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Characteristics of intracellular propagation of mitochondrial BAX recruitment during apoptosis

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Abstract

Recent advancements in live cell imaging technologies have identified the phenomenon of intracellular propagation of late apoptotic events, such as cytochrome c release and caspase activation. The mechanism, prevalence, and speed of apoptosis propagation remain unclear. Additionally, no studies have demonstrated propagation of the pro-apoptotic protein, BAX. To evaluate the role of BAX in intracellular apoptotic propagation, we used high speed live-cell imaging to visualize fluorescently tagged-BAX recruitment to mitochondria in four immortalized cell lines. We show that propagation of mitochondrial BAX recruitment occurs in parallel to cytochrome c and SMAC/Diablo release and is affected by cellular morphology, such that cells with processes are more likely to exhibit propagation. The initiation of propagation events is most prevalent in the distal tips of processes, while the rate of propagation is influenced by the 2-dimensional width of the process. Propagation was rarely observed in the cell soma, which exhibited near synchronous recruitment of BAX. Propagation velocity is not affected by mitochondrial volume in segments of processes, but is negatively affected by mitochondrial density. There was no evidence of a propagating wave of increased levels of intracellular calcium ions. Alternatively, we did observe a uniform increase in superoxide build-up in cellular mitochondria, which was released as a propagating wave simultaneously with the propagating recruitment of BAX to the mitochondrial outer membrane.

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Data availability

The datasets generated and analyzed for the current study are available from the corresponding author on reasonable request.

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Acknowledgements

This work was supported by National Institute of Health grants R01 EY030123, P30 EY016665, and T32 GM081061, an unrestricted research grant from Research to Prevent Blindness, Inc., and the Frederick A. Davis Endowment from the Department of Ophthalmology and Visual Sciences at the University of Wisconsin-Madison.

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Authors and Affiliations

  1. Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, 1300 University Ave, Madison, WI, 53706, USA

    Joshua A. Grosser & Robert W. Nickells

  2. Wake Forest University School of Medicine, Winston-Salem, NC, USA

    Joshua A. Grosser

  3. Institute of Science and Technology Austria, Klosterneuburg, Austria

    Margaret E. Maes

  4. McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, WI, USA

    Robert W. Nickells

Authors
  1. Joshua A. Grosser
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  2. Margaret E. Maes
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  3. Robert W. Nickells
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Contributions

J.G. and M.M. collected data. J.G. analyzed data. J.G. and R.N. designed experiments, interpreted results, and wrote the manuscript. All authors approved of the final draft.

Corresponding author

Correspondence to Robert W. Nickells.

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The authors declare no conflict of interest in the execution of this work. No patients or animals were used in these studies.

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Supplementary Information

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Supplementary file1 (jpg 1494 KB)

Live-cell imaging of the cell in Figure 1. Size bars are 5 µm (mov 4261 KB)

Example of the propagation of both BAX recruitment and SMAC/Diablo release. This cell was nucleofected with mCherry-BAX, SMAC/Diablo-GFP, and Mito-BFP and induced to die using 1 µM STS. BAX recruitment and SMAC release initiated 14.5 hrs post-STS on the left side of the image and propagated across the cellular chain over the course of approximately 15 minutes (mov 3916 KB)

Live-cell imaging of the cell in Figure 3. Size bars are 10 µm (mov 7104 KB)

Live-cell imaging of the cell in Figure 6. Size bars are 5 µm (mov 7708 KB)

Live-cell imaging of the cell in Figure 7. Size bars are 5 µm (mov 12484 KB)

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Grosser, J.A., Maes, M.E. & Nickells, R.W. Characteristics of intracellular propagation of mitochondrial BAX recruitment during apoptosis. Apoptosis 26, 132–145 (2021). https://doi.org/10.1007/s10495-020-01654-w

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