NEWS & VIEWS
doi:10.1038/nature13213
CE LL B IO LO GY
The disassembly of death During the cell-death program known as apoptosis, cells break up into membrane-bound fragments. It emerges that this process is controlled by the protein pannexin 1 and can be deregulated by an antibiotic. CHRISTOPHER D. GREGORY
C
ell death is a feature of normal life. Around the clock, cells are disassembled by an evolutionarily conserved controlled-death program called apoptosis, which ensures proper regulation of the size and quality of cell populations in tissues. Much is known about the molecular mechanisms that trigger this program, but it is unclear how dying cells are dismantled in a controlled manner once apoptosis is under way. In a paper published on Nature’s website today, Poon et al.1 demonstrate a central role for a cellmembrane channel, the pannexin 1 protein, in inhibiting cell disassembly during apoptosis, and report that, unexpectedly, a member of a commonly prescribed class of antibiotic is able to modulate the activity of such channels. Pannexin 1 (PANX1) channels are complex structures that span the plasma membrane of cells, regulating the movement of small molecules of various sizes between the intracellular and extracellular milieux. These channels are best known for controlling the release of ATP, a multifunctional energy-carrying and signalling molecule. It was previously shown2,3 that PANX1 channels seemingly open up to release ATP from the cell during apoptosis. This release acts as a ‘find-me’ signal for phagocytes — cells that seek out, engulf and digest apoptotic cells and their fragments (Fig. 1a). In addition to ATP, PANX1 channels are likely to control the efflux and influx of a wide variety of bioactive molecules from many cell types in various states of activation and differentiation, as well as cell death4. Conveniently, PANX1channel opening can be monitored using surrogate markers, because fluorescent DNAbinding dyes such as TO-PRO-3 are among the molecules that selectively enter cells through these channels1,3. In their groundbreaking paper, Poon and colleagues demonstrate that rigorous molecular screening, when coupled with the power of observation in a well-regulated setting, can uncover surprising and invaluable findings. The authors designed an assay in which uptake
of TO-PRO-3 into apoptotic human cells was monitored as an indicator of PANX1-channel activity. They subjected this assay to a widely available library of 1,280 small molecules, including the quinolone-based antibiotic, trovafloxacin. Unexpectedly, trovafloxacin proved to be a potent and direct pharmacological inhibitor of PANX1 channels. The authors demonstrated that this role was independent of apoptosis, because trovafloxacin was able to inhibit individual, non-apoptotic channels.
Poon and co-workers went on to study the effects of trovafloxacin on apoptotic cells and discovered that PANX1 channels not only regulate ATP release during apoptosis, but also control the formation of apoptotic bodies, membrane-bound cell fragments that bud off from the cell during apoptosis. Inhibition of normal PANX1-channel function with trovafloxacin resulted in a large increase in the number of apoptotic bodies formed, causing dysregulated fragmentation of the cell (Fig. 1b). The authors also found that active PANX1 channels prevent the formation of string-like structures (which they term apoptopodia) that arise during inhibition of channel activity in apoptotic cells. Somehow apoptopodia prevent the detachment of blebs — irregular bulges in the plasma membrane — that commonly appear at cell surfaces during the early stages of apoptosis. The purpose of apoptotic bodies is currently unknown. Each apoptotic body has a diverse mixture of components, and may contain DNA, fragments of the cell nucleus, other intact organelles and a wide array of
a PANX1 channel Viable cell
Nucleus
Nuclear fragments
ATP ‘find-me’ signal
Apoptosis TO-PRO-3 Apoptotic bodies
b
Phagocyte
Trovafloxacin Nuclear fragments Trovafloxacin
Apoptosis Dysregulated fragmentation
Apoptopodia
Figure 1 | PANX1 channels regulate cell disassembly. a, PANX1 channels control the passage of molecules across the membrane in viable cells (green arrows). When cells die by apoptosis, membranebound fragments called apoptotic bodies bud off from the cell. PANX1 channels release ATP molecules from the cell and allow entry of selected molecules such as the fluorescent dye TO-PRO-3 (blue arrows). ATP acts as a ‘find-me’ signal to phagocytes that engulf both the dying cell and the apoptotic bodies. b, Poon et al.1 report that PANX1-channel function is inhibited by the antibiotic trovafloxacin, which modulates the channel’s activity in viable cells (red arrows). Channel inhibition also results in the formation of many more apoptotic bodies during apoptosis, leading to dysregulated fragmentation of the cell. The study reveals that apoptotic bodies form in association with string-like structures, termed apoptopodia. | NAT U R E | 1
© 2014 Macmillan Publishers Limited. All rights reserved
RESEARCH NEWS & VIEWS intracellular proteins and other molecules, all encapsulated by portions of the membrane that previously surrounded the living cell. Although the bodies are rapidly ‘gobbled up’ by phagocytes, whole dying cells can also be engulfed effectively; in fact, not all apoptotic cells break up. Perhaps apoptotic bodies are more efficiently degraded than whole apoptotic cells within the phagocyte, or maybe they are targeted or degraded differently. Apoptotic bodies may also be important in controlling immune responses to apoptotic cells5. It is possible that apoptotic bodies retain biological functions and act at sites well away from the dying cell. Given that apoptosis can drive cell regeneration in tissues6 — an attractive replacement of the old with the new — it is particularly tempting to suggest that apoptotic-cell-derived bodies transport active components that contribute to these regenerative processes. Small vesicles have been found by others to export bioactive cargoes from nonapoptotic cells in response to various stimuli7. Although such vesicles are generally smaller than apoptotic bodies, the two types have certain structural characteristics in common. Exactly how PANX1 channels contribute to
the functional (including possible pathological) attributes of apoptotic bodies, and perhaps other extracellular vesicles, will be valuable avenues of future investigation. Poon and colleagues’ work raises an issue of relevance to improved antibiotic design. Trovaf loxacin, a highly effective, broadspectrum fluoroquinolone antibiotic, was withdrawn because of adverse side effects in humans8. Fluoroquinolones in general represent some of the most commonly prescribed antibiotics, but they carry the risk of severe side effects that can harm multiple organ systems9. Could abrogating unwanted targeting of PANX1 channels by these drugs offer an opportunity to develop improved antibiotics? As yet, we do not know whether dysregulated production of apoptotic bodies has pathological consequences, or whether another, unidentified mechanism could cause adverse clinical effects when patients are treated with trovafloxacin — a mechanism that might be dependent or independent of PANX1channel inhibition. What is crucial to determine is the functional diversity of PANX1 channels, and the pathological consequences of their blockade.
2 | NAT U R E |
© 2014 Macmillan Publishers Limited. All rights reserved
As always, the unexpected is the most fascinating. There is no doubt that this work will stimulate researchers in the seemingly disparate areas of apoptosis and antibiotics to find out more about PANX1. The results may improve not only scientific understanding but also health care. ■ Christopher D. Gregory is at the MRC/University of Edinburgh Centre for Inflammation Research, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh EH16 4TJ, UK. e-mail: [email protected] 1. Poon, I. K. H. et al. Nature http://dx.doi. org/10.1038/nature13147 (2014). 2. Elliott, M. R. et al. Nature 461, 282–286 (2009). 3. Chekeni, F. B. et al. Nature 467, 863–867 (2010). 4. Sandilos, J. K. & Bayliss, D. A. J. Physiol. (Lond.) 590, 6257–6266 (2012). 5. Wickman, G., Julian, L. & Olson, M. F. Cell Death Differ. 19, 735–742 (2012). 6. King, R. S. & Newmark, P. A. J. Cell Biol. 196, 553–562 (2012). 7. Hugel, B., Martínez, M. C., Kunzelmann, C. & Freyssinet, J. M. Physiology 20, 22–27, (2005). 8. Lucena, M. I. et al. Clin. Infect. Dis. 30, 400–401 (2000). 9. Liu, H. H. Drug Safety 33, 353–369 (2010).
doi:10.1038/nature13213
CE LL B IO LO GY
The disassembly of death During the cell-death program known as apoptosis, cells break up into membrane-bound fragments. It emerges that this process is controlled by the protein pannexin 1 and can be deregulated by an antibiotic. CHRISTOPHER D. GREGORY
C
ell death is a feature of normal life. Around the clock, cells are disassembled by an evolutionarily conserved controlled-death program called apoptosis, which ensures proper regulation of the size and quality of cell populations in tissues. Much is known about the molecular mechanisms that trigger this program, but it is unclear how dying cells are dismantled in a controlled manner once apoptosis is under way. In a paper published on Nature’s website today, Poon et al.1 demonstrate a central role for a cellmembrane channel, the pannexin 1 protein, in inhibiting cell disassembly during apoptosis, and report that, unexpectedly, a member of a commonly prescribed class of antibiotic is able to modulate the activity of such channels. Pannexin 1 (PANX1) channels are complex structures that span the plasma membrane of cells, regulating the movement of small molecules of various sizes between the intracellular and extracellular milieux. These channels are best known for controlling the release of ATP, a multifunctional energy-carrying and signalling molecule. It was previously shown2,3 that PANX1 channels seemingly open up to release ATP from the cell during apoptosis. This release acts as a ‘find-me’ signal for phagocytes — cells that seek out, engulf and digest apoptotic cells and their fragments (Fig. 1a). In addition to ATP, PANX1 channels are likely to control the efflux and influx of a wide variety of bioactive molecules from many cell types in various states of activation and differentiation, as well as cell death4. Conveniently, PANX1channel opening can be monitored using surrogate markers, because fluorescent DNAbinding dyes such as TO-PRO-3 are among the molecules that selectively enter cells through these channels1,3. In their groundbreaking paper, Poon and colleagues demonstrate that rigorous molecular screening, when coupled with the power of observation in a well-regulated setting, can uncover surprising and invaluable findings. The authors designed an assay in which uptake
of TO-PRO-3 into apoptotic human cells was monitored as an indicator of PANX1-channel activity. They subjected this assay to a widely available library of 1,280 small molecules, including the quinolone-based antibiotic, trovafloxacin. Unexpectedly, trovafloxacin proved to be a potent and direct pharmacological inhibitor of PANX1 channels. The authors demonstrated that this role was independent of apoptosis, because trovafloxacin was able to inhibit individual, non-apoptotic channels.
Poon and co-workers went on to study the effects of trovafloxacin on apoptotic cells and discovered that PANX1 channels not only regulate ATP release during apoptosis, but also control the formation of apoptotic bodies, membrane-bound cell fragments that bud off from the cell during apoptosis. Inhibition of normal PANX1-channel function with trovafloxacin resulted in a large increase in the number of apoptotic bodies formed, causing dysregulated fragmentation of the cell (Fig. 1b). The authors also found that active PANX1 channels prevent the formation of string-like structures (which they term apoptopodia) that arise during inhibition of channel activity in apoptotic cells. Somehow apoptopodia prevent the detachment of blebs — irregular bulges in the plasma membrane — that commonly appear at cell surfaces during the early stages of apoptosis. The purpose of apoptotic bodies is currently unknown. Each apoptotic body has a diverse mixture of components, and may contain DNA, fragments of the cell nucleus, other intact organelles and a wide array of
a PANX1 channel Viable cell
Nucleus
Nuclear fragments
ATP ‘find-me’ signal
Apoptosis TO-PRO-3 Apoptotic bodies
b
Phagocyte
Trovafloxacin Nuclear fragments Trovafloxacin
Apoptosis Dysregulated fragmentation
Apoptopodia
Figure 1 | PANX1 channels regulate cell disassembly. a, PANX1 channels control the passage of molecules across the membrane in viable cells (green arrows). When cells die by apoptosis, membranebound fragments called apoptotic bodies bud off from the cell. PANX1 channels release ATP molecules from the cell and allow entry of selected molecules such as the fluorescent dye TO-PRO-3 (blue arrows). ATP acts as a ‘find-me’ signal to phagocytes that engulf both the dying cell and the apoptotic bodies. b, Poon et al.1 report that PANX1-channel function is inhibited by the antibiotic trovafloxacin, which modulates the channel’s activity in viable cells (red arrows). Channel inhibition also results in the formation of many more apoptotic bodies during apoptosis, leading to dysregulated fragmentation of the cell. The study reveals that apoptotic bodies form in association with string-like structures, termed apoptopodia. | NAT U R E | 1
© 2014 Macmillan Publishers Limited. All rights reserved
RESEARCH NEWS & VIEWS intracellular proteins and other molecules, all encapsulated by portions of the membrane that previously surrounded the living cell. Although the bodies are rapidly ‘gobbled up’ by phagocytes, whole dying cells can also be engulfed effectively; in fact, not all apoptotic cells break up. Perhaps apoptotic bodies are more efficiently degraded than whole apoptotic cells within the phagocyte, or maybe they are targeted or degraded differently. Apoptotic bodies may also be important in controlling immune responses to apoptotic cells5. It is possible that apoptotic bodies retain biological functions and act at sites well away from the dying cell. Given that apoptosis can drive cell regeneration in tissues6 — an attractive replacement of the old with the new — it is particularly tempting to suggest that apoptotic-cell-derived bodies transport active components that contribute to these regenerative processes. Small vesicles have been found by others to export bioactive cargoes from nonapoptotic cells in response to various stimuli7. Although such vesicles are generally smaller than apoptotic bodies, the two types have certain structural characteristics in common. Exactly how PANX1 channels contribute to
the functional (including possible pathological) attributes of apoptotic bodies, and perhaps other extracellular vesicles, will be valuable avenues of future investigation. Poon and colleagues’ work raises an issue of relevance to improved antibiotic design. Trovaf loxacin, a highly effective, broadspectrum fluoroquinolone antibiotic, was withdrawn because of adverse side effects in humans8. Fluoroquinolones in general represent some of the most commonly prescribed antibiotics, but they carry the risk of severe side effects that can harm multiple organ systems9. Could abrogating unwanted targeting of PANX1 channels by these drugs offer an opportunity to develop improved antibiotics? As yet, we do not know whether dysregulated production of apoptotic bodies has pathological consequences, or whether another, unidentified mechanism could cause adverse clinical effects when patients are treated with trovafloxacin — a mechanism that might be dependent or independent of PANX1channel inhibition. What is crucial to determine is the functional diversity of PANX1 channels, and the pathological consequences of their blockade.
2 | NAT U R E |
© 2014 Macmillan Publishers Limited. All rights reserved
As always, the unexpected is the most fascinating. There is no doubt that this work will stimulate researchers in the seemingly disparate areas of apoptosis and antibiotics to find out more about PANX1. The results may improve not only scientific understanding but also health care. ■ Christopher D. Gregory is at the MRC/University of Edinburgh Centre for Inflammation Research, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh EH16 4TJ, UK. e-mail: [email protected] 1. Poon, I. K. H. et al. Nature http://dx.doi. org/10.1038/nature13147 (2014). 2. Elliott, M. R. et al. Nature 461, 282–286 (2009). 3. Chekeni, F. B. et al. Nature 467, 863–867 (2010). 4. Sandilos, J. K. & Bayliss, D. A. J. Physiol. (Lond.) 590, 6257–6266 (2012). 5. Wickman, G., Julian, L. & Olson, M. F. Cell Death Differ. 19, 735–742 (2012). 6. King, R. S. & Newmark, P. A. J. Cell Biol. 196, 553–562 (2012). 7. Hugel, B., Martínez, M. C., Kunzelmann, C. & Freyssinet, J. M. Physiology 20, 22–27, (2005). 8. Lucena, M. I. et al. Clin. Infect. Dis. 30, 400–401 (2000). 9. Liu, H. H. Drug Safety 33, 353–369 (2010).
