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Engineering a polarity-sensitive biosensor for time-lapse imaging of apoptotic processes and degeneration

Nature Methods volume 7, pages 67–73 (2010)Cite this article

Abstract

Apoptosis is of central importance to many areas of biological research, but there is a lack of methods that permit continuous monitoring of apoptosis or cell viability in a nontoxic and noninvasive manner. Here we report the development of a tool applicable to live-cell imaging that facilitates the visualization of real-time apoptotic changes without perturbing the cellular environment. We designed a polarity-sensitive annexin-based biosensor (pSIVA) with switchable fluorescence states, which allows detection only when bound to apoptotic cells. Using pSIVA with live-cell imaging, we observed dynamic local changes in individual rat neurons during degeneration in vitro and in vivo. Furthermore, we observed that pSIVA binding was reversible and clearly defined the critical period for neurons to be rescued. We anticipate pSIVA can be widely applied to address questions concerning spatiotemporal events in apoptotic processes, its reversibility and the general viability of cells in culture.

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Figure 1: Structure-based design and fluorescence properties of annexin-based pSIVA biosensors.
Figure 2: Application of pSIVAm to real-time live-cell imaging of apoptosis in COS-7 cells.
Figure 3: Application of pSIVAm to monitoring the degeneration of DRG neurons.
Figure 4: In vivo imaging of axonal degeneration using pSIVAm.
Figure 5: Rescue of neuronal degeneration visualized by pSIVAm.

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Acknowledgements

We thank the members of the Chan lab for technical assistance and M. Cayouette for commenting on the manuscript. This work was partially supported by the US National Multiple Sclerosis Society Career Transition Award (J.R.C.), EY12155 (J.C.). J.R.C. is a Harry Weaver Neuroscience Scholar (TA 3008A/T, JF 2142-A-2).

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Authors and Affiliations

  1. Departments of Biochemistry and Molecular Biology, Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, California, USA

    Yujin E Kim, Jonah R Chan & Ralf Langen

  2. Department of Cell and Neurobiology, Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, California, USA

    Jeannie Chen

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  1. Yujin E Kim
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Contributions

R.L. conceived the study and, together with J.C. and J.R.C., supervised the project. Y.E.K. designed and performed the experiments and, together with J.R.C., designed and performed the experiments involving neurons. R.L., J.C., J.R.C. and Y.E.K. analyzed the data. Y.E.K. wrote the paper.

Corresponding authors

Correspondence to Jonah R Chan or Ralf Langen.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 and Supplementary Table 1 (PDF 7788 kb)

Supplementary Video 1

Time-lapse imaging of DRG neurons undergoing apoptosis (axon terminals). pSIVA staining of PS exposure on degenerating axons is shown in green, and PI staining of nuclei after loss of membrane integrity is shown in red. Images were taken 9 h after initial NGF deprivation, and the movie was compiled at a speed of 6 frames per second, with a 20 min interval between frames (images). (MOV 383 kb)

Supplementary Video 2

Time-lapse imaging of DRG neurons undergoing apoptosis. pSIVA staining of PS exposure is shown in green, and PI staining of nuclei after loss of membrane integrity is shown in red. Images were taken 9 h after initial NGF deprivation, and the movie was compiled at a speed of 6 frames per second, with a 20 min interval between frames (images). (MOV 702 kb)

Supplementary Video 3

Recovery of DRG neurons from 10 h of NGF deprivation. Neurons were deprived of NGF for 10 h to induce apoptosis before NGF was subsequently added back to the culture medium to induce rescue. pSIVA staining of PS exposure on degenerating axons is shown in green, and PI staining of nuclei after loss of membrane integrity is shown in red. Images were taken 7.5 h after initial NGF deprivation, and the movie was compiled at a speed of 4 frames per second, with a 30 min interval between frames (images). (MOV 710 kb)

Supplementary Video 4

Recovery of DRG neurons from 15 h of NGF deprivation. Neurons were deprived of NGF for 15 h to induce apoptosis before NGF was subsequently added back to the culture medium to induce rescue. pSIVA staining of PS exposure on degenerating axons is shown in green, and PI staining of nuclei after loss of membrane integrity is shown in red. Images were taken 7.5 h after initial NGF deprivation, and the movie was compiled at a speed of 4 frames per second, with a 30 min interval between frames (images). Boxes highlight specific areas where PS exposure occurs on the axon and reverses. (MOV 636 kb)

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Kim, Y., Chen, J., Chan, J. et al. Engineering a polarity-sensitive biosensor for time-lapse imaging of apoptotic processes and degeneration. Nat Methods 7, 67–73 (2010). https://doi.org/10.1038/nmeth.1405

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