Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Single-molecule analysis of cell surface dynamics in Caenorhabditis elegans embryos

Nature Methods volume 11, pages 677–682 (2014)Cite this article

Subjects

Abstract

We describe a general, versatile and minimally invasive method to image single molecules near the cell surface that can be applied to any GFP-tagged protein in Caenorhabditis elegans embryos. We exploited tunable expression via RNAi and a dynamically exchanging monomer pool to achieve fast, continuous single-molecule imaging at optimal densities with signal-to-noise ratios adequate for robust single-particle tracking (SPT). We introduce a method called smPReSS, single-molecule photobleaching relaxation to steady state, that infers exchange rates from quantitative analysis of single-molecule photobleaching kinetics without using SPT. Combining SPT and smPReSS allowed for spatially and temporally resolved measurements of protein mobility and exchange kinetics. We used these methods to (i) resolve distinct mobility states and spatial variation in exchange rates of the polarity protein PAR-6 and (ii) measure spatiotemporal modulation of actin filament assembly and disassembly. These methods offer a promising avenue to investigate dynamic mechanisms that pattern the embryonic cell surface.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Imaging single molecules at the cortex in living embryos.
Figure 2: Long-term sampling of single cell-surface molecules in vivo.
Figure 3: Analysis of PAR-6∷GFP mobility.
Figure 4: Analysis of actin dynamics in nmy-2 RNAi embryos.

Similar content being viewed by others

References

  1. Lord, S.J., Lee, H.L. & Moerner, W.E. Single-molecule spectroscopy and imaging of biomolecules in living cells. Anal. Chem. 82, 2192–2203 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Xia, T., Li, N. & Fang, X. Single-molecule fluorescence imaging in living cells. Annu. Rev. Phys. Chem. 64, 459–480 (2013).

    Article  CAS  PubMed  Google Scholar 

  3. Sako, Y. et al. Live cell single-molecule detection in systems biology. Wiley Interdiscip. Rev. Syst. Biol. Med. 4, 183–192 (2012).

    Article  CAS  PubMed  Google Scholar 

  4. Waterman-Storer, C.M., Desai, A., Bulinski, J.C. & Salmon, E.D. Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells. Curr. Biol. 8, 1227–1230 (1998).

    Article  CAS  PubMed  Google Scholar 

  5. Ponti, A., Machacek, M., Gupton, S.L., Waterman-Storer, C.M. & Danuser, G. Two distinct actin networks drive the protrusion of migrating cells. Science 305, 1782–1786 (2004).

    Article  CAS  PubMed  Google Scholar 

  6. Watanabe, N. & Mitchison, T.J. Single-molecule speckle analysis of actin filament turnover in lamellipodia. Science 295, 1083–1086 (2002).

    Article  CAS  PubMed  Google Scholar 

  7. Douglass, A.D. & Vale, R.D. Single-molecule imaging of fluorescent proteins. Methods Cell Biol. 85, 113–125 (2008).

    Article  CAS  PubMed  Google Scholar 

  8. Douglass, A.D. & Vale, R.D. Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signaling molecules in T cells. Cell 121, 937–950 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Millius, A., Watanabe, N. & Weiner, O.D. Diffusion, capture and recycling of SCAR/WAVE and Arp2/3 complexes observed in cells by single-molecule imaging. J. Cell Sci. 125, 1165–1176 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Ueda, M., Sako, Y., Tanaka, T., Devreotes, P. & Yanagida, T. Single-molecule analysis of chemotactic signaling in Dictyostelium cells. Science 294, 864–867 (2001).

    Article  CAS  PubMed  Google Scholar 

  11. Uyemura, T., Takagi, H., Yanagida, T. & Sako, Y. Single-molecule analysis of epidermal growth factor signaling that leads to ultrasensitive calcium response. Biophys. J. 88, 3720–3730 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Jaqaman, K. et al. Cytoskeletal control of CD36 diffusion promotes its receptor and signaling function. Cell 146, 593–606 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Giannone, G. et al. Dynamic superresolution imaging of endogenous proteins on living cells at ultra-high density. Biophys. J. 99, 1303–1310 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Manley, S. et al. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat. Methods 5, 155–157 (2008).

    Article  CAS  PubMed  Google Scholar 

  15. Burnette, D.T. et al. A role for actin arcs in the leading-edge advance of migrating cells. Nat. Cell Biol. 13, 371–381 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Niu, L. & Yu, J. Investigating intracellular dynamics of FtsZ cytoskeleton with photoactivation single-molecule tracking. Biophys. J. 95, 2009–2016 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Sarov, M. et al. A genome-scale resource for in vivo tag-based protein function exploration in C. elegans. Cell 150, 855–866 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Bird, A.F. & Bird, J. The Structure of Nematodes 2nd edn. (Academic Press, 1991).

  19. Rappleye, C.A., Paredez, A.R., Smith, C.W., McDonald, K.L. & Aroian, R.V. The coronin-like protein POD-1 is required for anterior-posterior axis formation and cellular architecture in the nematode Caenorhabditis elegans. Genes Dev. 13, 2838–2851 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Tokunaga, M., Imamoto, N. & Sakata-Sogawa, K. Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat. Methods 5, 159–161 (2008).

    Article  CAS  PubMed  Google Scholar 

  21. Salbreux, G., Charras, G. & Paluch, E. Actin cortex mechanics and cellular morphogenesis. Trends Cell Biol. 22, 536–545 (2012).

    Article  CAS  PubMed  Google Scholar 

  22. Beers, M. & Kemphues, K. Depletion of the co-chaperone CDC-37 reveals two modes of PAR-6 cortical association in C. elegans embryos. Development 133, 3745–3754 (2006).

    Article  CAS  PubMed  Google Scholar 

  23. Nakayama, Y. et al. Dynamin participates in the maintenance of anterior polarity in the Caenorhabditis elegans embryo. Dev. Cell 16, 889–900 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Cheeks, R.J. et al. C. elegans PAR proteins function by mobilizing and stabilizing asymmetrically localized protein complexes. Curr. Biol. 14, 851–862 (2004).

    Article  CAS  PubMed  Google Scholar 

  25. Goehring, N.W., Hoege, C., Grill, S.W. & Hyman, A.A. PAR proteins diffuse freely across the anterior-posterior boundary in polarized C. elegans embryos. J. Cell Biol. 193, 583–594 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Pelletier, V., Gal, N., Fournier, P. & Kilfoil, M.L. Microrheology of microtubule solutions and actin-microtubule composite networks. Phys. Rev. Lett. 102, 188303 (2009).

    Article  PubMed  Google Scholar 

  27. Jaqaman, K. et al. Robust single-particle tracking in live-cell time-lapse sequences. Nat. Methods 5, 695–702 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Monnier, N. et al. Bayesian approach to MSD-based analysis of particle motion in live cells. Biophys. J. 103, 616–626 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Persson, F., Lindén, M., Unoson, C. & Elf, J. Extracting intracellular diffusive states and transition rates from single-molecule tracking data. Nat. Methods 10, 265–269 (2013).

    Article  PubMed  Google Scholar 

  30. Hoege, C. & Hyman, A.A. Principles of PAR polarity in Caenorhabditis elegans embryos. Nat. Rev. Mol. Cell Biol. 14, 315–322 (2013).

    Article  CAS  PubMed  Google Scholar 

  31. Kumfer, K.T. et al. CGEF-1 and CHIN-1 regulate CDC-42 activity during asymmetric division in the Caenorhabditis elegans embryo. Mol. Biol. Cell 21, 266–277 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Pollard, T.D. Mechanics of cytokinesis in eukaryotes. Curr. Opin. Cell Biol. 22, 50–56 (2010).

    Article  CAS  PubMed  Google Scholar 

  33. Guha, M., Zhou, M. & Wang, Y.L. Cortical actin turnover during cytokinesis requires myosin II. Curr. Biol. 15, 732–736 (2005).

    Article  CAS  PubMed  Google Scholar 

  34. Murthy, K. & Wadsworth, P. Myosin II-dependent localization and dynamics of F-actin during cytokinesis. Curr. Biol. 15, 724–731 (2005).

    Article  CAS  PubMed  Google Scholar 

  35. Danuser, G. & Waterman-Storer, C.M. Quantitative fluorescent speckle microscopy of cytoskeleton dynamics. Annu. Rev. Biophys. Biomol. Struct. 35, 361–387 (2006).

    Article  CAS  PubMed  Google Scholar 

  36. Giannone, G. et al. Dynamic superresolution imaging of endogenous proteins on living cells at ultra-high density. Biophys. J. 99, 1303–1310 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Dickinson, D.J., Ward, J.D., Reiner, D.J. & Goldstein, B. Engineering the Caenorhabditis genome using Cas9-triggered homologous recombination. Nat. Methods 10, 1028–1034 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Motegi, F. et al. Microtubules induce self-organization of polarized PAR domains in Caenorhabditis elegans zygotes. Nat. Cell Biol. 13, 1361–1367 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Tse, Y.C. et al. RhoA activation during polarization and cytokinesis of the early Caenorhabditis elegans embryo is differentially dependent on NOP-1 and CYK-4. Mol. Biol. Cell 23, 4020–4031 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Totong, R., Achilleos, A. & Nance, J. PAR-6 is required for junction formation but not apicobasal polarization in C. elegans embryonic epithelial cells. Development 134, 1259–1268 (2007).

    Article  CAS  PubMed  Google Scholar 

  42. Munro, E., Nance, J. & Priess, J.R. Cortical flows powered by asymmetrical contraction transport PAR proteins to establish and maintain anterior-posterior polarity in the early C. elegans embryo. Dev. Cell 7, 413–424 (2004).

    Article  CAS  PubMed  Google Scholar 

  43. Timmons, L. & Fire, A. Specific interference by ingested dsRNA. Nature 395, 854 (1998).

    Article  CAS  PubMed  Google Scholar 

  44. Kamath, R.S. et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231–237 (2003).

    Article  CAS  PubMed  Google Scholar 

  45. Sijen, T. et al. On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 107, 465–476 (2001).

    Article  CAS  PubMed  Google Scholar 

  46. Alder, M.N., Dames, S., Gaudet, J. & Mango, S.E. Gene silencing in Caenorhabditis elegans by transitive RNA interference. RNA 9, 25–32 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Oegema, K. & Hyman, A.A. in WormBook (ed. The C. elegans Research Community) 10.1895/wormbook.1.72.1 (2006).

  48. Crocker, J.C. & Grier, D.G. Methods of digital video microscopy for colloidal studies. J. Colloid Interface Sci. 179, 298–310 (1996).

    Article  CAS  Google Scholar 

  49. Saxton, M.J. Lateral diffusion in an archipelago. Biophys. J. 64, 1766–1780 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Huang, B., Wang, W., Bates, M. & Zhuang, X. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This research was supported by the US National Institutes of Health R01 grant GM098441 (to E.M.M.) and by the University of Chicago Materials Research Science & Engineering Center. We thank G. Seydoux (Johns Hopkins University), J. Nance (NYU School of Medicine), K. Kemphues (Cornell University), M. Glotzer and K. Longhini (University of Chicago) for strains; A. Sailer for technical support; N. Bartley, X. Zhang and W. Dong for their contributions to the initial stages of this project; and the Glotzer lab for sharing strains and reagents.

Author information

Authors and Affiliations

  1. Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois, USA

    François B Robin, William M McFadden, Baixue Yao, Edwin M Munro & Edwin M Munro

  2. Biophysics Graduate Program, University of Chicago, Chicago, Illinois, USA

    William M McFadden

  3. Institute for Biophysical Dynamics, University of Chicago, Chicago, Illinois, USA

    Edwin M Munro

  4. Computation Institute, University of Chicago, Chicago, Illinois, USA

    Edwin M Munro

Authors
  1. François B Robin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. William M McFadden
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Baixue Yao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Edwin M Munro
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

E.M.M. conceived the imaging approach and provided overall guidance. F.B.R. conceived and developed the smPReSS approach and, with E.M.M., developed the tracking-based turnover analysis. F.B.R. and W.M.M. acquired the data for actin; B.Y. and F.B.R. acquired the data for PAR-6; W.M.M., F.B.R. and B.Y. performed the nonlinear fitting; E.M.M., F.B.R., B.Y. and W.M.M. developed and performed the mobility analysis. F.B.R. performed the tracking-based turnover analysis, and F.B.R., W.M.M., B.Y. and E.M.M. made the figures. E.M.M. and F.B.R. wrote the text.

Corresponding author

Correspondence to Edwin M Munro.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Notes 1 and 2 (PDF 2656 kb)

Single molecule movie of PAR-6::GFP

A one cell embryo during maintenance phase expressing PAR-6::GFP at single molecule levels and imaged for 20 s at 30 frames per second, at 100% laser power. Replay speed is 1×. Image size = 331 × 231 pixels, pixel size = 107 nm. Anterior is to the left. Note the range of particle behaviors and mobilities are readily apparent (see main text, and Supplementary Video 3,4). (MOV 13657 kb)

Single molecule movie of GFP::Actin.

A one-cell embryo expressing GFP::Actin at single molecule levels and depleted of NMY-2 by RNAi. The embryo was imaged for 500s at 10 frames per second, at 30% laser power, beginning in maintenance phase and proceeding through anaphase and into telophase. Replay speed is 12×. Image size = 424 × 267 pixels, pixel size = 107 nm. (MOV 22442 kb)

Single molecule movie of GFP::Actin.

Same embryo as in Video 2, showing the entire acquisition sequence. Replay speed is 48×. Image size = 424 × 267 pixels, pixel size = 107 nm. Note that actin is initially evenly distributed along the anterior posterior axis, but during anaphase and telophase, it accumulates at the equator and is depleted from the poles. (MOV 21895 kb)

A single PAR-6::GFP molecule displaying simple diffusive behavior.

Original image size: 45 × 45 pixels, pixel size: 107 nm. Imaging conditions as in Supplementary Video 1. Transient failures to detect the particle in a given frame due to motion blur are indicated by the absence of a red circle. (MOV 2140 kb)

A single PAR-6::GFP molecule displaying sub-diffusive behavior.

Original image size: 45 × 45 pixels, pixel size: 107 nm. Imaging conditions as in Supplementary Video 1. For this slower moving particle, there are no detection failures. (MOV 1577 kb)

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Robin, F., McFadden, W., Yao, B. et al. Single-molecule analysis of cell surface dynamics in Caenorhabditis elegans embryos. Nat Methods 11, 677–682 (2014). https://doi.org/10.1038/nmeth.2928

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.2928

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing