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Is Ca2+ Essential for Synaptic Vesicle Endocytosis?
TINS 1564 No. of Pages 2
Trends in Neurosciences
Spotlight
Is Ca2+ Essential for Synaptic Vesicle Endocytosis? Natali L. Chanaday1 and Ege T. Kavalali1,2,* Synaptic vesicle fusion is coupled to swift retrieval of vesicle components from the synaptic plasma membrane. Ca2+ has been assumed to be a key mediator of this coupling. In a recent study, Orlando et al. unequivocally demonstrate that Ca2+ is not essential for synaptic vesicle retrieval. Neurotransmitters are released from presynaptic terminals following regulated fusion of synaptic vesicles with the plasma membrane at the active zone. Synaptic vesicles are small distinct membranous organelles with an average diameter around 40 nm and they harbor a number of specific molecular components that are critical for their function. For neurotransmission to be sustained over time, synaptic vesicle components, lipids and proteins, need to be retrieved from the plasma membrane after fusion and synaptic vesicles need to be regenerated [1]. The mechanisms mediating synaptic vesicle recycling have been a matter of intense research during the last 50 years. Extensive evidence supports the notion that synaptic vesicle exocytosis and endocytosis are coupled processes suggesting some form of signaling that activates endocytotic machinery following synaptic vesicle fusion. This central property of synaptic vesicle release makes it technically challenging to experimentally assess the molecules and factors mediating endocytosis, since most manipulations to alter or probe endocytosis also affect exocytosis. This strong coupling also makes it difficult to discriminate between factors that trigger or modulate endocytosis, giving rise to
discrepancies and contradictions in the field. While the essential role of Ca2+ in triggering action potential evoked fusion of synaptic vesicles is undisputed, the exact role of Ca2+ in synaptic vesicle endocytosis is far from clear [2]. Several studies have suggested an essential role for Ca2+ in synaptic vesicle retrieval. For instance, in early experiments, Ceccarelli and colleagues took advantage of low doses of crude black widow spider venom or purified α-latrotoxin to trigger synaptic vesicle fusion in a Ca2+independent manner and examined synaptic vesicle retrieval using electron microscopy [3]. At the frog neuromuscular junction, when these agents were applied in a Ca2+-free solution, neurotransmission was irreversibly blocked within 1 h, coupled with a complete depletion of vesicles from the terminals. In contrast, when these toxins were applied in the presence of Ca2+, neurotransmission was sustained throughout the 1-h exposure, and morphological attributes of synaptic terminals as well as numbers of synaptic vesicles were preserved. These initial observations suggested that Ca2+ plays a critical role in maintenance of synaptic vesicle endocytosis at the frog neuromuscular junction. More recent experiments have demonstrated that Ca2+ influx into the presynaptic terminal can elicit different modes of endocytosis by targeting calmodulin and the Ca2+-calmodulin-dependent phosphatase calcineurin [4]. However, these proposals have been difficult to reconcile with the observation that synaptic vesicle fusion events triggered in a Ca2+-independent fashion, such as hypertonic sucrose driven release or spontaneous release, are also tightly coupled to subsequent endocytosis [5,6]. Furthermore, depending on the type of synapse used as a model system, the temperature, and the intensity and mode of stimulation, Ca2+ has been found to either accelerate, slow down, or have no impact at all on endocytosis at presynaptic terminals [2,7].
A recent elegant study by Orlando and colleagues [8], took advantage of a wellknown Ca2+-independent mode of release of neurotransmitters, hypertonic sucrose stimulation [9], to address whether Ca2+ is indeed essential for synaptic vesicle retrieval. For this purpose, they stimulated hippocampal neurons with hypertonic sucrose application followed by high pressure freezing or cryofixation and analyzed synaptic ultrastructure with transmission electron microscopy. Using this approach, the authors demonstrated that endocytic pits (plasma membrane invaginations), the first step in the initiation of the endocytic process, can form in the absence of Ca2+ influx into the presynaptic terminal [8]. This direct visualization of calcium-independent endocytosis using electron microscopy agrees well with previous functional studies using optical and electrophysiological methods [5,7], and supports the notion that Ca2+ is not essential for the triggering of endocytosis (Figure 1). A follow-up question that arises is whether endocytosis, once initiated, can also proceed in the absence of presynaptic Ca2+ influx. Orlando and colleagues observed that the endocytic pits were halted at the periactive zone, and endocytosis could only progress after removal of sucrose. This observation indicates that Ca2+ dependence of endocytosis can be divided into two components: the triggering step and the progression step. This result agrees with previous reports of recovery of synaptic vesicles in a Ca2+independent manner after sucrose stimulation, but with kinetics N10 times slower than in the presence of Ca2+ [5]. Thus, while initiation of endocytosis does not rely on Ca2+, progression of endocytosis is modulated by Ca2+. Through the work of Orlando and colleagues, as well as others, tension and other biophysical properties of biological membranes are emerging as strong candidates for fast coupling of exocytosis and endocytosis Trends in Neurosciences, Month 2019, Vol. xx, No. xx
1
Trends in Neurosciences
of release to retrieval of synaptic vesicle components, but it rather plays a modulatory role [10]. Moreover, biophysical membrane properties as well as actin dynamics likely mediate the actual coupling of exocytosis and endocytosis. In intact nerve terminals, a localized and shortlasting Ca2+ increase triggers the fusion of a synaptic vesicle after action potential arrival, so a physical coupling mechanism to quickly and simultaneously activate compensatory endocytosis possibly provides a more robust solution to this problem than Ca2+ itself.
1
Department of Pharmacology, Vanderbilt University, Nashville, TN, 37240-7933, USA Vanderbilt Brain Institute, Vanderbilt University, Nashville, TN, 37240-7933, USA
2
*Correspondence: [email protected] (E.T. Kavalali). https://doi.org/10.1016/j.tins.2019.12.001 © 2019 Elsevier Ltd. All rights reserved.
Trends in Neurosciences
Figure 1. Ca2+-Independent Progression of Synaptic Vesicle Endocytosis during Hypertonic Sucrose Stimulation. Top to bottom: cartoon depicting the sequence of events following hypertonic sucrose stimulation leading to Ca2+-independent retrieval of fused synaptic vesicles, as analyzed by Orlando and colleagues [8]. While initial endocytic pits form during hypertonic stimulation, vesicle budding proceeds only after removal of hypertonic solution. The latter process can be facilitated by Ca2+. Abbreviations: RRP, readily releasable pool.
of synaptic vesicles, while other factors including Ca2+ and certain proteins may work downstream as regulators. Ca2+-independent triggering of endocytosis is also insensitive to temperature changes [8]. What are the molecules regulating initiation of endocytosis? Orlando and colleagues show that actin dynamics have a key role in pit formation, probably through modulation of membrane tension and curvature. Moreover, Ca2+-independent endocytic pit formation is dependent on dynamin [8],
2
Trends in Neurosciences, Month 2019, Vol. xx, No. xx
a GTPase that facilitates membrane scission catalyzing the physical separation of the plasma membrane and the endocytic vesicle, but independent of clathrin, agreeing with recent reports on clathrin-independent retrieval of synaptic vesicle proteins [1]. When considered in the context of this long-standing debate about the molecular mechanisms underlying synaptic vesicle endocytosis, the work by Orlando and colleagues provides a clear answer: Ca2+ is not an essential factor for the coupling
References 1. Chanaday, N.L. and Kavalali, E.T. (2017) How do you recognize and reconstitute a synaptic vesicle after fusion? F1000Res. 6, 1734 2. Leitz, J. and Kavalali, E.T. (2016) Ca2+ Dependence of synaptic vesicle endocytosis. Neuroscientist 22, 464–476 3. Ceccarelli, B. and Hurlbut, W.P. (1980) Ca2+-dependent recycling of synaptic vesicles at the frog neuromuscular junction. J. Cell Biol. 87, 297–303 4. Wu, X.S. et al. (2009) Ca(2+) and calmodulin initiate all forms of endocytosis during depolarization at a nerve terminal. Nat. Neurosci. 12, 1003–1010 5. Pyle, J.L. et al. (2000) Rapid reuse of readily releasable pool vesicles at hippocampal synapses. Neuron 28, 221–231 6. Leitz, J. and Kavalali, E.T. (2014) Fast retrieval and autonomous regulation of single spontaneously recycling synaptic vesicles. eLife 3, e03658 7. Miyano, R. et al. (2019) Ca-dependence of synaptic vesicle exocytosis and endocytosis at the hippocampal mossy fibre terminal. J. Physiol. 597, 4373–4386 8. Orlando, M. et al. (2019) Calcium-independent exoendocytosis coupling at small central synapses. Cell Rep. 29, 3767–3774.e3. https://doi.org/10.1016/j. celrep.2019.11.060 9. Rosenmund, C. and Stevens, C.F. (1996) Definition of the readily releasable pool of vesicles at hippocampal synapses. Neuron 16, 1197–1207 10. Li, Y.C. et al. (2017) Synaptotagmin-1 and synaptotagmin7-dependent fusion mechanisms target synaptic vesicles to kinetically distinct endocytic pathways. Neuron 93, 616–631
Trends in Neurosciences
Spotlight
Is Ca2+ Essential for Synaptic Vesicle Endocytosis? Natali L. Chanaday1 and Ege T. Kavalali1,2,* Synaptic vesicle fusion is coupled to swift retrieval of vesicle components from the synaptic plasma membrane. Ca2+ has been assumed to be a key mediator of this coupling. In a recent study, Orlando et al. unequivocally demonstrate that Ca2+ is not essential for synaptic vesicle retrieval. Neurotransmitters are released from presynaptic terminals following regulated fusion of synaptic vesicles with the plasma membrane at the active zone. Synaptic vesicles are small distinct membranous organelles with an average diameter around 40 nm and they harbor a number of specific molecular components that are critical for their function. For neurotransmission to be sustained over time, synaptic vesicle components, lipids and proteins, need to be retrieved from the plasma membrane after fusion and synaptic vesicles need to be regenerated [1]. The mechanisms mediating synaptic vesicle recycling have been a matter of intense research during the last 50 years. Extensive evidence supports the notion that synaptic vesicle exocytosis and endocytosis are coupled processes suggesting some form of signaling that activates endocytotic machinery following synaptic vesicle fusion. This central property of synaptic vesicle release makes it technically challenging to experimentally assess the molecules and factors mediating endocytosis, since most manipulations to alter or probe endocytosis also affect exocytosis. This strong coupling also makes it difficult to discriminate between factors that trigger or modulate endocytosis, giving rise to
discrepancies and contradictions in the field. While the essential role of Ca2+ in triggering action potential evoked fusion of synaptic vesicles is undisputed, the exact role of Ca2+ in synaptic vesicle endocytosis is far from clear [2]. Several studies have suggested an essential role for Ca2+ in synaptic vesicle retrieval. For instance, in early experiments, Ceccarelli and colleagues took advantage of low doses of crude black widow spider venom or purified α-latrotoxin to trigger synaptic vesicle fusion in a Ca2+independent manner and examined synaptic vesicle retrieval using electron microscopy [3]. At the frog neuromuscular junction, when these agents were applied in a Ca2+-free solution, neurotransmission was irreversibly blocked within 1 h, coupled with a complete depletion of vesicles from the terminals. In contrast, when these toxins were applied in the presence of Ca2+, neurotransmission was sustained throughout the 1-h exposure, and morphological attributes of synaptic terminals as well as numbers of synaptic vesicles were preserved. These initial observations suggested that Ca2+ plays a critical role in maintenance of synaptic vesicle endocytosis at the frog neuromuscular junction. More recent experiments have demonstrated that Ca2+ influx into the presynaptic terminal can elicit different modes of endocytosis by targeting calmodulin and the Ca2+-calmodulin-dependent phosphatase calcineurin [4]. However, these proposals have been difficult to reconcile with the observation that synaptic vesicle fusion events triggered in a Ca2+-independent fashion, such as hypertonic sucrose driven release or spontaneous release, are also tightly coupled to subsequent endocytosis [5,6]. Furthermore, depending on the type of synapse used as a model system, the temperature, and the intensity and mode of stimulation, Ca2+ has been found to either accelerate, slow down, or have no impact at all on endocytosis at presynaptic terminals [2,7].
A recent elegant study by Orlando and colleagues [8], took advantage of a wellknown Ca2+-independent mode of release of neurotransmitters, hypertonic sucrose stimulation [9], to address whether Ca2+ is indeed essential for synaptic vesicle retrieval. For this purpose, they stimulated hippocampal neurons with hypertonic sucrose application followed by high pressure freezing or cryofixation and analyzed synaptic ultrastructure with transmission electron microscopy. Using this approach, the authors demonstrated that endocytic pits (plasma membrane invaginations), the first step in the initiation of the endocytic process, can form in the absence of Ca2+ influx into the presynaptic terminal [8]. This direct visualization of calcium-independent endocytosis using electron microscopy agrees well with previous functional studies using optical and electrophysiological methods [5,7], and supports the notion that Ca2+ is not essential for the triggering of endocytosis (Figure 1). A follow-up question that arises is whether endocytosis, once initiated, can also proceed in the absence of presynaptic Ca2+ influx. Orlando and colleagues observed that the endocytic pits were halted at the periactive zone, and endocytosis could only progress after removal of sucrose. This observation indicates that Ca2+ dependence of endocytosis can be divided into two components: the triggering step and the progression step. This result agrees with previous reports of recovery of synaptic vesicles in a Ca2+independent manner after sucrose stimulation, but with kinetics N10 times slower than in the presence of Ca2+ [5]. Thus, while initiation of endocytosis does not rely on Ca2+, progression of endocytosis is modulated by Ca2+. Through the work of Orlando and colleagues, as well as others, tension and other biophysical properties of biological membranes are emerging as strong candidates for fast coupling of exocytosis and endocytosis Trends in Neurosciences, Month 2019, Vol. xx, No. xx
1
Trends in Neurosciences
of release to retrieval of synaptic vesicle components, but it rather plays a modulatory role [10]. Moreover, biophysical membrane properties as well as actin dynamics likely mediate the actual coupling of exocytosis and endocytosis. In intact nerve terminals, a localized and shortlasting Ca2+ increase triggers the fusion of a synaptic vesicle after action potential arrival, so a physical coupling mechanism to quickly and simultaneously activate compensatory endocytosis possibly provides a more robust solution to this problem than Ca2+ itself.
1
Department of Pharmacology, Vanderbilt University, Nashville, TN, 37240-7933, USA Vanderbilt Brain Institute, Vanderbilt University, Nashville, TN, 37240-7933, USA
2
*Correspondence: [email protected] (E.T. Kavalali). https://doi.org/10.1016/j.tins.2019.12.001 © 2019 Elsevier Ltd. All rights reserved.
Trends in Neurosciences
Figure 1. Ca2+-Independent Progression of Synaptic Vesicle Endocytosis during Hypertonic Sucrose Stimulation. Top to bottom: cartoon depicting the sequence of events following hypertonic sucrose stimulation leading to Ca2+-independent retrieval of fused synaptic vesicles, as analyzed by Orlando and colleagues [8]. While initial endocytic pits form during hypertonic stimulation, vesicle budding proceeds only after removal of hypertonic solution. The latter process can be facilitated by Ca2+. Abbreviations: RRP, readily releasable pool.
of synaptic vesicles, while other factors including Ca2+ and certain proteins may work downstream as regulators. Ca2+-independent triggering of endocytosis is also insensitive to temperature changes [8]. What are the molecules regulating initiation of endocytosis? Orlando and colleagues show that actin dynamics have a key role in pit formation, probably through modulation of membrane tension and curvature. Moreover, Ca2+-independent endocytic pit formation is dependent on dynamin [8],
2
Trends in Neurosciences, Month 2019, Vol. xx, No. xx
a GTPase that facilitates membrane scission catalyzing the physical separation of the plasma membrane and the endocytic vesicle, but independent of clathrin, agreeing with recent reports on clathrin-independent retrieval of synaptic vesicle proteins [1]. When considered in the context of this long-standing debate about the molecular mechanisms underlying synaptic vesicle endocytosis, the work by Orlando and colleagues provides a clear answer: Ca2+ is not an essential factor for the coupling
References 1. Chanaday, N.L. and Kavalali, E.T. (2017) How do you recognize and reconstitute a synaptic vesicle after fusion? F1000Res. 6, 1734 2. Leitz, J. and Kavalali, E.T. (2016) Ca2+ Dependence of synaptic vesicle endocytosis. Neuroscientist 22, 464–476 3. Ceccarelli, B. and Hurlbut, W.P. (1980) Ca2+-dependent recycling of synaptic vesicles at the frog neuromuscular junction. J. Cell Biol. 87, 297–303 4. Wu, X.S. et al. (2009) Ca(2+) and calmodulin initiate all forms of endocytosis during depolarization at a nerve terminal. Nat. Neurosci. 12, 1003–1010 5. Pyle, J.L. et al. (2000) Rapid reuse of readily releasable pool vesicles at hippocampal synapses. Neuron 28, 221–231 6. Leitz, J. and Kavalali, E.T. (2014) Fast retrieval and autonomous regulation of single spontaneously recycling synaptic vesicles. eLife 3, e03658 7. Miyano, R. et al. (2019) Ca-dependence of synaptic vesicle exocytosis and endocytosis at the hippocampal mossy fibre terminal. J. Physiol. 597, 4373–4386 8. Orlando, M. et al. (2019) Calcium-independent exoendocytosis coupling at small central synapses. Cell Rep. 29, 3767–3774.e3. https://doi.org/10.1016/j. celrep.2019.11.060 9. Rosenmund, C. and Stevens, C.F. (1996) Definition of the readily releasable pool of vesicles at hippocampal synapses. Neuron 16, 1197–1207 10. Li, Y.C. et al. (2017) Synaptotagmin-1 and synaptotagmin7-dependent fusion mechanisms target synaptic vesicles to kinetically distinct endocytic pathways. Neuron 93, 616–631