Developmental Cell
Previews effects on spontaneous release, in agreement with results obtained upon insertion of similar linkers into synaptobrevin, as also reported in Zhou et al. (2013) and previous studies (e.g., Kesavan et al., 2007). These results show that the continuity of the SNARE motif helices into the TMRs may facilitate evoked release with the WT proteins but is not an essential feature of the fusion mechanism. They also show that the close membrane proximity caused by zippering of the SNARE complex is important for evoked release but is much less critical for spontaneous release, likely because the evoked release imposes more tight demands on the release machinery. However, Zhou et al. (2013) also show that syntaxin-1 DTMR rescued evoked release in syntaxin-1-
deficient neurons more fully in the presence than in the absence of a seven-residue flexible linker between the SNARE motif and the lipid anchor. These results emphasize that much remains to be learned about how the SNAREs apply force on the membranes to induce membrane fusion. Ingenious studies that disentangle which features are essential for fusion and which are not, such as that presented by Zhou et al. (2013), will be necessary to answer this question.
Kesavan, J., Borisovska, M., and Bruns, D. (2007). Cell 131, 351–363. Langosch, D., Crane, J.M., Brosig, B., Hellwig, A., Tamm, L.K., and Reed, J. (2001). J. Mol. Biol. 311, 709–721. Ma, C., Su, L., Seven, A.B., Xu, Y., and Rizo, J. (2013). Science 339, 421–425. McNew, J.A., Weber, T., Parlati, F., Johnston, R.J., Melia, T.J., So¨llner, T.H., and Rothman, J.E. (2000). J. Cell Biol. 150, 105–117. Rizo, J., and Su¨dhof, T.C. (2012). Annu. Rev. Cell Dev. Biol. 28, 279–308.
REFERENCES
Stein, A., Weber, G., Wahl, M.C., and Jahn, R. (2009). Nature 460, 525–528.
Han, X., Wang, C.T., Bai, J., Chapman, E.R., and Jackson, M.B. (2004). Science 304, 289–292.
Xu, H., Zick, M., Wickner, W.T., and Jun, Y. (2011). Proc. Natl. Acad. Sci. USA 108, 17325–17330.
Jun, Y., Xu, H., Thorngren, N., and Wickner, W. (2007). EMBO J. 26, 4935–4945.
Zhou, P., Bacaj, T., Yang, X., Pang, Z.P., and Sudhof, T.C. (2013). Neuron 80, in press.
Cilia Grow by Taking a Bite out of the Cell Nathan W. Pierce1 and Maxence V. Nachury1,* 1Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2013.10.013
Autophagy and primary cilium assembly have long been known to be induced by the same conditions in cultured cells. Two recent studies in Nature—Tang et al. (2013) and Pampliega et al. (2013)—link the two processes, suggesting that a specialized autophagy pathway near the basal body regulates cilium assembly. The primary cilium is a microtubule-based protrusion present on the surface of most mammalian cells that relays a diverse set of developmental, homeostatic, and sensory signals to the rest of the cell. The assembly of primary cilia is a dynamic process initiated once cells exit the cell cycle to enter quiescence (Seeley and Nachury, 2010). In cultured cells, primary cilium assembly is triggered by withdrawal of growth factors, i.e., serum starvation. However, when researchers remove serum from their medium, they also trigger the self-digestive process of autophagy. While autophagy is classically induced by removing both serum and amino acids, it was long known that serum deprivation alone triggers autophagic processes (Hershko and Tomkins, 1971). Yet, despite a common physiological trigger, autophagy and ciliogenesis
were largely seen as independent processes. Two studies from Pampliega et al. (2013) and Tang et al. (2013), published recently in Nature, now provide evidence that link these processes together biochemically, cytologically, and functionally. The work from Pampliega et al. (2013) showed that a subpopulation of the autophagy machinery is present near the basal body (the differentiated centriole that forms the base of a cilium). To digest cellular contents, a series of dedicated autophagy (ATG) proteins organizes the growth of a double-membrane sheet around specific regions of the cytoplasm, protein aggregates, or organelles to isolate them inside a vesicular structure (the autophagosome) that subsequently fuses with the lysosome. While most of the early autophagic processes are found
126 Developmental Cell 27, October 28, 2013 ª2013 Elsevier Inc.
to take place near endoplasmic reticulum membranes (Itakura and Mizushima, 2010), the proteins ATG16L and ATG5 (which function to extend the isolation membrane) are enriched near the basal body. Moreover, the presence of a functional cilium appears to be required for the recruitment of ATG16L and ATG5 to the vicinity of the basal body. The cytological connection between autophagy proteins and ciliogenesis may be of functional importance, as genetic ablation of Atg5, Atg7, and Atg14 slightly ameliorates ciliation in the presence of serum. This functional connection between autophagy and the cilium may in fact be a twoway street, as Pampliega et al. (2013) find that Hedgehog signaling, a ciliumdependent developmental pathway that patterns the skeleton and the neural tube, potently activates autophagy. While
Developmental Cell
Previews potentially important, the connection between Hedgehog signaling and autophagy is currently controversial, with several groups arriving at opposite conclusions regarding the influence of Hh pathway on autophagy (see Pampliega et al., 2013 for references). The study by Tang et al. (2013) was initiated by an unbiased search for biochemical interactors of a central component of the autophagy machinery. A key step driving autophagosome formation is the conjugation of LC3 to the phospholipid phosphatidylethanolamine (PE). Concordant with its prominent enrichment on preautophagosome membranes, LC3PE (also called LC3-II) recruits a series of adaptors (such as p62, NIX, and NBR1) for cargoes destined to be autophagocytosed. In addition to expected interactors (i.e., p62), Tang et al. found that tandem affinity purification of LC3 also recovered the centriolar satellites proteins PCM1, OFD1, and CEP131. Despite having been described more than 50 years ago, centriolar satellites still remain enigmatic structures (Ba¨renz et al., 2011). They consist of 80-nm electron-dense granules devoid of limiting membranes that gravitate around basal bodies and centrioles. It has been proposed that centriolar satellites play important roles in cilium formation by delivering ciliadestined proteins to the basal body. Congruently, a variety of ciliary proteins is found at centriolar satellites. Of particular interest are the disease proteins BBS4, CEP290/JBTS5, and OFD1. These are located at centriolar satellites as well as cilia (BBS4), basal body (OFD1), or transition zone (CEP290), and their dysfunction underlies the ciliopathies Bardet-Biedl syndrome (BBS), Joubert syndrome (JBTS), and oral-facial-digital syndrome (OFD). The interaction between LC3 and PCM1, OFD1, and CEP131 adds a new piece to the centriolar satellites puzzle. Given their morphological resemblance to protein aggregates, it was tempting to consider that centriolar satellites may become digested by autophagy. Yet, while the global levels of OFD1 decrease upon serum-starvation-induced
autophagy, other markers of centriolar satellites are unaffected. Moreover, autophagy leads to the disappearance of OFD1 from satellites without affecting the levels of OFD1 at basal bodies or the overall distribution and number of PCM1-marked satellites. Thus, it appears that LC3 specifically targets OFD1 to autophagosomes, possibly using OFD1 as an autophagy adaptor for some insofarunknown cargoes. In this sense, it will be very interesting to explore OFD1-interacting proteins as potential substrates of the autophagy pathway. In a series of provocative experiments, Tang et al. (2013) go on to suggest that forced removal of OFD1 from satellites is sufficient to trigger cilium assembly in cells that normally assemble cilia very inefficiently (i.e., serum-fed cells) or not at all (i.e., transformed cells such as MCF7). A plausible model thus becomes that autophagic removal of OFD1 from satellites is a necessary and sufficient step for cilium assembly. Concordantly, genetic ablation of autophagy upregulates the levels of OFD1 at satellites and inhibits serum-starvation-induced cilium assembly. Because centriolar satellites move toward the centrosome/basal body in a microtubule- and dynein-dependent manner, and given the newly found pool of autophagy proteins in the vicinity of the basal body, it is tempting to speculate that centriolar satellites may deliver OFD1 to the autophagy machinery at the basal body. This new model for the interplay between autophagy and ciliogenesis poses several exciting questions. First, how is the autophagic removal of OFD1 permissive to ciliogenesis? The bestcharacterized inhibitor of ciliogenesis is the protein CP110, which sits at the very tip of the centriole, very close to where OFD1 is found at centrioles. While the bulk of CP110 is removed immediately prior to cilium assembly by ubiquitindependent degradation (D’Angiolella et al., 2010), it is conceivable that a specific pool of CP110 could be eliminated by autophagy using OFD1 as an adaptor. Second, while cilia are known to harbor a
specific lipidome, the source of ciliary lipids is presently unclear. Could autophagic processes provide membranes to the growing cilium? In this context, it is worth mentioning the tantalizing parallels between cilia and melanosomes, which are lysosome-related organelles whose biogenesis is largely dependent on the autophagy pathway (Sarmah et al., 2007; Yen et al., 2006). The possibility that ciliary signaling pathways regulate autophagy is a fascinating one. While amino acid deprivation is used nearly universally to induce autophagy in cultured cells, the circulating amounts of amino acids in the body vary very little. Instead, the sensing of the nutritional status is carried out at the level of the whole organism, and autophagy is under hormonal control, with the glucagon/insulin system functioning as a potent pair of activators/ inhibitors of autophagy in hepatocytes. Whether cilia are themselves sensing hormonal cues (e.g., Hh) to regulate autophagy will remain an exciting possibility for future investigations. REFERENCES Ba¨renz, F., Mayilo, D., and Gruss, O.J. (2011). Eur. J. Cell Biol. 90, 983–989. D’Angiolella, V., Donato, V., Vijayakumar, S., Saraf, A., Florens, L., Washburn, M.P., Dynlacht, B., and Pagano, M. (2010). Nature 466, 138–142. Hershko, A., and Tomkins, G.M. (1971). J. Biol. Chem. 246, 710–714. Itakura, E., and Mizushima, N. (2010). Autophagy 6, 764–776. Pampliega, O., Orhon, I., Patel, B., Sridhar, S., Dı´az-Carretero, A., Beau, I., Codogno, P., Satir, B.H., Satir, P., and Cuervo, A.M. (2013). Nature 502, 194–200. Sarmah, B., Winfrey, V.P., Olson, G.E., Appel, B., and Wente, S.R. (2007). Proc. Natl. Acad. Sci. USA 104, 19843–19848. Seeley, E.S., and Nachury, M.V. (2010). J. Cell Sci. 123, 511–518. Tang, Z., Lin, M.G., Stowe, T.R., Chen, S., Zhu, M., Stearns, T., Franco, B., and Zhong, Q. (2013). Nature 502, 254–257. Yen, H.-J., Tayeh, M.K., Mullins, R.F., Stone, E.M., Sheffield, V.C., and Slusarski, D.C. (2006). Hum. Mol. Genet. 15, 667–677.
Developmental Cell 27, October 28, 2013 ª2013 Elsevier Inc. 127
Previews effects on spontaneous release, in agreement with results obtained upon insertion of similar linkers into synaptobrevin, as also reported in Zhou et al. (2013) and previous studies (e.g., Kesavan et al., 2007). These results show that the continuity of the SNARE motif helices into the TMRs may facilitate evoked release with the WT proteins but is not an essential feature of the fusion mechanism. They also show that the close membrane proximity caused by zippering of the SNARE complex is important for evoked release but is much less critical for spontaneous release, likely because the evoked release imposes more tight demands on the release machinery. However, Zhou et al. (2013) also show that syntaxin-1 DTMR rescued evoked release in syntaxin-1-
deficient neurons more fully in the presence than in the absence of a seven-residue flexible linker between the SNARE motif and the lipid anchor. These results emphasize that much remains to be learned about how the SNAREs apply force on the membranes to induce membrane fusion. Ingenious studies that disentangle which features are essential for fusion and which are not, such as that presented by Zhou et al. (2013), will be necessary to answer this question.
Kesavan, J., Borisovska, M., and Bruns, D. (2007). Cell 131, 351–363. Langosch, D., Crane, J.M., Brosig, B., Hellwig, A., Tamm, L.K., and Reed, J. (2001). J. Mol. Biol. 311, 709–721. Ma, C., Su, L., Seven, A.B., Xu, Y., and Rizo, J. (2013). Science 339, 421–425. McNew, J.A., Weber, T., Parlati, F., Johnston, R.J., Melia, T.J., So¨llner, T.H., and Rothman, J.E. (2000). J. Cell Biol. 150, 105–117. Rizo, J., and Su¨dhof, T.C. (2012). Annu. Rev. Cell Dev. Biol. 28, 279–308.
REFERENCES
Stein, A., Weber, G., Wahl, M.C., and Jahn, R. (2009). Nature 460, 525–528.
Han, X., Wang, C.T., Bai, J., Chapman, E.R., and Jackson, M.B. (2004). Science 304, 289–292.
Xu, H., Zick, M., Wickner, W.T., and Jun, Y. (2011). Proc. Natl. Acad. Sci. USA 108, 17325–17330.
Jun, Y., Xu, H., Thorngren, N., and Wickner, W. (2007). EMBO J. 26, 4935–4945.
Zhou, P., Bacaj, T., Yang, X., Pang, Z.P., and Sudhof, T.C. (2013). Neuron 80, in press.
Cilia Grow by Taking a Bite out of the Cell Nathan W. Pierce1 and Maxence V. Nachury1,* 1Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2013.10.013
Autophagy and primary cilium assembly have long been known to be induced by the same conditions in cultured cells. Two recent studies in Nature—Tang et al. (2013) and Pampliega et al. (2013)—link the two processes, suggesting that a specialized autophagy pathway near the basal body regulates cilium assembly. The primary cilium is a microtubule-based protrusion present on the surface of most mammalian cells that relays a diverse set of developmental, homeostatic, and sensory signals to the rest of the cell. The assembly of primary cilia is a dynamic process initiated once cells exit the cell cycle to enter quiescence (Seeley and Nachury, 2010). In cultured cells, primary cilium assembly is triggered by withdrawal of growth factors, i.e., serum starvation. However, when researchers remove serum from their medium, they also trigger the self-digestive process of autophagy. While autophagy is classically induced by removing both serum and amino acids, it was long known that serum deprivation alone triggers autophagic processes (Hershko and Tomkins, 1971). Yet, despite a common physiological trigger, autophagy and ciliogenesis
were largely seen as independent processes. Two studies from Pampliega et al. (2013) and Tang et al. (2013), published recently in Nature, now provide evidence that link these processes together biochemically, cytologically, and functionally. The work from Pampliega et al. (2013) showed that a subpopulation of the autophagy machinery is present near the basal body (the differentiated centriole that forms the base of a cilium). To digest cellular contents, a series of dedicated autophagy (ATG) proteins organizes the growth of a double-membrane sheet around specific regions of the cytoplasm, protein aggregates, or organelles to isolate them inside a vesicular structure (the autophagosome) that subsequently fuses with the lysosome. While most of the early autophagic processes are found
126 Developmental Cell 27, October 28, 2013 ª2013 Elsevier Inc.
to take place near endoplasmic reticulum membranes (Itakura and Mizushima, 2010), the proteins ATG16L and ATG5 (which function to extend the isolation membrane) are enriched near the basal body. Moreover, the presence of a functional cilium appears to be required for the recruitment of ATG16L and ATG5 to the vicinity of the basal body. The cytological connection between autophagy proteins and ciliogenesis may be of functional importance, as genetic ablation of Atg5, Atg7, and Atg14 slightly ameliorates ciliation in the presence of serum. This functional connection between autophagy and the cilium may in fact be a twoway street, as Pampliega et al. (2013) find that Hedgehog signaling, a ciliumdependent developmental pathway that patterns the skeleton and the neural tube, potently activates autophagy. While
Developmental Cell
Previews potentially important, the connection between Hedgehog signaling and autophagy is currently controversial, with several groups arriving at opposite conclusions regarding the influence of Hh pathway on autophagy (see Pampliega et al., 2013 for references). The study by Tang et al. (2013) was initiated by an unbiased search for biochemical interactors of a central component of the autophagy machinery. A key step driving autophagosome formation is the conjugation of LC3 to the phospholipid phosphatidylethanolamine (PE). Concordant with its prominent enrichment on preautophagosome membranes, LC3PE (also called LC3-II) recruits a series of adaptors (such as p62, NIX, and NBR1) for cargoes destined to be autophagocytosed. In addition to expected interactors (i.e., p62), Tang et al. found that tandem affinity purification of LC3 also recovered the centriolar satellites proteins PCM1, OFD1, and CEP131. Despite having been described more than 50 years ago, centriolar satellites still remain enigmatic structures (Ba¨renz et al., 2011). They consist of 80-nm electron-dense granules devoid of limiting membranes that gravitate around basal bodies and centrioles. It has been proposed that centriolar satellites play important roles in cilium formation by delivering ciliadestined proteins to the basal body. Congruently, a variety of ciliary proteins is found at centriolar satellites. Of particular interest are the disease proteins BBS4, CEP290/JBTS5, and OFD1. These are located at centriolar satellites as well as cilia (BBS4), basal body (OFD1), or transition zone (CEP290), and their dysfunction underlies the ciliopathies Bardet-Biedl syndrome (BBS), Joubert syndrome (JBTS), and oral-facial-digital syndrome (OFD). The interaction between LC3 and PCM1, OFD1, and CEP131 adds a new piece to the centriolar satellites puzzle. Given their morphological resemblance to protein aggregates, it was tempting to consider that centriolar satellites may become digested by autophagy. Yet, while the global levels of OFD1 decrease upon serum-starvation-induced
autophagy, other markers of centriolar satellites are unaffected. Moreover, autophagy leads to the disappearance of OFD1 from satellites without affecting the levels of OFD1 at basal bodies or the overall distribution and number of PCM1-marked satellites. Thus, it appears that LC3 specifically targets OFD1 to autophagosomes, possibly using OFD1 as an autophagy adaptor for some insofarunknown cargoes. In this sense, it will be very interesting to explore OFD1-interacting proteins as potential substrates of the autophagy pathway. In a series of provocative experiments, Tang et al. (2013) go on to suggest that forced removal of OFD1 from satellites is sufficient to trigger cilium assembly in cells that normally assemble cilia very inefficiently (i.e., serum-fed cells) or not at all (i.e., transformed cells such as MCF7). A plausible model thus becomes that autophagic removal of OFD1 from satellites is a necessary and sufficient step for cilium assembly. Concordantly, genetic ablation of autophagy upregulates the levels of OFD1 at satellites and inhibits serum-starvation-induced cilium assembly. Because centriolar satellites move toward the centrosome/basal body in a microtubule- and dynein-dependent manner, and given the newly found pool of autophagy proteins in the vicinity of the basal body, it is tempting to speculate that centriolar satellites may deliver OFD1 to the autophagy machinery at the basal body. This new model for the interplay between autophagy and ciliogenesis poses several exciting questions. First, how is the autophagic removal of OFD1 permissive to ciliogenesis? The bestcharacterized inhibitor of ciliogenesis is the protein CP110, which sits at the very tip of the centriole, very close to where OFD1 is found at centrioles. While the bulk of CP110 is removed immediately prior to cilium assembly by ubiquitindependent degradation (D’Angiolella et al., 2010), it is conceivable that a specific pool of CP110 could be eliminated by autophagy using OFD1 as an adaptor. Second, while cilia are known to harbor a
specific lipidome, the source of ciliary lipids is presently unclear. Could autophagic processes provide membranes to the growing cilium? In this context, it is worth mentioning the tantalizing parallels between cilia and melanosomes, which are lysosome-related organelles whose biogenesis is largely dependent on the autophagy pathway (Sarmah et al., 2007; Yen et al., 2006). The possibility that ciliary signaling pathways regulate autophagy is a fascinating one. While amino acid deprivation is used nearly universally to induce autophagy in cultured cells, the circulating amounts of amino acids in the body vary very little. Instead, the sensing of the nutritional status is carried out at the level of the whole organism, and autophagy is under hormonal control, with the glucagon/insulin system functioning as a potent pair of activators/ inhibitors of autophagy in hepatocytes. Whether cilia are themselves sensing hormonal cues (e.g., Hh) to regulate autophagy will remain an exciting possibility for future investigations. REFERENCES Ba¨renz, F., Mayilo, D., and Gruss, O.J. (2011). Eur. J. Cell Biol. 90, 983–989. D’Angiolella, V., Donato, V., Vijayakumar, S., Saraf, A., Florens, L., Washburn, M.P., Dynlacht, B., and Pagano, M. (2010). Nature 466, 138–142. Hershko, A., and Tomkins, G.M. (1971). J. Biol. Chem. 246, 710–714. Itakura, E., and Mizushima, N. (2010). Autophagy 6, 764–776. Pampliega, O., Orhon, I., Patel, B., Sridhar, S., Dı´az-Carretero, A., Beau, I., Codogno, P., Satir, B.H., Satir, P., and Cuervo, A.M. (2013). Nature 502, 194–200. Sarmah, B., Winfrey, V.P., Olson, G.E., Appel, B., and Wente, S.R. (2007). Proc. Natl. Acad. Sci. USA 104, 19843–19848. Seeley, E.S., and Nachury, M.V. (2010). J. Cell Sci. 123, 511–518. Tang, Z., Lin, M.G., Stowe, T.R., Chen, S., Zhu, M., Stearns, T., Franco, B., and Zhong, Q. (2013). Nature 502, 254–257. Yen, H.-J., Tayeh, M.K., Mullins, R.F., Stone, E.M., Sheffield, V.C., and Slusarski, D.C. (2006). Hum. Mol. Genet. 15, 667–677.
Developmental Cell 27, October 28, 2013 ª2013 Elsevier Inc. 127
