1. Biochemistry and Chemical Biology
  2. Physics of Living Systems
Download icon

Optical estimation of absolute membrane potential using fluorescence lifetime imaging

  1. Julia Rose Lazzari-Dean
  2. Anneliese M M Gest
  3. Evan W Miller  Is a corresponding author
  1. University of California, Berkeley, United States
Tools and Resources
  • Cited 4
  • Views 3,752
  • Annotations
Cite this article as: eLife 2019;8:e44522 doi: 10.7554/eLife.44522

Abstract

All cells maintain ionic gradients across their plasma membranes, producing transmembrane potentials (Vmem). Mounting evidence suggests a relationship between resting Vmem and the physiology of non-excitable cells with implications in diverse areas, including cancer, cellular differentiation, and body patterning. A lack of non-invasive methods to record absolute Vmem limits our understanding of this fundamental signal. To address this need, we developed a fluorescence lifetime-based approach (VF-FLIM) to visualize and optically quantify Vmem with single-cell resolution in mammalian cell culture. Using VF-FLIM, we report Vmem distributions over thousands of cells, a 100-fold improvement relative to electrophysiological approaches. In human carcinoma cells, we visualize the voltage response to growth factor stimulation, stably recording a 10-15 mV hyperpolarization over minutes. Using pharmacological inhibitors, we identify the source of the hyperpolarization as the Ca2+-activated K+ channel KCa3.1. The ability to optically quantify absolute Vmem with cellular resolution will allow a re-examination of its signaling roles.

Article and author information

Author details

  1. Julia Rose Lazzari-Dean

    Department of Chemistry, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2971-5379

  2. Anneliese M M Gest

    Department of Chemistry, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.

  3. Evan W Miller

    Department of Chemistry, University of California, Berkeley, Berkeley, United States
    For correspondence
    evanwmiller@berkeley.edu
    Competing interests
    Evan W Miller, is listed as an inventor on a patent describing voltage-sensitive fluorophores. This patent (US20170315059) is owned by the Regents of the University of California.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6556-7679

Funding

National Science Foundation (GRFP)

  • Julia Rose Lazzari-Dean

National Institutes of Health (R35GM119855)

  • Evan W Miller

Alfred P. Sloan Foundation (FG-2016-6359)

  • Evan W Miller

March of Dimes Foundation (5-FY-16-65)

  • Evan W Miller

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

Reviewing Editor

  1. Lawrence Cohen, Yale, United States

Publication history

  1. Received: February 14, 2019
  2. Accepted: September 16, 2019
  3. Accepted Manuscript published: September 23, 2019 (version 1)
  4. Version of Record published: October 25, 2019 (version 2)

Copyright

© 2019, Lazzari-Dean 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

  • 3,752
    Page views
  • 568
    Downloads
  • 4
    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. Biochemistry and Chemical Biology
    2. Cell Biology

    SON and SRRM2 are essential for nuclear speckle formation

    İbrahim Avşar Ilik et al.
    Research Article

    The nucleus of higher eukaryotes is a highly compartmentalized and dynamic organelle consisting of several biomolecular condensates that regulate gene expression at multiple levels (Banani et al., 2017; Shin and Brangwynne, 2017). First reported more than 100 years ago by Ramón y Cajal, nuclear speckles (NS) are among the most prominent of such condensates (Spector and Lamond, 2011). Despite their prevalence, research on the function of NS is virtually restricted to colocalization analyses, since an organizing core, without which NS cannot form, remains unidentified (Chen and Belmont, 2019; Galganski et al., 2017). The monoclonal antibody SC35, which was raised against a spliceosomal extract, is a frequently used reagent to mark NS since its debut in 1990 (Fu and Maniatis, 1990). Unexpectedly, we found that this antibody has been misidentified and the main target of SC35 mAb is SRRM2, a large (~300 kDa), spliceosome-associated (Jia and Sun, 2018) protein with prominent intrinsically disordered regions (IDRs) that sharply localizes to NS (Blencowe et al., 1994). Here we show that, the core of NS is likely formed by SON and SRRM2, since depletion of SON leads only to a partial disassembly of NS as reported previously (Ahn et al., 2011; Fei et al., 2017; Sharma et al., 2010), in contrast, combined depletion of SON together with SRRM2, but not other NS associated factors, or depletion of SON in a cell line where IDRs of SRRM2 are genetically deleted, leads to a near-complete dissolution of NS. This work, therefore, paves the way to study the role of NS under diverse physiological and stress conditions.

    1. Cell Biology
    2. Biochemistry and Chemical Biology

    Feedback control of Wnt signaling based on ultrastable histidine cluster co-aggregation between Naked/NKD and Axin

    Melissa V Gammons et al.
    Research Article Updated

    Feedback control is a universal feature of cell signaling pathways. Naked/NKD is a widely conserved feedback regulator of Wnt signaling which controls animal development and tissue homeostasis. Naked/NKD destabilizes Dishevelled, which assembles Wnt signalosomes to inhibit the β-catenin destruction complex via recruitment of Axin. Here, we discover that the molecular mechanism underlying Naked/NKD function relies on its assembly into ultra-stable decameric core aggregates via its conserved C-terminal histidine cluster (HisC). HisC aggregation is facilitated by Dishevelled and depends on accumulation of Naked/NKD during prolonged Wnt stimulation. Naked/NKD HisC cores co-aggregate with a conserved histidine cluster within Axin, to destabilize it along with Dishevelled, possibly via the autophagy receptor p62, which binds to HisC aggregates. Consistent with this, attenuated Wnt responses are observed in CRISPR-engineered flies and human epithelial cells whose Naked/NKD HisC has been deleted. Thus, HisC aggregation by Naked/NKD provides context-dependent feedback control of prolonged Wnt responses.

    1. Biochemistry and Chemical Biology

    A novel DNA primase-helicase pair encoded by SCCmec elements

    Aleksandra Bebel et al.
    Research Article Updated

    Mobile genetic elements (MGEs) are a rich source of new enzymes, and conversely, understanding the activities of MGE-encoded proteins can elucidate MGE function. Here, we biochemically characterize three proteins encoded by a conserved operon carried by the Staphylococcal Cassette Chromosome (SCCmec), an MGE that confers methicillin resistance to Staphylococcus aureus, creating MRSA strains. The first of these proteins, CCPol, is an active A-family DNA polymerase. The middle protein, MP, binds tightly to CCPol and confers upon it the ability to synthesize DNA primers de novo. The CCPol-MP complex is therefore a unique primase-polymerase enzyme unrelated to either known primase family. The third protein, Cch2, is a 3’-to-5’ helicase. Cch2 additionally binds specifically to a dsDNA sequence downstream of its gene that is also a preferred initiation site for priming by CCPol-MP. Taken together, our results suggest that this is a functional replication module for SCCmec.