1. Cell Biology
Download icon

Stable transplantation of human mitochondrial DNA by high-throughput, pressurized isolated mitochondrial delivery

  1. Alexander J Sercel
  2. Alexander N Patananan
  3. Tianxing Man
  4. Ting-Hsiang Wu
  5. Amy K Yu
  6. Garret W Guyot
  7. Shahrooz Rabizadeh
  8. Kayvan R Niazi
  9. Pei-Yu Chiou
  10. Michael A Teitell  Is a corresponding author
  1. UCLA, United States
  2. NanoCav, LLC, NantBio, Inc, and ImmunityBio, Inc, United States
  3. NanoCav, LLC, NantBio, Inc, and ImmunityBio, Inc, and NantOmics, LLC, United States
Tools and Resources
  • Cited 0
  • Views 104
  • Annotations
Cite this article as: eLife 2021;10:e63102 doi: 10.7554/eLife.63102

Abstract

Generating mammalian cells with specific mtDNA-nDNA combinations is desirable but difficult to achieve and would be enabling for studies of mitochondrial-nuclear communication and coordination in controlling cell fates and functions. We developed 'MitoPunch', a pressure-driven mitochondrial transfer device, to deliver isolated mitochondria into numerous target mammalian cells simultaneously. MitoPunch and MitoCeption, a previously described force-based mitochondrial transfer approach, both yield stable isolated mitochondrial recipient (SIMR) cells that permanently retain exogenous mtDNA, whereas coincubation of mitochondria with cells does not yield SIMR cells. Although a typical MitoPunch or MitoCeption delivery results in dozens of immortalized SIMR clones with restored oxidative phosphorylation, only MitoPunch can produce replication-limited, non-immortal human SIMR clones. The MitoPunch device is versatile, inexpensive to assemble, and easy to use for engineering mtDNA-nDNA combinations to enable fundamental studies and potential translational applications.

Article and author information

Author details

  1. Alexander J Sercel

    Molecular Biology Institute Interdepartmental Program, UCLA, Los Angeles, United States
    Competing interests
    No competing interests declared.

  2. Alexander N Patananan

    Pathology and Laboratory Medicine, UCLA, Los Angeles, United States
    Competing interests
    No competing interests declared.

  3. Tianxing Man

    Mechanical and Aerospace Engineering, UCLA, Los Angeles, United States
    Competing interests
    No competing interests declared.

  4. Ting-Hsiang Wu

    NanoCav, LLC, NantBio, Inc, and ImmunityBio, Inc, Culver City, United States
    Competing interests
    Ting-Hsiang Wu, T.-H.W. was an employee of NanoCav, LLC, and is currently employed by NantBio, Inc and ImmunityBio, Inc..

  5. Amy K Yu

    Molecular Biology Institute Interdepartmental Program, UCLA, Los Angeles, United States
    Competing interests
    No competing interests declared.

  6. Garret W Guyot

    Pathology and Laboratory Medicine, UCLA, Los Angeles, United States
    Competing interests
    No competing interests declared.

  7. Shahrooz Rabizadeh

    NanoCav, LLC, NantBio, Inc, and ImmunityBio, Inc, and NantOmics, LLC, Culver City, United States
    Competing interests
    Shahrooz Rabizadeh, S.R. is a board member of NanoCav, LLC, and employed by NantBio, Inc, ImmunityBio, Inc, and NantOmics, LLC..

  8. Kayvan R Niazi

    NanoCav, LLC, NantBio, Inc, and ImmunityBio, Inc, Culver City, United States
    Competing interests
    Kayvan R Niazi, K.R.N. is a board member of NanoCav, LLC, and employed by NantBio, Inc and ImmunityBio, Inc..

  9. Pei-Yu Chiou

    Mechanical and Aerospace Engineering, UCLA, Los Angeles, United States
    Competing interests
    Pei-Yu Chiou, P.-Y.C. is a co-founder, board member, shareholder, and consultant for NanoCav, LLC, a private start-up company working on mitochondrial transfer techniques and applications..

  10. Michael A Teitell

    Pathology and Laboratory Medicine, UCLA, Los Angeles, United States
    For correspondence
    mteitell@mednet.ucla.edu
    Competing interests
    Michael A Teitell, M.A.T. is a co-founder, board member, shareholder, and consultant for NanoCav, LLC, a private start-up company working on mitochondrial transfer techniques and applications..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4495-8750

Funding

National Institutes of Health (T32CA009120)

  • Alexander J Sercel
  • Alexander N Patananan

National Institutes of Health (R21CA227480)

  • Michael A Teitell

National Institutes of Health (P30CA016042)

  • Michael A Teitell

CIRM (RT3-07678)

  • Michael A Teitell

National Institutes of Health (T32GM007185)

  • Alexander J Sercel

American Heart Association (18POST34080342)

  • Alexander N Patananan

National Institutes of Health (T32GM008042)

  • Amy K Yu

National Science Foundation (CBET 1404080)

  • Pei-Yu Chiou

National Institutes of Health (R01GM114188)

  • Pei-Yu Chiou
  • Michael A Teitell

Air Force Office of Scientific Research (FA9550-15-1-0406)

  • Pei-Yu Chiou
  • Michael A Teitell

National Institutes of Health (R01GM073981)

  • Michael A Teitell

National Institutes of Health (R01CA185189)

  • Michael A Teitell

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

Reviewing Editor

  1. Simon C Johnson, University of Washington, United States

Publication history

  1. Received: September 15, 2020
  2. Accepted: January 12, 2021
  3. Accepted Manuscript published: January 13, 2021 (version 1)

Copyright

© 2021, Sercel 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

  • 104
    Page views
  • 25
    Downloads
  • 0
    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)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Categories and tags

Research organism

Further reading

    1. Cell Biology
    2. Evolutionary Biology

    A flagellate-to-amoeboid switch in the closest living relatives of animals

    Thibaut Brunet et al.
    Research Article

    Amoeboid cells are fundamental to animal biology and broadly distributed across animal diversity, but their evolutionary origin is unclear. The closest living relatives of animals, the choanoflagellates, display a polarized cell architecture (with an apical flagellum encircled by microvilli) that closely resembles that of epithelial cells and suggests homology, but this architecture differs strikingly from the deformable phenotype of animal amoeboid cells. Here, we show that choanoflagellates subjected to confinement differentiate into an amoeboid form by retracting their flagella and activating myosin-based motility. This switch allows escape from confinement and is conserved across choanoflagellate diversity. The conservation of the amoeboid cell phenotype across animals and choanoflagellates, together with the conserved role of myosin, is consistent with the homology of amoeboid motility in both lineages. We hypothesize that the differentiation between animal epithelial and crawling cells might have evolved from a stress-induced phenotypic switch between flagellate and amoeboid forms in their single-celled ancestors.

    1. Cell Biology
    2. Structural Biology and Molecular Biophysics

    Ca2+ signaling driving pacemaker activity in submucosal interstitial cells of Cajal in the murine colon

    Salah A Baker et al.
    Research Article Updated

    Interstitial cells of Cajal (ICC) generate pacemaker activity responsible for phasic contractions in colonic segmentation and peristalsis. ICC along the submucosal border (ICC-SM) contribute to mixing and more complex patterns of colonic motility. We show the complex patterns of Ca2+ signaling in ICC-SM and the relationship between ICC-SM Ca2+ transients and activation of smooth muscle cells (SMCs) using optogenetic tools. ICC-SM displayed rhythmic firing of Ca2+transients ~ 15 cpm and paced adjacent SMCs. The majority of spontaneous activity occurred in regular Ca2+ transients clusters (CTCs) that propagated through the network. CTCs were organized and dependent upon Ca2+ entry through voltage-dependent Ca2+ conductances, L- and T-type Ca2+ channels. Removal of Ca2+ from the external solution abolished CTCs. Ca2+ release mechanisms reduced the duration and amplitude of Ca2+ transients but did not block CTCs. These data reveal how colonic pacemaker ICC-SM exhibit complex Ca2+-firing patterns and drive smooth muscle activity and overall colonic contractions.

    1. Biochemistry and Chemical Biology
    2. Cell Biology

    ARL3 activation requires the co-GEF BART and effector-mediated turnover

    Yasmin ElMaghloob et al.
    Research Article

    The ADP-ribosylation factor-like 3 (ARL3) is a ciliopathy G-protein which regulates the ciliary trafficking of several lipid-modified proteins. ARL3 is activated by its guanine exchange factor (GEF) ARL13B via an unresolved mechanism. BART is described as an ARL3 effector which has also been implicated in ciliopathies, although the role of its ARL3 interaction is unknown. Here we show that, at physiological GTP:GDP levels, human ARL3GDP is weakly activated by ARL13B. However, BART interacts with nucleotide-free ARL3 and, in concert with ARL13B, efficiently activates ARL3. In addition, BART binds ARL3GTP and inhibits GTP dissociation, thereby stabilising the active G-protein; the binding of ARL3 effectors then releases BART. Finally, using live cell imaging, we show that BART accesses the primary cilium and colocalises with ARL13B. We propose a model wherein BART functions as a bona fide co-GEF for ARL3 and maintains the active ARL3GTP, until it is recycled by ARL3 effectors.