The Nesprin-1/-2 ortholog ANC-1 regulates organelle positioning in C. elegans independently from its KASH or actin-binding domains

  1. Hongyan Hao
  2. Shilpi Kalra
  3. Laura E Jameson
  4. Leslie A Guerrero
  5. Natalie E Cain
  6. Jessica Bolivar
  7. Daniel A Starr  Is a corresponding author
  1. University of California, Davis, United States

Abstract

KASH proteins in the outer nuclear membrane comprise the cytoplasmic half of LINC complexes that connect nuclei to the cytoskeleton. Caenorhabditis elegans ANC-1, an ortholog of Nesprin-1/2, contains actin-binding and KASH domains at opposite ends of a long spectrin-like region. Deletion of either the KASH or calponin homology (CH) domains does not completely disrupt nuclear positioning, suggesting neither KASH nor CH domains are essential. Deletions in the spectrin-like region of ANC-1 led to significant defects, but only recapitulated the null phenotype in combination with mutations in the trans-membrane span. In anc-1 mutants, the ER, mitochondria, and lipid droplets were unanchored, moving throughout the cytoplasm. The data presented here support a cytoplasmic integrity model where ANC-1 localizes to the ER membrane and extends into the cytoplasm to position nuclei, ER, mitochondria, and likely other organelles in place.

Data availability

The list of strains generated is detailed in Table 1. All data points are displayed in the histograms in the figures.

Article and author information

Author details

  1. Hongyan Hao

    Molecular and Cellular Biology, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Shilpi Kalra

    Molecular and Cellular Biology, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Laura E Jameson

    Molecular and Cellular Biology, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Leslie A Guerrero

    Molecular and Cellular Biology, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Natalie E Cain

    Molecular and Cellular Biology, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1391-404X
  6. Jessica Bolivar

    Molecular and Cellular Biology, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Daniel A Starr

    Molecular and Cellular Biology, University of California, Davis, Davis, United States
    For correspondence
    dastarr@ucdavis.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7339-6606

Funding

National Institutes of Health (R01GM073874)

  • Daniel A Starr

National Institutes of Health (R35GM134859)

  • Daniel A Starr

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

Reviewing Editor

  1. Maddy Parsons, King's College London, United Kingdom

Publication history

  1. Received: July 14, 2020
  2. Accepted: April 11, 2021
  3. Accepted Manuscript published: April 16, 2021 (version 1)
  4. Version of Record published: May 21, 2021 (version 2)

Copyright

© 2021, Hao 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

  • 1,655
    Page views
  • 215
    Downloads
  • 6
    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. Hongyan Hao
  2. Shilpi Kalra
  3. Laura E Jameson
  4. Leslie A Guerrero
  5. Natalie E Cain
  6. Jessica Bolivar
  7. Daniel A Starr
(2021)
The Nesprin-1/-2 ortholog ANC-1 regulates organelle positioning in C. elegans independently from its KASH or actin-binding domains
eLife 10:e61069.
https://doi.org/10.7554/eLife.61069
  1. Further reading

Further reading

    1. Cell Biology
    Willa Wen-You Yim et al.
    Tools and Resources Updated

    Monitoring autophagic flux is necessary for most autophagy studies. The autophagic flux assays currently available for mammalian cells are generally complicated and do not yield highly quantitative results. Yeast autophagic flux is routinely monitored with the green fluorescence protein (GFP)-based processing assay, whereby the amount of GFP proteolytically released from GFP-containing reporters (e.g. GFP-Atg8), detected by immunoblotting, reflects autophagic flux. However, this simple and effective assay is typically inapplicable to mammalian cells because GFP is efficiently degraded in lysosomes while the more proteolytically resistant red fluorescent protein (RFP) accumulates in lysosomes under basal conditions. Here, we report a HaloTag (Halo)-based reporter processing assay to monitor mammalian autophagic flux. We found that Halo is sensitive to lysosomal proteolysis but becomes resistant upon ligand binding. When delivered into lysosomes by autophagy, pulse-labeled Halo-based reporters (e.g. Halo-LC3 and Halo-GFP) are proteolytically processed to generate Haloligand when delivered into lysosomes by autophagy. Hence, the amount of free Haloligand detected by immunoblotting or in-gel fluorescence imaging reflects autophagic flux. We demonstrate the applications of this assay by monitoring the autophagy pathways, macroautophagy, selective autophagy, and even bulk nonselective autophagy. With the Halo-based processing assay, mammalian autophagic flux and lysosome-mediated degradation can be monitored easily and precisely.

    1. Cell Biology
    2. Computational and Systems Biology
    Théo Aspert et al.
    Tools and Resources

    Automating the extraction of meaningful temporal information from sequences of microscopy images represents a major challenge to characterize dynamical biological processes. So far, strong limitations in the ability to quantitatively analyze single-cell trajectories have prevented large-scale investigations to assess the dynamics of entry into replicative senescence in yeast. Here, we have developed DetecDiv, a microfluidic-based image acquisition platform combined with deep learning-based software for high-throughput single-cell division tracking. We show that DetecDiv can automatically reconstruct cellular replicative lifespans with high accuracy and performs similarly with various imaging platforms and geometries of microfluidic traps. In addition, this methodology provides comprehensive temporal cellular metrics using time-series classification and image semantic segmentation. Last, we show that this method can be further applied to automatically quantify the dynamics of cellular adaptation and real-time cell survival upon exposure to environmental stress. Hence, this methodology provides an all-in-one toolbox for high-throughput phenotyping for cell cycle, stress response, and replicative lifespan assays.