compartments. By applying genetic complementation tests we estimate that about two-thirds of the tagged proteins are functional. Moreover, these tagged proteins enable interaction proteomics from developing pupae and adult flies. Taken together, this resource will boost systematic analysis of protein expression and localisation in various cellular and developmental contexts.
Article and author information
Author details
Reviewing Editor
- Hugo J Bellen, Howard Hughes Medical Institute, Baylor College of Medicine, United States
Version history
- Received: October 3, 2015
- Accepted: February 19, 2016
- Accepted Manuscript published: February 20, 2016 (version 1)
- Version of Record published: March 14, 2016 (version 2)
Copyright
© 2016, Sarov et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 21,684
- views
-
- 3,937
- downloads
-
- 226
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
A genome-wide resource for the analysis of protein localisation in Drosophila
eLife 5:e12068.
https://doi.org/10.7554/eLife.12068
Further reading
-
- Developmental Biology
Inhibitory G alpha (GNAI or Gαi) proteins are critical for the polarized morphogenesis of sensory hair cells and for hearing. The extent and nature of their actual contributions remains unclear, however, as previous studies did not investigate all GNAI proteins and included non-physiological approaches. Pertussis toxin can downregulate functionally redundant GNAI1, GNAI2, GNAI3, and GNAO proteins, but may also induce unrelated defects. Here, we directly and systematically determine the role(s) of each individual GNAI protein in mouse auditory hair cells. GNAI2 and GNAI3 are similarly polarized at the hair cell apex with their binding partner G protein signaling modulator 2 (GPSM2), whereas GNAI1 and GNAO are not detected. In Gnai3 mutants, GNAI2 progressively fails to fully occupy the sub-cellular compartments where GNAI3 is missing. In contrast, GNAI3 can fully compensate for the loss of GNAI2 and is essential for hair bundle morphogenesis and auditory function. Simultaneous inactivation of Gnai2 and Gnai3 recapitulates for the first time two distinct types of defects only observed so far with pertussis toxin: (1) a delay or failure of the basal body to migrate off-center in prospective hair cells, and (2) a reversal in the orientation of some hair cell types. We conclude that GNAI proteins are critical for hair cells to break planar symmetry and to orient properly before GNAI2/3 regulate hair bundle morphogenesis with GPSM2.
-
- Developmental Biology
- Evolutionary Biology
Despite rapid evolution across eutherian mammals, the X-linked MIR-506 family miRNAs are located in a region flanked by two highly conserved protein-coding genes (SLITRK2 and FMR1) on the X chromosome. Intriguingly, these miRNAs are predominantly expressed in the testis, suggesting a potential role in spermatogenesis and male fertility. Here, we report that the X-linked MIR-506 family miRNAs were derived from the MER91C DNA transposons. Selective inactivation of individual miRNAs or clusters caused no discernible defects, but simultaneous ablation of five clusters containing 19 members of the MIR-506 family led to reduced male fertility in mice. Despite normal sperm counts, motility, and morphology, the KO sperm were less competitive than wild-type sperm when subjected to a polyandrous mating scheme. Transcriptomic and bioinformatic analyses revealed that these X-linked MIR-506 family miRNAs, in addition to targeting a set of conserved genes, have more targets that are critical for spermatogenesis and embryonic development during evolution. Our data suggest that the MIR-506 family miRNAs function to enhance sperm competitiveness and reproductive fitness of the male by finetuning gene expression during spermatogenesis.
