Single-cell proteomics reveals changes in expression during hair-cell development

  1. Ying Zhu
  2. Mirko Scheibinger
  3. Daniel Christian Ellwanger
  4. Jocelyn F Krey
  5. Dongseok Choi
  6. Ryan T Kelly
  7. Stefan Heller
  8. Peter G Barr-Gillespie  Is a corresponding author
  1. Pacific Northwest National Laboratory, United States
  2. Stanford University, United States
  3. Oregon Health and Science University, United States
  4. Brigham Young University, United States

Abstract

Hearing and balance rely on small sensory hair cells that reside in the inner ear. To explore dynamic changes in the abundant proteins present in differentiating hair cells, we used nanoliter-scale shotgun mass spectrometry of single cells, each ~1 picoliter, from utricles of embryonic day 15 chickens. We identified unique constellations of proteins or protein groups from presumptive hair cells and from progenitor cells. The single-cell proteomes enabled the de novo reconstruction of a developmental trajectory using protein expression levels, revealing proteins that greatly increased in expression during differentiation of hair cells (e.g., OCM, CRABP1, GPX2, AK1, GSTO1) and those that decreased during differentiation (e.g., TMSB4X, AGR3). Complementary single-cell transcriptome profiling showed corresponding changes in mRNA during maturation of hair cells. Single-cell proteomics data thus can be mined to reveal features of cellular development that may be missed with transcriptomics.

Data availability

The mass spectrometry proteomics data, including raw data from the mass spectrometry runs, have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD014256. The analyzed data are reported in Figure 1-source data 1. The analyzed single-cell RNA-seq data are reported in Figure 5-source data 1. The complete analysis of the single-cell RNA-seq will be reported elsewhere

The following data sets were generated

Article and author information

Author details

  1. Ying Zhu

    Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, United States
    Competing interests
    No competing interests declared.
  2. Mirko Scheibinger

    Department of Otolaryngology, Stanford University, Stanford, United States
    Competing interests
    No competing interests declared.
  3. Daniel Christian Ellwanger

    Department of Otolaryngology, Stanford University, Stanford, United States
    Competing interests
    Daniel Christian Ellwanger, is affiliated with Amgen Inc.. The author has no other competing interests to declare.
  4. Jocelyn F Krey

    Oregon Hearing Research Center, Oregon Health and Science University, Portland, United States
    Competing interests
    No competing interests declared.
  5. Dongseok Choi

    OHSU-PSU School of Public Health, Oregon Health and Science University, Portland, United States
    Competing interests
    No competing interests declared.
  6. Ryan T Kelly

    Department of Chemistry and Biochemistry, Brigham Young University, Provo, United States
    Competing interests
    No competing interests declared.
  7. Stefan Heller

    Department of Otolaryngology, Stanford University, Stanford, United States
    Competing interests
    No competing interests declared.
  8. Peter G Barr-Gillespie

    Oregon Hearing Research Center, Oregon Health and Science University, Portland, United States
    For correspondence
    gillespp@ohsu.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9787-5860

Funding

National Institutes of Health (R01 DC011034)

  • Peter G Barr-Gillespie

National Institutes of Health (R01 DC015201)

  • Stefan Heller

National Institutes of Health (R33 CA225248)

  • Ryan T Kelly

Laboratory Directed Research and Development Program at PNNL (Earth & Biological Sciences Directorate Mission Seed)

  • Ying Zhu

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

Copyright

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Metrics

  • 7,954
    views
  • 1,187
    downloads
  • 91
    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. Ying Zhu
  2. Mirko Scheibinger
  3. Daniel Christian Ellwanger
  4. Jocelyn F Krey
  5. Dongseok Choi
  6. Ryan T Kelly
  7. Stefan Heller
  8. Peter G Barr-Gillespie
(2019)
Single-cell proteomics reveals changes in expression during hair-cell development
eLife 8:e50777.
https://doi.org/10.7554/eLife.50777

Share this article

https://doi.org/10.7554/eLife.50777

Further reading

    1. Cell Biology
    Fabian Link, Sisco Jung ... Brooke Morriswood
    Research Article

    The actin cytoskeleton is a ubiquitous feature of eukaryotic cells, yet its complexity varies across different taxa. In the parasitic protist Trypanosoma brucei, a rudimentary actomyosin system consisting of one actin gene and two myosin genes has been retained despite significant investment in the microtubule cytoskeleton. The functions of this highly simplified actomyosin system remain unclear, but appear to centre on the endomembrane system. Here, advanced light and electron microscopy imaging techniques, together with biochemical and biophysical assays, were used to explore the relationship between the actomyosin and endomembrane systems. The class I myosin (TbMyo1) had a large cytosolic pool and its ability to translocate actin filaments in vitro was shown here for the first time. TbMyo1 exhibited strong association with the endosomal system and was additionally found on glycosomes. At the endosomal membranes, TbMyo1 colocalised with markers for early and late endosomes (TbRab5A and TbRab7, respectively), but not with the marker associated with recycling endosomes (TbRab11). Actin and myosin were simultaneously visualised for the first time in trypanosomes using an anti-actin chromobody. Disruption of the actomyosin system using the actin-depolymerising drug latrunculin A resulted in a delocalisation of both the actin chromobody signal and an endosomal marker, and was accompanied by a specific loss of endosomal structure. This suggests that the actomyosin system is required for maintaining endosomal integrity in T. brucei.

    1. Cell Biology
    Georgia Maria Sagia, Xenia Georgiou ... Sofia Dimou
    Research Article Updated

    Membrane proteins are sorted to the plasma membrane via Golgi-dependent trafficking. However, our recent studies challenged the essentiality of Golgi in the biogenesis of specific transporters. Here, we investigate the trafficking mechanisms of membrane proteins by following the localization of the polarized R-SNARE SynA versus the non-polarized transporter UapA, synchronously co-expressed in wild-type or isogenic genetic backgrounds repressible for conventional cargo secretion. In wild-type, the two cargoes dynamically label distinct secretory compartments, highlighted by the finding that, unlike SynA, UapA does not colocalize with the late-Golgi. In line with early partitioning into distinct secretory carriers, the two cargoes collapse in distinct ER-Exit Sites (ERES) in a sec31ts background. Trafficking via distinct cargo-specific carriers is further supported by showing that repression of proteins essential for conventional cargo secretion does not affect UapA trafficking, while blocking SynA secretion. Overall, this work establishes the existence of distinct, cargo-dependent, trafficking mechanisms, initiating at ERES and being differentially dependent on Golgi and SNARE interactions.