Imaging cytoplasmic lipid droplets in vivo with fluorescent perilipin 2 and perilipin 3 knock-in zebrafish

  1. Meredith H Wilson
  2. Stephen C Ekker
  3. Steven Arthur Farber  Is a corresponding author
  1. Carnegie Institution for Science, United States
  2. Mayo Clinic, United States

Abstract

Cytoplasmic lipid droplets are highly dynamic storage organelles that are critical for cellular lipid homeostasis. While the molecular details of lipid droplet dynamics are a very active area of investigation, this work has been primarily performed in cultured cells. Taking advantage of the powerful transgenic and in vivo imaging opportunities available in zebrafish, we built a suite of tools to study lipid droplets in real-time from the subcellular to the whole organism level. Fluorescently tagging the lipid-droplet-associated proteins, perilipin 2 and perilipin 3, in the endogenous loci permits visualization of lipid droplets in the intestine, liver, and adipose tissue. Using these tools, we found that perilipin 3 is rapidly loaded on intestinal lipid droplets following a high-fat meal and later replaced by perilipin 2. These powerful new tools will facilitate studies on the role of lipid droplets in different tissues, under different genetic and physiological manipulations, and in a variety of human disease models.

Data availability

All data generated during this study are included in the manuscript and supporting files; Source Data files have been provided for Figures 1, 2, 3, 4, 5, and 6.

Article and author information

Author details

  1. Meredith H Wilson

    Embryology, Carnegie Institution for Science, Baltimore, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6152-7127
  2. Stephen C Ekker

    Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, United States
    Competing interests
    Stephen C Ekker, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0726-4212
  3. Steven Arthur Farber

    Embryology, Carnegie Institution for Science, Baltimore, Maryland, United States
    For correspondence
    farber@carnegiescience.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8037-7312

Funding

National Institutes of Health (R01 DK093399)

  • Steven Arthur Farber

National Institutes of Health (R01 GM63904)

  • Stephen C Ekker
  • Steven Arthur Farber

National Institutes of Health (F32DK109592)

  • Meredith H Wilson

G. Harold and Leila Y. Mathers Foundation

  • Steven Arthur Farber

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

Ethics

Animal experimentation: All procedures using zebrafish (Danio rerio) were approved by the Carnegie Institution Department of Embryology Animal Care and Use Committee (Protocol #139).

Reviewing Editor

  1. Amnon Schlegel, University of Utah School of Medicine, United States

Publication history

  1. Received: January 8, 2021
  2. Preprint posted: January 10, 2021 (view preprint)
  3. Accepted: August 10, 2021
  4. Accepted Manuscript published: August 13, 2021 (version 1)
  5. Version of Record published: September 2, 2021 (version 2)

Copyright

© 2021, Wilson 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

  • 1,681
    Page views
  • 292
    Downloads
  • 7
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

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. Meredith H Wilson
  2. Stephen C Ekker
  3. Steven Arthur Farber
(2021)
Imaging cytoplasmic lipid droplets in vivo with fluorescent perilipin 2 and perilipin 3 knock-in zebrafish
eLife 10:e66393.
https://doi.org/10.7554/eLife.66393

Further reading

    1. Cell Biology
    Lei Yuan et al.
    Research Article

    The phagocytic receptor CED-1 mediates apoptotic cell recognition by phagocytic cells, enabling cell corpse clearance in Caenorhabditis elegans. Whether appropriate levels of CED-1 are maintained for executing the engulfment function remains unknown. Here, we identified the C. elegans E3 ubiquitin ligase tripartite motif containing-21 (TRIM-21) as a component of the CED-1 pathway for apoptotic cell clearance. When the NPXY motif of CED-1 was bound to the adaptor protein CED-6 or the YXXL motif of CED-1 was phosphorylated by tyrosine kinase SRC-1 and subsequently bound to the adaptor protein NCK-1 containing the SH2 domain, TRIM-21 functioned in conjunction with UBC-21 to catalyze K48-linked poly-ubiquitination on CED-1, targeting it for proteasomal degradation. In the absence of TRIM-21, CED-1 accumulated post-translationally and drove cell corpse degradation defects, as evidenced by direct binding to VHA-10. These findings reveal a unique mechanism for the maintenance of appropriate levels of CED-1 to regulate apoptotic cell clearance.

    1. Cell Biology
    2. Genetics and Genomics
    Anthony J Veltri et al.
    Research Article Updated

    Key protein adapters couple translation to mRNA decay on specific classes of problematic mRNAs in eukaryotes. Slow decoding on non-optimal codons leads to codon-optimality-mediated decay (COMD) and prolonged arrest at stall sites leads to no-go decay (NGD). The identities of the decay factors underlying these processes and the mechanisms by which they respond to translational distress remain open areas of investigation. We use carefully designed reporter mRNAs to perform genetic screens and functional assays in Saccharomyces cerevisiae. We characterize the roles of Hel2, Syh1, and Smy2 in coordinating translational repression and mRNA decay on NGD reporter mRNAs, finding that Syh1 and, to a lesser extent its paralog Smy2, act in a distinct pathway from Hel2. This Syh1/Smy2-mediated pathway acts as a redundant, compensatory pathway to elicit NGD when Hel2-dependent NGD is impaired. Importantly, we observe that these NGD factors are not involved in the degradation of mRNAs enriched in non-optimal codons. Further, we establish that a key factor previously implicated in COMD, Not5, contributes modestly to the degradation of an NGD-targeted mRNA. Finally, we use ribosome profiling to reveal distinct ribosomal states associated with each reporter mRNA that readily rationalize the contributions of NGD and COMD factors to degradation of these reporters. Taken together, these results provide new insight into the role of Syh1 and Smy2 in NGD and into the ribosomal states that correlate with the activation of distinct pathways targeting mRNAs for degradation in yeast.