- Email: [email protected]
Phagocytosis: Coupling of Mitochondrial Uncoupling and Engulfment
Current Biology Vol 21 No 20 R852
agreement with this notion, Silva et al. [12] observed that reprogramming of fibroblasts from a 129/MF1 hybrid strain of mice gives rise mostly to partially reprogrammed iPSCs that rarely progress to pluripotency unless treated with 2i. Similarly, the establishment of rat iPSCs requires 2i culture. What consequences might these findings have for human ESC/iPSC research? Human ESCs are fundamentally different from mouse ESCs in that they require bFgf and Activin A for their stable propagation [1]. Interestingly, exposure of mouse blastocysts, ESCs or postimplantation embryos to bFgf and Activin A gives rise to so-called epiblast stem cells (EpiSCs) [13,14], which are very similar to human ESCs and seem to represent a developmentally more advanced or ‘primed’ state compared with the more primitive or ‘naive’ state of mouse ESCs [15]. The findings by Chu et al. [6] thus raise the interesting possibility that progression of epiblast cells towards a germ-cell fate, either by enforced expression of certain transcription factors [1,9] or by exposure of cells to germ-cell-inducing cytokines, might be sufficient to derive stable naive ESC/iPSC lines in humans and other species. Recent exciting progress in
identifying molecules that coax pluripotent cells into germ cells may aid in these efforts [16]. References 1. Stadtfeld, M., and Hochedlinger, K. (2010). Induced pluripotency: history, mechanisms, and applications. Genes Dev. 24, 2239–2263. 2. Zwaka, T.P., and Thomson, J.A. (2005). A germ cell origin of embryonic stem cells? Development 132, 227–233. 3. Buehr, M., and Smith, A. (2003). Genesis of embryonic stem cells. Phil. Trans. R. Soc. Lond. 358, 1397–1402, discussion 1402. 4. Brook, F.A., and Gardner, R.L. (1997). The origin and efficient derivation of embryonic stem cells in the mouse. Proc. Natl. Acad. Sci. USA 94, 5709–5712. 5. Durcova-Hills, G., Tang, F., Doody, G., Tooze, R., and Surani, M.A. (2008). Reprogramming primordial germ cells into pluripotent stem cells. PLoS One 3, e3531. 6. Chu, L.F., Surani, M.A., Jaenisch, R., and Zwaka, T.P. (2011). Blimp1 expression predicts embryonic stem cell development in vitro. Curr. Biol. 21, 1759–1765. 7. Saitou, M., Payer, B., O’Carroll, D., Ohinata, Y., and Surani, M.A. (2005). Blimp1 and the emergence of the germ line during development in the mouse. Cell Cycle 4, 1736–1740. 8. Ying, Q.L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J., Cohen, P., and Smith, A. (2008). The ground state of embryonic stem cell self-renewal. Nature 453, 519–523. 9. Nichols, J., and Smith, A. (2011). The origin and identity of embryonic stem cells. Development 138, 3–8. 10. Buehr, M., Meek, S., Blair, K., Yang, J., Ure, J., Silva, J., McLay, R., Hall, J., Ying, Q.L., and Smith, A. (2008). Capture of authentic embryonic stem cells from rat blastocysts. Cell 135, 1287–1298. 11. Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse
Phagocytosis: Coupling of Mitochondrial Uncoupling and Engulfment Clearance of apoptotic cells by phagocytes avoids triggering an inflammatory response. A new study reveals that phagocytes dissipate their mitochondrial proton electrochemical gradient to allow for the ingestion of more apoptotic corpses. Mitochondria are therefore involved in all aspects of apoptosis, from its activation through to the phagocytosis of dead cells. Grazia M. Cereghetti and Luca Scorrano Sustained cell proliferation during development, tissue renewal or in the course of the immune response is accompanied by the production of excess or damaged cells that die by apoptosis. The accumulation of these cells may lead to tissue damage and inflammation: specialized systems therefore efficiently remove them [1,2].
Phagocytes are deputed to the clearance of apoptotic cells and are able to engulf multiple cells in order to adapt their ‘cleaning efficiency’ to the rate of apoptotic cell accumulation. In recent years, some of the crucial steps in the phagocytosis of apoptotic cells, as well as the principal players in the phagocytic process, have been elucidated [3,4]. Dying cells release signals to attract the motile phagocytes. The two cells make
12.
13.
14.
15.
16.
embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676. Silva, J., Barrandon, O., Nichols, J., Kawaguchi, J., Theunissen, T.W., and Smith, A. (2008). Promotion of reprogramming to ground state pluripotency by signal inhibition. PLoS Biol. 6, e253. Brons, I.G., Smithers, L.E., Trotter, M.W., Rugg-Gunn, P., Sun, B., Chuva de Sousa Lopes, S.M., Howlett, S.K., Clarkson, A., Ahrlund-Richter, L., Pedersen, R.A., et al. (2007). Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448, 191–195. Tesar, P.J., Chenoweth, J.G., Brook, F.A., Davies, T.J., Evans, E.P., Mack, D.L., Gardner, R.L., and McKay, R.D. (2007). New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196–199. Nichols, J., and Smith, A. (2009). Naive and primed pluripotent states. Cell Stem Cell 4, 487–492. Hayashi, K., Ohta, H., Kurimoto, K., Aramaki, S., and Saitou, M. (2011). Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 146, 519–532.
1Howard Hughes Medical Institute and Department of Stem Cell and Regenerative Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA. 2Massachusetts General Hospital Cancer Center and Center for Regenerative Medicine, 185 Cambridge Street, Boston, MA 02114, USA. 3Harvard Stem Cell Institute, 42 Church Street, Cambridge, MA 02138, USA. E-mail: [email protected]. edu
DOI: 10.1016/j.cub.2011.09.024
physical contact via markers that are released from the apoptotic cell and bind to receptors on the phagocyte, inducing a signaling cascade that prepares the phagocyte membrane for the internalization of the dead cell. Several molecules are involved in engulfment by phagocytes, including: brain angiogenesis inhibitor 1 (BAI1), a transmembrane protein highly expressed in the brain; Rac GTPases, which remodel the cytoskeleton; ELMO, an evolutionarily conserved cytoplasmic engulfment protein; and the unconventional guanine nucleotide exchange factor and Rho GTPase activator Dock180 [5]. Despite our knowledge of some key molecular steps in the engulfment cascade, how a single phagocyte can serially internalize many apoptotic cells is unclear. Park et al. [6] have now shown, in a recent issue of Nature, that serial internalization of apoptotic cells unexpectedly depends on
Dispatch R853
mitochondrial activity in the phagocyte, and that a key player in this process is uncoupling protein 2 (Ucp2) [6]. Mitochondria are important organelles with several cellular functions besides energy conversion, ranging from metabolism to calcium signaling to their key role in amplification of apoptosis. Their unique ability to efficiently synthesize ATP depends on the accumulation of free energy in the form of a proton w electrochemical gradient (Dm H+ ) across the inner mitochondrial membrane that is then converted by the ATP synthase into the chemical energy of the thiophosphoric bond. The second law of thermodynamics dictates that futile dissipation of this gradient will result in its conversion into other forms of energy, such as thermal energy. Nature clearly exploited this possibility in a regulated fashion, by means of uncoupling proteins. Ucp1, also known as thermogenin, is a 32 kDa inner mitochondrial membrane protein present almost exclusively in brown adipose tissue, where it participates in thermogenesis by catalysing the net transfer of protons from the mitochondrial intermembrane space to the matrix [7]. The Ucp1 homologue Ucp2 is also a carrier for protons and small molecules across the inner mitochondrial membrane. Unlike Ucp1, Ucp2 and Ucp3 are expressed in multiple tissues, leading to questions about their role: they have indeed been reported to be involved in different processes, such as modulation of insulin secretion [8] and of mitochondrial calcium uptake [9]. In particular, functions attributed to Ucp2 are related to its ability to uncouple ATP production from the proton electrochemical gradient [10–13]. In the new study, Park et al. [6] asked whether mitochondrial metabolism could play a role in the phagocytosis of apoptotic cells and analyzed the mitochondrial membrane potential (Dcm), i.e. the electrical component w of the Dm H+ , in LR73 phagocytes upon internalization of an apoptotic cell. Surprisingly, Dcm increased specifically when an apoptotic corpse was being phagocytosed: other synthetic targets did not alter Dcm, in spite of a complete activation of the engulfment machinery. While it is not clear what drives this increase in Dcm, its subsequent return to baseline is very slow and is not connected to a net increase in total cellular ATP levels
Phagocyte
3 Ucp2 Tim4 Apoptotic cell
4 H+
Mitochondrion
5 Δψm 1 Apoptotic cell recognition
2 Δψ m
Current Biology
Figure 1. Apoptotic cell engulfment capacity relies on the mitochondrial membrane potential (Dcm), which is influenced by Ucp2. Internalization of apoptotic cells by phagocytes (1) is followed by phagocyte mitochondrial membrane hyperpolarization (2), which blocks the engulfment of further cells. Upregulation of Ucp2 (3) and dissipation of the proton gradient as a consequence of proton leak in the mitochondrial matrix (4) lowers Dcm (5), resulting in an as yet unidentified ‘continue-to-eat’ signal.
but is associated with increased expression of Ucp2. Moreover, artificial overexpression of Ucp2 increased the rate and capacity of internalization by phagocytes, suggesting a specific role for Ucp2 and for dissipation of the electrochemical gradient in this process of increased ‘phagocytic capacitance’ (Figure 1). Ravichandran and coworkers [6] exclude that Ucp2 regulates phagocytosis by altering production of reactive oxygen species (ROS) or lipid oxidation [11] and were able to recapitulate the stimulation of phagocytosis by using synthetic uncouplers like 2,4-dinitrophenol (2,4-DNP) and carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), further substantiating the proposal that Ucp2 (unlike its homologues Ucp1 and Ucp3) modulates phagocytosis via its primary w effect on DmH+ . While the result strongly argues for a key role for mitochondria in this process, it should be noted that the synthetic uncouplers have powerful side effects, and it is surprising that the phagocytes can survive upon complete uncoupling or upon poisoning of the respiratory chain with azide. It is likely that the excess
glucose in the culture medium allows cells to rely solely on glycolysis to generate ATP. Would the role of Ucp2 w (and of Dm H+ ) be maintained under more physiological conditions in which mitochondria are used as the main ATP source by the cell? A first answer to this question comes from the analysis of the effects of Ucp2 ablation on waste elimination in tissues. Ucp2-/- mice are viable but present higher ROS production and altered macrophagemediated immunity [14]. The Dcm of bone-marrow-derived macrophages (BMDMs) from Ucp2-/- mice is higher and these cells also have a decreased ability to engulf apoptotic cells compared with control cells. Dexamethasone injection induces rapid death of thymocytes in vivo [15], followed by clearance of dead cells in control mice, but not in Ucp2-/- animals. Similarly, induction of germ cell apoptosis via testicular torsion is followed by accumulation of dying germ cells in seminiferous tubules in Ucp2-/- mice. How is Ucp2 upregulated upon engulfment of apoptotic corpses? What is the mechanism granting selectivity, i.e discriminating between dead cells and other phagocytosed
Current Biology Vol 21 No 20 R854
material? Overexpression of the specific engulfment receptor Tim-4 improves the ability of phagocytes to clear apoptotic cells [16–18] and this also increases the upregulation of Ucp2 [6]. While the pathways emanating from Tim-4 and leading to specific Ucp2 upregulation are at present unknown, this piece of evidence connects a plasma membrane receptor with a specific metabolic event at the mitochondrial level, showing that these organelles are integrated with the cellular signaling cascades. The work by Ravichandran and colleagues [6] adds a novel function for mitochondria in the sequential activation of the engulfment machinery in phagocytes. We expect that this paper will open new exciting avenues of research that will address the many questions raised by these findings. For example, how is ingestion of apoptotic cells coupled to the reported increase in Dcm in phagocytes? How can Ucp2 overexpression augment the engulfment ability of these specialized cells? Is this simply linked to energy dissipation and heat generation, or is it a consequence of the ensuing local depletion of ATP that is consumed by mitochondria in a futile attempt to maintain their membrane potential in the presence of a proton leak? Ravichandran and colleagues [6] for now help us in placing mitochondria not only as key regulators of apoptosis execution, but also as essential
modulators of the clearance of apoptotic cells. 12.
References 1. Elliott, M.R., and Ravichandran, K.S. (2010). Clearance of apoptotic cells: implications in health and disease. J. Cell Biol. 189, 1059–1070. 2. Savill, J., Dransfield, I., Gregory, C., and Haslett, C. (2002). A blast from the past: Clearance of apoptotic cells regulates immune responses. Nat. Rev. Immunol. 2, 965–975. 3. Erwig, L.P., and Henson, P.M. (2008). Clearance of apoptotic cells by phagocytes. Cell Death Differentiation 15, 243–250. 4. Lauber, K., Blumenthal, S.G., Waibel, M., and Wesselborg, S. (2004). Clearance of apoptotic cells: Getting rid of the corpses. Mol. Cell 14, 277–287. 5. Park, D., Tosello-Trampont, A.C., Elliott, M.R., Lu, M.J., Haney, L.B., Ma, Z., Klibanov, A.L., Mandell, J.W., and Ravichandran, K.S. (2007). BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module. Nature 450, 430–434. 6. Park, D., Han, C.Z., Elliott, M.R., Kinchen, J.M., Trampont, P.C., Das, S., Collins, S., Lysiak, J.J., Hoehn, K.L., and Ravichandran, K.S. (2011). Continued clearance of apoptotic cells critically depends on the phagocyte Ucp2 protein. Nature 477, 220–224. 7. Klingenberg, M. (1999). Uncoupling protein - A useful energy dissipator. J. Bioenerg. Biomemb. 31, 419–430. 8. Chan, C., De Leo, D., Joseph, J., McQuaid, T., Saleh, M., Xu, F., et al. (2001). UCP2 regulates insulin secretion in beta-cells. Diabetes 50, A51–A52. 9. Trenker, M., Malli, R., Fertschai, I., Levak-Frank, S., and Graier, W.F. (2007). Uncoupling proteins 2 and 3 are fundamental for mitochondrial Ca2+ uniport. Nat. Cell Biol. 9, 445–U156. 10. Andrews, Z.B., Liu, Z.W., Walllingford, N., Erion, D.M., Borok, E., Friedman, J.M., Tscho¨p, M.H., Shanabrough, M., Cline, G., Shulman, G.I., et al. (2008). UCP2 mediates ghrelin’s action on NPY/AgRP neurons by lowering free radicals. Nature 454, 846–851. 11. Krauss, S., Zhang, C.Y., and Lowell, B.B. (2002). A significant portion of mitochondrial proton
Developmental Biology: Physics Adds a Twist to Gut Looping Much of the effort in understanding the dynamic process of development has focused on dissecting biochemical pathways. Recent studies illustrate that simple physical forces are also important in patterning organs. Rima Arnaout1,2,* and Didier Y.R. Stainier1 ‘‘Cell and tissue, shell and bone, leaf and flower, are so many portions of matter, and it is in obedience to the laws of physics that their particles have been moved, moulded and conformed.’’ — D’Arcy Wentworth Thompson, On Growth and Form (1917)
Nearly a century ago D’Arcy Wentworth Thompson argued that the
morphological variations seen among species obey basic physical and mathematical laws (Figure 1). The fantastic spectrum of form and function has inspired the study of allometry — differential growth of tissues from a basic body plan — in many contexts, from tree height to the design of carnivores’ footpads [1,2]. As different as these examples are, all of these organisms start as a single fertilized cell. Development
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leak in intact thymocytes depends on expression of UCP2. Proc. Natl. Acad. Sci. USA 99, 118–122. Krauss, S., Zhang, C.Y., and Lowell, B.B. (2005). The mitochondrial uncoupling-protein homologues. Nat. Rev. Mol. Cell Biol. 6, 248–261. Zhang, C.Y., Baffy, G., Perret, P., Krauss, S., Peroni, O., Grujic, D., Hagen, T., Vidal-Puig, A.J., Boss, O., Kim, Y.B., et al. (2001). Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, beta cell dysfunction, and type 2 diabetes. Cell 105, 745–755. Arsenijevic, D., Onuma, H., Pecqueur, C., Raimbault, S., Manning, B.S., Miroux, B., Couplan, E., Alves-Guerra, M.C., Goubern, M., Surwit, R., et al. (2000). Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production. Nat. Genet. 26, 435–439. Elliott, M.R., Chekeni, F.B., Trampont, P.C., Lazarowski, E.R., Kadl, A., Walk, S.F., Park, D., Woodson, R.I., Ostankovich, M., Sharma, P., et al. (2009). Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance. Nature 461, 282–286. Kobayashi, N., Karisola, P., Pena-Cruz, V., Dorfman, D.M., Jinushi, M., Umetsu, S.E., Butte, M.J., Nagumo, H., Chernova, I., Zhu, B., et al. (2007). TIM-1 and TIM-4 glycoproteins bind phosphatidylserine and mediate uptake of apoptotic cells. Immunity 27, 927–940. Miyanishi, M., Tada, K., Koike, M., Uchiyama, Y., Kitamura, T., and Nagata, S. (2007). Identification of Tim4 as a phosphatidylserine receptor. Nature 450, 435–439. Park, D., Hochreiter-Hufford, A., and Ravichandran, K.S. (2009). The phosphatidylserine receptor TIM-4 does not mediate direct signaling. Curr. Biol. 19, 346–351.
Department of Cell Physiology and Metabolism, University of Geneva Medical School, 1 Rue M. Servet, 1206 Gene`ve, Switzerland. E-mail: [email protected] DOI: 10.1016/j.cub.2011.09.007
is the process that exhibits the dynamic movements and morphological changes that underlie Thompson’s observations. Over more than one hundred years, biologists have made considerable progress in understanding development. They have discovered many biochemical regulatory networks that are highly conserved among species and that, taken together, are beginning to provide a coherent genetic blueprint for development [3,4]. They haven’t forgotten, however, that development — and life — takes place in a physical world and, as Thompson wrote, obeys physical laws. Examples can be seen in heart tube looping, brain folding, airway branching and gut looping [5–9].
agreement with this notion, Silva et al. [12] observed that reprogramming of fibroblasts from a 129/MF1 hybrid strain of mice gives rise mostly to partially reprogrammed iPSCs that rarely progress to pluripotency unless treated with 2i. Similarly, the establishment of rat iPSCs requires 2i culture. What consequences might these findings have for human ESC/iPSC research? Human ESCs are fundamentally different from mouse ESCs in that they require bFgf and Activin A for their stable propagation [1]. Interestingly, exposure of mouse blastocysts, ESCs or postimplantation embryos to bFgf and Activin A gives rise to so-called epiblast stem cells (EpiSCs) [13,14], which are very similar to human ESCs and seem to represent a developmentally more advanced or ‘primed’ state compared with the more primitive or ‘naive’ state of mouse ESCs [15]. The findings by Chu et al. [6] thus raise the interesting possibility that progression of epiblast cells towards a germ-cell fate, either by enforced expression of certain transcription factors [1,9] or by exposure of cells to germ-cell-inducing cytokines, might be sufficient to derive stable naive ESC/iPSC lines in humans and other species. Recent exciting progress in
identifying molecules that coax pluripotent cells into germ cells may aid in these efforts [16]. References 1. Stadtfeld, M., and Hochedlinger, K. (2010). Induced pluripotency: history, mechanisms, and applications. Genes Dev. 24, 2239–2263. 2. Zwaka, T.P., and Thomson, J.A. (2005). A germ cell origin of embryonic stem cells? Development 132, 227–233. 3. Buehr, M., and Smith, A. (2003). Genesis of embryonic stem cells. Phil. Trans. R. Soc. Lond. 358, 1397–1402, discussion 1402. 4. Brook, F.A., and Gardner, R.L. (1997). The origin and efficient derivation of embryonic stem cells in the mouse. Proc. Natl. Acad. Sci. USA 94, 5709–5712. 5. Durcova-Hills, G., Tang, F., Doody, G., Tooze, R., and Surani, M.A. (2008). Reprogramming primordial germ cells into pluripotent stem cells. PLoS One 3, e3531. 6. Chu, L.F., Surani, M.A., Jaenisch, R., and Zwaka, T.P. (2011). Blimp1 expression predicts embryonic stem cell development in vitro. Curr. Biol. 21, 1759–1765. 7. Saitou, M., Payer, B., O’Carroll, D., Ohinata, Y., and Surani, M.A. (2005). Blimp1 and the emergence of the germ line during development in the mouse. Cell Cycle 4, 1736–1740. 8. Ying, Q.L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J., Cohen, P., and Smith, A. (2008). The ground state of embryonic stem cell self-renewal. Nature 453, 519–523. 9. Nichols, J., and Smith, A. (2011). The origin and identity of embryonic stem cells. Development 138, 3–8. 10. Buehr, M., Meek, S., Blair, K., Yang, J., Ure, J., Silva, J., McLay, R., Hall, J., Ying, Q.L., and Smith, A. (2008). Capture of authentic embryonic stem cells from rat blastocysts. Cell 135, 1287–1298. 11. Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse
Phagocytosis: Coupling of Mitochondrial Uncoupling and Engulfment Clearance of apoptotic cells by phagocytes avoids triggering an inflammatory response. A new study reveals that phagocytes dissipate their mitochondrial proton electrochemical gradient to allow for the ingestion of more apoptotic corpses. Mitochondria are therefore involved in all aspects of apoptosis, from its activation through to the phagocytosis of dead cells. Grazia M. Cereghetti and Luca Scorrano Sustained cell proliferation during development, tissue renewal or in the course of the immune response is accompanied by the production of excess or damaged cells that die by apoptosis. The accumulation of these cells may lead to tissue damage and inflammation: specialized systems therefore efficiently remove them [1,2].
Phagocytes are deputed to the clearance of apoptotic cells and are able to engulf multiple cells in order to adapt their ‘cleaning efficiency’ to the rate of apoptotic cell accumulation. In recent years, some of the crucial steps in the phagocytosis of apoptotic cells, as well as the principal players in the phagocytic process, have been elucidated [3,4]. Dying cells release signals to attract the motile phagocytes. The two cells make
12.
13.
14.
15.
16.
embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676. Silva, J., Barrandon, O., Nichols, J., Kawaguchi, J., Theunissen, T.W., and Smith, A. (2008). Promotion of reprogramming to ground state pluripotency by signal inhibition. PLoS Biol. 6, e253. Brons, I.G., Smithers, L.E., Trotter, M.W., Rugg-Gunn, P., Sun, B., Chuva de Sousa Lopes, S.M., Howlett, S.K., Clarkson, A., Ahrlund-Richter, L., Pedersen, R.A., et al. (2007). Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448, 191–195. Tesar, P.J., Chenoweth, J.G., Brook, F.A., Davies, T.J., Evans, E.P., Mack, D.L., Gardner, R.L., and McKay, R.D. (2007). New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196–199. Nichols, J., and Smith, A. (2009). Naive and primed pluripotent states. Cell Stem Cell 4, 487–492. Hayashi, K., Ohta, H., Kurimoto, K., Aramaki, S., and Saitou, M. (2011). Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 146, 519–532.
1Howard Hughes Medical Institute and Department of Stem Cell and Regenerative Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA. 2Massachusetts General Hospital Cancer Center and Center for Regenerative Medicine, 185 Cambridge Street, Boston, MA 02114, USA. 3Harvard Stem Cell Institute, 42 Church Street, Cambridge, MA 02138, USA. E-mail: [email protected]. edu
DOI: 10.1016/j.cub.2011.09.024
physical contact via markers that are released from the apoptotic cell and bind to receptors on the phagocyte, inducing a signaling cascade that prepares the phagocyte membrane for the internalization of the dead cell. Several molecules are involved in engulfment by phagocytes, including: brain angiogenesis inhibitor 1 (BAI1), a transmembrane protein highly expressed in the brain; Rac GTPases, which remodel the cytoskeleton; ELMO, an evolutionarily conserved cytoplasmic engulfment protein; and the unconventional guanine nucleotide exchange factor and Rho GTPase activator Dock180 [5]. Despite our knowledge of some key molecular steps in the engulfment cascade, how a single phagocyte can serially internalize many apoptotic cells is unclear. Park et al. [6] have now shown, in a recent issue of Nature, that serial internalization of apoptotic cells unexpectedly depends on
Dispatch R853
mitochondrial activity in the phagocyte, and that a key player in this process is uncoupling protein 2 (Ucp2) [6]. Mitochondria are important organelles with several cellular functions besides energy conversion, ranging from metabolism to calcium signaling to their key role in amplification of apoptosis. Their unique ability to efficiently synthesize ATP depends on the accumulation of free energy in the form of a proton w electrochemical gradient (Dm H+ ) across the inner mitochondrial membrane that is then converted by the ATP synthase into the chemical energy of the thiophosphoric bond. The second law of thermodynamics dictates that futile dissipation of this gradient will result in its conversion into other forms of energy, such as thermal energy. Nature clearly exploited this possibility in a regulated fashion, by means of uncoupling proteins. Ucp1, also known as thermogenin, is a 32 kDa inner mitochondrial membrane protein present almost exclusively in brown adipose tissue, where it participates in thermogenesis by catalysing the net transfer of protons from the mitochondrial intermembrane space to the matrix [7]. The Ucp1 homologue Ucp2 is also a carrier for protons and small molecules across the inner mitochondrial membrane. Unlike Ucp1, Ucp2 and Ucp3 are expressed in multiple tissues, leading to questions about their role: they have indeed been reported to be involved in different processes, such as modulation of insulin secretion [8] and of mitochondrial calcium uptake [9]. In particular, functions attributed to Ucp2 are related to its ability to uncouple ATP production from the proton electrochemical gradient [10–13]. In the new study, Park et al. [6] asked whether mitochondrial metabolism could play a role in the phagocytosis of apoptotic cells and analyzed the mitochondrial membrane potential (Dcm), i.e. the electrical component w of the Dm H+ , in LR73 phagocytes upon internalization of an apoptotic cell. Surprisingly, Dcm increased specifically when an apoptotic corpse was being phagocytosed: other synthetic targets did not alter Dcm, in spite of a complete activation of the engulfment machinery. While it is not clear what drives this increase in Dcm, its subsequent return to baseline is very slow and is not connected to a net increase in total cellular ATP levels
Phagocyte
3 Ucp2 Tim4 Apoptotic cell
4 H+
Mitochondrion
5 Δψm 1 Apoptotic cell recognition
2 Δψ m
Current Biology
Figure 1. Apoptotic cell engulfment capacity relies on the mitochondrial membrane potential (Dcm), which is influenced by Ucp2. Internalization of apoptotic cells by phagocytes (1) is followed by phagocyte mitochondrial membrane hyperpolarization (2), which blocks the engulfment of further cells. Upregulation of Ucp2 (3) and dissipation of the proton gradient as a consequence of proton leak in the mitochondrial matrix (4) lowers Dcm (5), resulting in an as yet unidentified ‘continue-to-eat’ signal.
but is associated with increased expression of Ucp2. Moreover, artificial overexpression of Ucp2 increased the rate and capacity of internalization by phagocytes, suggesting a specific role for Ucp2 and for dissipation of the electrochemical gradient in this process of increased ‘phagocytic capacitance’ (Figure 1). Ravichandran and coworkers [6] exclude that Ucp2 regulates phagocytosis by altering production of reactive oxygen species (ROS) or lipid oxidation [11] and were able to recapitulate the stimulation of phagocytosis by using synthetic uncouplers like 2,4-dinitrophenol (2,4-DNP) and carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), further substantiating the proposal that Ucp2 (unlike its homologues Ucp1 and Ucp3) modulates phagocytosis via its primary w effect on DmH+ . While the result strongly argues for a key role for mitochondria in this process, it should be noted that the synthetic uncouplers have powerful side effects, and it is surprising that the phagocytes can survive upon complete uncoupling or upon poisoning of the respiratory chain with azide. It is likely that the excess
glucose in the culture medium allows cells to rely solely on glycolysis to generate ATP. Would the role of Ucp2 w (and of Dm H+ ) be maintained under more physiological conditions in which mitochondria are used as the main ATP source by the cell? A first answer to this question comes from the analysis of the effects of Ucp2 ablation on waste elimination in tissues. Ucp2-/- mice are viable but present higher ROS production and altered macrophagemediated immunity [14]. The Dcm of bone-marrow-derived macrophages (BMDMs) from Ucp2-/- mice is higher and these cells also have a decreased ability to engulf apoptotic cells compared with control cells. Dexamethasone injection induces rapid death of thymocytes in vivo [15], followed by clearance of dead cells in control mice, but not in Ucp2-/- animals. Similarly, induction of germ cell apoptosis via testicular torsion is followed by accumulation of dying germ cells in seminiferous tubules in Ucp2-/- mice. How is Ucp2 upregulated upon engulfment of apoptotic corpses? What is the mechanism granting selectivity, i.e discriminating between dead cells and other phagocytosed
Current Biology Vol 21 No 20 R854
material? Overexpression of the specific engulfment receptor Tim-4 improves the ability of phagocytes to clear apoptotic cells [16–18] and this also increases the upregulation of Ucp2 [6]. While the pathways emanating from Tim-4 and leading to specific Ucp2 upregulation are at present unknown, this piece of evidence connects a plasma membrane receptor with a specific metabolic event at the mitochondrial level, showing that these organelles are integrated with the cellular signaling cascades. The work by Ravichandran and colleagues [6] adds a novel function for mitochondria in the sequential activation of the engulfment machinery in phagocytes. We expect that this paper will open new exciting avenues of research that will address the many questions raised by these findings. For example, how is ingestion of apoptotic cells coupled to the reported increase in Dcm in phagocytes? How can Ucp2 overexpression augment the engulfment ability of these specialized cells? Is this simply linked to energy dissipation and heat generation, or is it a consequence of the ensuing local depletion of ATP that is consumed by mitochondria in a futile attempt to maintain their membrane potential in the presence of a proton leak? Ravichandran and colleagues [6] for now help us in placing mitochondria not only as key regulators of apoptosis execution, but also as essential
modulators of the clearance of apoptotic cells. 12.
References 1. Elliott, M.R., and Ravichandran, K.S. (2010). Clearance of apoptotic cells: implications in health and disease. J. Cell Biol. 189, 1059–1070. 2. Savill, J., Dransfield, I., Gregory, C., and Haslett, C. (2002). A blast from the past: Clearance of apoptotic cells regulates immune responses. Nat. Rev. Immunol. 2, 965–975. 3. Erwig, L.P., and Henson, P.M. (2008). Clearance of apoptotic cells by phagocytes. Cell Death Differentiation 15, 243–250. 4. Lauber, K., Blumenthal, S.G., Waibel, M., and Wesselborg, S. (2004). Clearance of apoptotic cells: Getting rid of the corpses. Mol. Cell 14, 277–287. 5. Park, D., Tosello-Trampont, A.C., Elliott, M.R., Lu, M.J., Haney, L.B., Ma, Z., Klibanov, A.L., Mandell, J.W., and Ravichandran, K.S. (2007). BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module. Nature 450, 430–434. 6. Park, D., Han, C.Z., Elliott, M.R., Kinchen, J.M., Trampont, P.C., Das, S., Collins, S., Lysiak, J.J., Hoehn, K.L., and Ravichandran, K.S. (2011). Continued clearance of apoptotic cells critically depends on the phagocyte Ucp2 protein. Nature 477, 220–224. 7. Klingenberg, M. (1999). Uncoupling protein - A useful energy dissipator. J. Bioenerg. Biomemb. 31, 419–430. 8. Chan, C., De Leo, D., Joseph, J., McQuaid, T., Saleh, M., Xu, F., et al. (2001). UCP2 regulates insulin secretion in beta-cells. Diabetes 50, A51–A52. 9. Trenker, M., Malli, R., Fertschai, I., Levak-Frank, S., and Graier, W.F. (2007). Uncoupling proteins 2 and 3 are fundamental for mitochondrial Ca2+ uniport. Nat. Cell Biol. 9, 445–U156. 10. Andrews, Z.B., Liu, Z.W., Walllingford, N., Erion, D.M., Borok, E., Friedman, J.M., Tscho¨p, M.H., Shanabrough, M., Cline, G., Shulman, G.I., et al. (2008). UCP2 mediates ghrelin’s action on NPY/AgRP neurons by lowering free radicals. Nature 454, 846–851. 11. Krauss, S., Zhang, C.Y., and Lowell, B.B. (2002). A significant portion of mitochondrial proton
Developmental Biology: Physics Adds a Twist to Gut Looping Much of the effort in understanding the dynamic process of development has focused on dissecting biochemical pathways. Recent studies illustrate that simple physical forces are also important in patterning organs. Rima Arnaout1,2,* and Didier Y.R. Stainier1 ‘‘Cell and tissue, shell and bone, leaf and flower, are so many portions of matter, and it is in obedience to the laws of physics that their particles have been moved, moulded and conformed.’’ — D’Arcy Wentworth Thompson, On Growth and Form (1917)
Nearly a century ago D’Arcy Wentworth Thompson argued that the
morphological variations seen among species obey basic physical and mathematical laws (Figure 1). The fantastic spectrum of form and function has inspired the study of allometry — differential growth of tissues from a basic body plan — in many contexts, from tree height to the design of carnivores’ footpads [1,2]. As different as these examples are, all of these organisms start as a single fertilized cell. Development
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Department of Cell Physiology and Metabolism, University of Geneva Medical School, 1 Rue M. Servet, 1206 Gene`ve, Switzerland. E-mail: [email protected] DOI: 10.1016/j.cub.2011.09.007
is the process that exhibits the dynamic movements and morphological changes that underlie Thompson’s observations. Over more than one hundred years, biologists have made considerable progress in understanding development. They have discovered many biochemical regulatory networks that are highly conserved among species and that, taken together, are beginning to provide a coherent genetic blueprint for development [3,4]. They haven’t forgotten, however, that development — and life — takes place in a physical world and, as Thompson wrote, obeys physical laws. Examples can be seen in heart tube looping, brain folding, airway branching and gut looping [5–9].