Probing cellular protein complexes using single-molecule pull-down

Journal name:
Nature
Volume:
473,
Pages:
484–488
Date published:
DOI:
doi:10.1038/nature10016
Received
Accepted
Published online

Abstract

Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here we describe a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signalling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analysing protein assemblies in biological pathways.

At a glance

Figures

  1. Schematic for SiMPull assay.
    Figure 1: Schematic for SiMPull assay.

    a, b, Immunoprecipitated protein complexes are visualized using TIRF microscopy using fluorophore-labelled antibody (a) or fluorescent protein (FP) tags (b). TIR, total internal reflection. c, Multi-colour colocalization can distinguish between subcomplexes (for example, AB + AC versus ABC). d, Photobleaching analysis can provide stoichiometric information. A simulated photobleaching trajectory for a trimeric protein. e, TIRF images for YFP pulled down from cells expressing His6–YFP (YFP) and control cells (Con) using His-tag or a control (Flag-tag) antibody. Minus sign indicates no antibody or sample. IP, immunoprecipitate. Scale bar, 5μm. f, Average number of fluorescent molecules per imaging area, Nf. Error bars denote standard deviation (s.d.) (n>20).

  2. PKA pull-down.
    Figure 2: PKA pull-down.

    a, Schematic of PKA construct. In western blot, C–HA–YFP is pulled down via R–Flag–mCherry; on adding cAMP, PKA dissociates. IB, immunoblot. b, Nf for C–HA–YFP (C) as a function of lysates and antibodies demonstrate the specificity of pull-down. ce, PKA complex pull-down. c, Nf for YFP (C) and mCherry (R) spots. d, Images of single PKA complexes, YFP (left), mCherry (centre) and overlay (right). e, On adding cAMP, YFP spots decrease significantly. Scale bars, 5μm. f, g, Photobleaching step distribution (f) for C–HA–YFP-only lysate and (g) for C–HA–YFP pulled down via R–Flag–mCherry. Error bars denote s.d. (n>20).

  3. Applications of SiMPull assay.
    Figure 3: Applications of SiMPull assay.

    ac, β2AR–YFP pull-down. df, MAVS pull-down. Mitochondrial fraction (mito.) from cells overexpressing YFP–MAVS was added either directly or after detergent solubilization. gi, mTORC1 pull-down. Lysate from cells expressing Flag–mTOR, HA–Raptor or both was applied on chambers with Flag antibody, and probed through primary antibody against HA and labelled secondary antibody (Sec. Ab). jm, Endogenous PKA–AKAP complex pull-down from mouse brain extract. k, Western blot shows AKAP immunoprecipitation with PKA antibody. l, Immunofluorescence images of AKAP150 pulled down through PKA antibody. c, f, i, m show Nf. Scale bars, 5μm. Error bars denote s.d. (n>20).

  4. PcrA pull-down and activity.
    Figure 4: PcrA pull-down and activity.

    a, Schematic. b, c, Labelled DNA binding to immunoprecipitated PcrA. Scale bar, 5μm. Error bars represent s.d. (n>20). d, A typical time trace of repetitive reeling-in activity of PcrA monitored by FRET. a.u., arbitrary units. e, f, The distribution of translocation times, Δt, and its mean, <Δt>, for purified PcrA (e) and for PcrA pulled down from cell extracts (f), at 1mM ATP concentration.

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Author information

Affiliations

  1. Center for Biophysics and Computational Biology and Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA

    • Ankur Jain,
    • Kaushik Ragunathan,
    • Jeehae Park &
    • Taekjip Ha
  2. Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA

    • Ruijie Liu,
    • Biswarathan Ramani &
    • Yang K. Xiang
  3. Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA

    • Edwin Arauz &
    • Jie Chen
  4. Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA

    • Yuji Ishitsuka &
    • Taekjip Ha
  5. Howard Hughes Medical Institute, Urbana, Illinois 61801, USA

    • Yuji Ishitsuka &
    • Taekjip Ha

Contributions

A.J., Y.K.X. and T.H. designed the research. A.J., R.L. and Y.I. conducted experiments, R.L., B.R., E.A., J.C. and J.P. provided samples, K.R. and Y.I. contributed important ideas to the experiments, A.J. and R.L. analysed the data and A.J., Y.K.X. and T.H. wrote the paper with inputs from other authors.

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The authors declare no competing financial interests.

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Supplementary information

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  1. Supplementary Information (1.5M)

    This file contains a Supplementary Discussion, Supplementary Figures 1- 15 with legends and Supplementary Table 1.

Comments

  1. Report this comment #23213

    Joe Kaplinsky said:

    The accompanying 'news and views' notes that:

    'Potentially, the method might even be applied to single cells, thereby avoiding averaging over heterogeneous cell populations.'

    We have already used a similar system to make single cell measurements. See

    Ali Salehi-Reyhani, Joseph Kaplinsky, Edward Burgin, Miroslava Novakova, Andrew J. deMello, Richard H. Templer, Peter Parker, Mark A. A. Neil, Oscar Ces, Paul French, Keith R. Willison and David Klug,

    A first step towards practical single cell proteomics: a microfluidic antibody capture chip with TIRF detection

    Lab Chip, 2011, 11, 1256-1261
    DOI: 10.1039/C0LC00613K
    link

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