A genome engineering resource to uncover principles of cellular organization and tissue architecture by lipid signalling

Abstract

Phosphoinositides (PI) are key regulators of cellular organization in eukaryotes and genes that tune PI signalling are implicated in human disease mechanisms. Biochemical analyses and studies in cultured cells have identified a large number of proteins that can mediate PI signalling. However, the role of such proteins in regulating cellular processes in vivo and development in metazoans remains to be understood. Here we describe a set of CRISPR based genome engineering tools that allow the manipulation of each of these proteins with spatial and temporal control during metazoan development. We demonstrate the use of these reagents to deplete a set of 103 proteins individually in the Drosophila eye and identify several new molecules that control eye development. Our work demonstrates the power of this resource in uncovering the molecular basis of tissue homeostasis during normal development and in human disease biology.

Data availability

Full genome sequencing for isogenized Attp40 Stock submitted to NCBI (BioProject ID PRJNA606147). Full genome sequencing for S2R+ cells submitted to NCBI (Bioproject ID PRJNA606149). Images for PI signaling genetic screen saved at Open Source Frame https://osf.io/pt7zu/?view_only=14642fc3a5d74e408fb3766c2555393f

The following data sets were generated

Article and author information

Author details

  1. Deepti Trivedi

    Cellular Organization and Signalling, National Centre for Biological Sciences, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  2. Vinitha CM

    Cellular Organization and Signalling, National Centre for Biological Sciences, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  3. Karishma Bisht

    Cellular Organization and Signalling, National Centre for Biological Sciences, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9088-1141
  4. Vishnu Janardan

    Cellular Organization and Signalling, National Centre for Biological Sciences, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  5. Awadhesh Pandit

    Cellular Organization and Signalling, National Centre for Biological Sciences, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  6. Bishal Basak

    Cellular Organization and Signalling, National Centre for Biological Sciences, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  7. Shwetha H

    Cellular Organization and Signalling, National Centre for Biological Sciences, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  8. Navyashree Ramesh

    Cellular Organization and Signalling, National Centre for Biological Sciences, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  9. Padinjat Raghu

    Cellular Organization and Signalling, National Centre for Biological Sciences, Bangalore, India
    For correspondence
    praghu@ncbs.res.in
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3578-6413

Funding

Department of Biotechnology, Ministry of Science and Technology, India (BT/PRJ3748/GET/l 19/27/2015)

  • Deepti Trivedi
  • Vinitha CM
  • Karishma Bisht
  • Vishnu Janardan
  • Padinjat Raghu

Wellcome-DBT India Alliance (IA/S/14/2/501540)

  • Vinitha CM
  • Karishma Bisht
  • Vishnu Janardan
  • Bishal Basak
  • Padinjat Raghu

National Centre for Biological Sciences (core)

  • Deepti Trivedi
  • Vinitha CM
  • Karishma Bisht
  • Vishnu Janardan
  • Awadhesh Pandit
  • Bishal Basak
  • Padinjat Raghu

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2020, Trivedi 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

  • 3,208
    views
  • 355
    downloads
  • 16
    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)

  1. Deepti Trivedi
  2. Vinitha CM
  3. Karishma Bisht
  4. Vishnu Janardan
  5. Awadhesh Pandit
  6. Bishal Basak
  7. Shwetha H
  8. Navyashree Ramesh
  9. Padinjat Raghu
(2020)
A genome engineering resource to uncover principles of cellular organization and tissue architecture by lipid signalling
eLife 9:e55793.
https://doi.org/10.7554/eLife.55793

Share this article

https://doi.org/10.7554/eLife.55793

Further reading

    1. Developmental Biology
    2. Genetics and Genomics
    Debashish U Menon, Prabuddha Chakraborty ... Terry Magnuson
    Research Article

    We present evidence implicating the BAF (BRG1/BRM Associated Factor) chromatin remodeler in meiotic sex chromosome inactivation (MSCI). By immunofluorescence (IF), the putative BAF DNA binding subunit, ARID1A (AT-rich Interaction Domain 1 a), appeared enriched on the male sex chromosomes during diplonema of meiosis I. Germ cells showing a Cre-induced loss of ARID1A arrested in pachynema and failed to repress sex-linked genes, indicating a defective MSCI. Mutant sex chromosomes displayed an abnormal presence of elongating RNA polymerase II coupled with an overall increase in chromatin accessibility detectable by ATAC-seq. We identified a role for ARID1A in promoting the preferential enrichment of the histone variant, H3.3, on the sex chromosomes, a known hallmark of MSCI. Without ARID1A, the sex chromosomes appeared depleted of H3.3 at levels resembling autosomes. Higher resolution analyses by CUT&RUN revealed shifts in sex-linked H3.3 associations from discrete intergenic sites and broader gene-body domains to promoters in response to the loss of ARID1A. Several sex-linked sites displayed ectopic H3.3 occupancy that did not co-localize with DMC1 (DNA meiotic recombinase 1). This observation suggests a requirement for ARID1A in DMC1 localization to the asynapsed sex chromatids. We conclude that ARID1A-directed H3.3 localization influences meiotic sex chromosome gene regulation and DNA repair.

    1. Cell Biology
    2. Developmental Biology
    Sofía Suárez Freire, Sebastián Perez-Pandolfo ... Mariana Melani
    Research Article

    Eukaryotic cells depend on exocytosis to direct intracellularly synthesized material toward the extracellular space or the plasma membrane, so exocytosis constitutes a basic function for cellular homeostasis and communication between cells. The secretory pathway includes biogenesis of secretory granules (SGs), their maturation and fusion with the plasma membrane (exocytosis), resulting in release of SG content to the extracellular space. The larval salivary gland of Drosophila melanogaster is an excellent model for studying exocytosis. This gland synthesizes mucins that are packaged in SGs that sprout from the trans-Golgi network and then undergo a maturation process that involves homotypic fusion, condensation, and acidification. Finally, mature SGs are directed to the apical domain of the plasma membrane with which they fuse, releasing their content into the gland lumen. The exocyst is a hetero-octameric complex that participates in tethering of vesicles to the plasma membrane during constitutive exocytosis. By precise temperature-dependent gradual activation of the Gal4-UAS expression system, we have induced different levels of silencing of exocyst complex subunits, and identified three temporarily distinctive steps of the regulated exocytic pathway where the exocyst is critically required: SG biogenesis, SG maturation, and SG exocytosis. Our results shed light on previously unidentified functions of the exocyst along the exocytic pathway. We propose that the exocyst acts as a general tethering factor in various steps of this cellular process.