Regulation of presynaptic Ca2+ channel abundance at active zones through a balance of delivery and turnover
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Evaluation Summary:
The authors undertake a detailed investigation focused on how the abundance of the sole Cav2 Ca2+ channel Cac in Drosophila is regulated at active zones (AZs) using the larval neuromuscular junction (NMJ) as a model system. The larval NMJ is a particularly powerful system to address this question, and the authors have taken full advantage of the unique approaches available. This work makes important advances in our understanding of AZ Ca2+ channel regulation during development and will be of significant interest to the field.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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Abstract
Voltage-gated Ca 2+ channels (VGCCs) mediate Ca 2+ influx to trigger neurotransmitter release at specialized presynaptic sites termed active zones (AZs). The abundance of VGCCs at AZs regulates neurotransmitter release probability ( P r ), a key presynaptic determinant of synaptic strength. Although biosynthesis, delivery, and recycling cooperate to establish AZ VGCC abundance, experimentally isolating these distinct regulatory processes has been difficult. Here, we describe how the AZ levels of cacophony (Cac), the sole VGCC-mediating synaptic transmission in Drosophila , are determined. We also analyzed the relationship between Cac, the conserved VGCC regulatory subunit α2δ, and the core AZ scaffold protein Bruchpilot (BRP) in establishing a functional AZ. We find that Cac and BRP are independently regulated at growing AZs, as Cac is dispensable for AZ formation and structural maturation, and BRP abundance is not limiting for Cac accumulation. Additionally, AZs stop accumulating Cac after an initial growth phase, whereas BRP levels continue to increase given extended developmental time. AZ Cac is also buffered against moderate increases or decreases in biosynthesis, whereas BRP lacks this buffering. To probe mechanisms that determine AZ Cac abundance, intravital FRAP and Cac photoconversion were used to separately measure delivery and turnover at individual AZs over a multi-day period. Cac delivery occurs broadly across the AZ population, correlates with AZ size, and is rate-limited by α2δ. Although Cac does not undergo significant lateral transfer between neighboring AZs over the course of development, Cac removal from AZs does occur and is promoted by new Cac delivery, generating a cap on Cac accumulation at mature AZs. Together, these findings reveal how Cac biosynthesis, synaptic delivery, and recycling set the abundance of VGCCs at individual AZs throughout synapse development and maintenance.
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Evaluation Summary:
The authors undertake a detailed investigation focused on how the abundance of the sole Cav2 Ca2+ channel Cac in Drosophila is regulated at active zones (AZs) using the larval neuromuscular junction (NMJ) as a model system. The larval NMJ is a particularly powerful system to address this question, and the authors have taken full advantage of the unique approaches available. This work makes important advances in our understanding of AZ Ca2+ channel regulation during development and will be of significant interest to the field.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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Reviewer #3 (Public Review):
The authors undertake a detailed investigation focused on how the abundance of the sole Cav2 Ca2+ channel Cac in Drosophila is regulated at active zones (AZs) using the larval neuromuscular junction (NMJ) as a model system. The larval NMJ is a particularly powerful system to address this question, and the authors have taken full advantage of the unique approaches available. Specifically, using endogenously tagged Cac alleles and transgenes combined with cell biological, electrophysiological, and imaging approaches, the authors make several key findings. Most notably, the authors generated endogenously tagged, photoconvertible Cac alleles that enable the quantification of Cac turnover at specific AZs over time and during synaptic development. They find that the abundance of Cac vs the AZ scaffold BRP is …
Reviewer #3 (Public Review):
The authors undertake a detailed investigation focused on how the abundance of the sole Cav2 Ca2+ channel Cac in Drosophila is regulated at active zones (AZs) using the larval neuromuscular junction (NMJ) as a model system. The larval NMJ is a particularly powerful system to address this question, and the authors have taken full advantage of the unique approaches available. Specifically, using endogenously tagged Cac alleles and transgenes combined with cell biological, electrophysiological, and imaging approaches, the authors make several key findings. Most notably, the authors generated endogenously tagged, photoconvertible Cac alleles that enable the quantification of Cac turnover at specific AZs over time and during synaptic development. They find that the abundance of Cac vs the AZ scaffold BRP is independently regulated, that Cac does not appear to intermix between different AZs, and that Cac levels are buffered, where the alpha-2 delta subunit promotes new Cac delivery to AZs and removal of existing Cac without changing the apparent overall abundance.
Previous studies in Drosophila and in other systems have revealed considerable insight into Ca2+ channel regulation at AZs. For example, it is well established that the alpha-2 delta subunit is required for Cac trafficking to AZs, and in rodents, that Cav2 channels are not necessary for AZ or synapse assembly. There is, therefore, a question about how significant the new findings reported here are to the field. There is also a significant issue with the interpretation of the requirement of Cac in AZ assembly, where the authors have not demonstrated to what extent Cac and Ca2+ influx is eliminated in their conditional knockout in motor neurons. However, few studies have directly focused on how Cav2 channels are regulated and turned over at AZs, particularly during development and in baseline states. Most importantly, the photoconvertible Cac alleles and related imaging experiments reported in this study demonstrate significant new insights about the yin and yang of Ca2+ channel delivery and turnover at AZs, at a resolution and rigor rarely achieved. Furthermore, this study also provides an excellent foundation to unlock how Cav2 channel regulation at AZs is modified by such processes as neuronal activity and synaptic plasticity. Thus, in my opinion, this work will be of significant interest and importance to the field.
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Reviewer #2 (Public Review):
This manuscript represents a highly significant contribution to our understanding of synapse assembly and VGCC regulation. The authors generated a new Flpstop allele of Cac enabling them to assess the phenotypic consequences of loss of Cac at L3 NMJs for the first time. These data convincingly demonstrate that Cac is not required for either AZ nucleation or accumulation of cytomatrix proteins. Through a series of elegant experiments, they demonstrate that, unlike Brp, Cac levels are buffered at NMJ synapses. They then turn their attention to the auxiliary Ca2+ channel subunit a2d and show that it is limiting for Cac accumulation at AZs. Finally, they generate an endogenously tagged Cac-Maple enabling them to estimate Cac turnover and to argue that a2d is rate-limiting for Cac recycling. Strengths of this …
Reviewer #2 (Public Review):
This manuscript represents a highly significant contribution to our understanding of synapse assembly and VGCC regulation. The authors generated a new Flpstop allele of Cac enabling them to assess the phenotypic consequences of loss of Cac at L3 NMJs for the first time. These data convincingly demonstrate that Cac is not required for either AZ nucleation or accumulation of cytomatrix proteins. Through a series of elegant experiments, they demonstrate that, unlike Brp, Cac levels are buffered at NMJ synapses. They then turn their attention to the auxiliary Ca2+ channel subunit a2d and show that it is limiting for Cac accumulation at AZs. Finally, they generate an endogenously tagged Cac-Maple enabling them to estimate Cac turnover and to argue that a2d is rate-limiting for Cac recycling. Strengths of this paper include the importance of studying how AZ levels of VGCCs are regulated, the rigorous and logically presented experiments, and the exciting findings regarding the role of a2d in Cac delivery and turnover. Weaknesses are minor. (1) Regarding the novelty of the Cac findings, VGCCs have been shown to be dispensable for synapse assembly in mammalian neurons (Held et al., 2020). However, this is the first report to carefully approach this question in Drosophila, and the concurrence of the results suggests that it likely reflects an evolutionarily conserved design principle of presynaptic terminals. (2) The authors argue that a2d is rate-limiting for Cac delivery to AZs. This conclusion is based on Figures 6 and 7, where they show defects in delivery/turnover in homozygous and heterozygous mutant a2d NMJs. Whether a2d overexpression is sufficient to drive delivery/turnover was not investigated. Overall, this is an impressive paper that will have a lasting impact on the field of cellular and molecular neuroscience.
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Reviewer #1 (Public Review):
In this study, the authors set out to investigate mechanisms regulating voltage-gated calcium channel abundance at synaptic active zones. As action-potential induced calcium influx into presynaptic terminals drives neurotransmitter release and because the number of participating calcium channels has a major influence on this signaling, insights into principles regulating calcium channel abundance are highly relevant for our understanding of mechanisms controlling neural excitability. Despite the pivotal relevance, relatively little is known about how this abundance is controlled.
The study here provides some novel insights into this regulation during synapse development and upon genetic challenges in Drosophila melanogaster. The Drosophila neuromuscular junction is one of the few model synapses where channel …
Reviewer #1 (Public Review):
In this study, the authors set out to investigate mechanisms regulating voltage-gated calcium channel abundance at synaptic active zones. As action-potential induced calcium influx into presynaptic terminals drives neurotransmitter release and because the number of participating calcium channels has a major influence on this signaling, insights into principles regulating calcium channel abundance are highly relevant for our understanding of mechanisms controlling neural excitability. Despite the pivotal relevance, relatively little is known about how this abundance is controlled.
The study here provides some novel insights into this regulation during synapse development and upon genetic challenges in Drosophila melanogaster. The Drosophila neuromuscular junction is one of the few model synapses where channel levels, delivery, and turnover can be investigated using light microscopy. Previous studies have relied on the over-expression of a GFP-tagged obligatory subunit (Cacophony, Cac) of the synaptic voltage-gated channel and concluded on a consecutive arrival of calcium channels and active zone scaffolding proteins such as Bruchpilot (BRP)(Fouquet et al., 2009). The prevailing view is that BRP and other AZ scaffolds (such as BRP) then stabilize the channels at the AZs (Kittel et al., 2006; Liu et al., 2011). The current study provides further insights into this and uses genetically more advanced endogenously GFP-tagged Cac (one developed by the O'Gilles lab, one developed for this study) to evaluate the relation between Cac and BRP. Additional, well-suited genetic tools were applied to study these questions: Cac gene expression was knocked out in individual cells to study its loss while avoiding lethality, and development was delayed to study its adaptation during synaptic maturation in greater detail. In vivo imaging experiments (photobleaching and conversion) used to investigate Cac´s local, synaptic turnover and (for the first time) stability.
While the authors challenge the view that Cac is required for BRP AZ incorporation and that BRP abundance limits Cac levels, the study also lends support to conclusions obtained in previous studies after Cac overexpression as they show that this overexpression does not result in excessive over-population at synaptic AZs, an important finding in the light of previous studies. In fact, a major and unexpected finding is that the local abundance of Cac may be particularly well regulated and buffered against changes of gene expression to a much larger extent than the AZ scaffolding protein BRP. A remarkable finding is the high stability of these proteins at the synapse (days) and that this stability can be further increased to maintain local synaptic levels if protein synthesis or -delivery are impaired. Overall, the study provides some novel, unexpected and important insights regarding stable calcium channel abundance at synapses. However, the current manuscript may be further improved because despite the wealth of high-quality data, some of the measurements and comparisons are indirect and some alternative interpretations possible.
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