Allosteric stabilization of calcium and phosphoinositide dual binding engages three synaptotagmins in fast exocytosis
Listed in
 Evaluated articles (eLife)
 Neuroscience (eLife)
Abstract
Synaptic communication relies on the fusion of synaptic vesicles with the plasma membrane, which leads to neurotransmitter release. This exocytosis is triggered by brief and local elevations of intracellular Ca ^{2+} with remarkably high sensitivity. How this is molecularly achieved is unknown. While synaptotagmins confer the Ca ^{2+} sensitivity of neurotransmitter exocytosis, biochemical measurements reported Ca ^{2+} affinities too low to account for synaptic function. However, synaptotagmin’s Ca ^{2+} affinity increases upon binding the plasma membrane phospholipid PI(4,5)P _{2} and, vice versa, Ca ^{2+} binding increases synaptotagmin’s PI(4,5)P _{2} affinity, indicating a stabilization of the Ca ^{2+} /PI(4,5)P _{2} dualbound syt. We here devise a molecular exocytosis model based on this positive allosteric stabilization and the assumptions that (1.) synaptotagmin Ca ^{2+} /PI(4,5)P _{2} dual binding lowers the energy barrier for vesicle fusion and that (2.) the effect of multiple synaptotagmins on the energy barrier is additive. The model, which relies on biochemically measured Ca ^{2+} /PI(4,5)P _{2} affinities and protein copy numbers, reproduced the steep Ca ^{2+} dependency of neurotransmitter release. Our results indicate that each synaptotagmin dual binding Ca ^{2+} /PI(4,5)P _{2} lowers the energy barrier for vesicle fusion by 4.85 k _{B} T and that allosteric stabilization of this state enables the synchronized engagement of three synaptotagmins for fast exocytosis. Furthermore, we show that mutations altering synaptotagmin’s allosteric properties may show dominantnegative effects, even though synaptotagmins act independently on the energy barrier, and that dynamic changes of local PI(4,5)P _{2} (e.g. upon vesicle movement) dramatically impact synaptic responses. We conclude that allosterically stabilized Ca ^{2+} /PI(4,5)P _{2} dual binding enables synaptotagmins to exert their coordinated function in neurotransmission.
Article activity feed

Evaluation Summary:
Synaptotagmins are Ca2+ sensors for synchronous neurotransmitter release. However, despite intense study it remains unclear exactly how the binding of 5 Ca2+ ions to Synaptotagmin's two C2 domains leads to the observed Ca2+ dependence of vesicle fusion. This study puts forward a novel mechanistic model of neurotransmitter vesicle fusion (vesicle exocytosis) which is relatively simple but significantly more detailed than the widely used phenomenological models of calciumdependent fast exocytosis.
(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 #1 agreed to share their name with the authors.)

Reviewer #1 (Public Review):
This study puts forward a novel mechanistic model of neurotransmitter vesicle fusion (vesicle exocytosis) which is relatively simple but significantly more detailed than the widely used phenomenological models of calciumdependent fast exocytosis. The main goal of the Authors is to account more fully and holistically for the known properties of neurotransmitter release, namely: (1) the highly nonlinear dependence of neurotransmitter release rate and latency on calcium ion (Ca2+) concentration, based on the published recordings obtained from the giant calyx of Held auditory synaptic terminals, (2) the low intrinsic Ca2+ affinity of the main sensor for Ca2+ triggered exocytosis, synaptotagmin (Syt); (3) evidence that synaptotagmin's Ca2+ affinity is greatly increased after is binds to PIP2 phospholipids that …
Reviewer #1 (Public Review):
This study puts forward a novel mechanistic model of neurotransmitter vesicle fusion (vesicle exocytosis) which is relatively simple but significantly more detailed than the widely used phenomenological models of calciumdependent fast exocytosis. The main goal of the Authors is to account more fully and holistically for the known properties of neurotransmitter release, namely: (1) the highly nonlinear dependence of neurotransmitter release rate and latency on calcium ion (Ca2+) concentration, based on the published recordings obtained from the giant calyx of Held auditory synaptic terminals, (2) the low intrinsic Ca2+ affinity of the main sensor for Ca2+ triggered exocytosis, synaptotagmin (Syt); (3) evidence that synaptotagmin's Ca2+ affinity is greatly increased after is binds to PIP2 phospholipids that are concentrated at the interface between the vesicle and synaptic membranes.
The main conclusions of this study is that the increase in Syt sensor's affinity to Ca2+ upon allosteric binding to PIP2 phospholipids is sufficient to account for the observed high sensitivity of neurotransmitter release rate and latency to Ca2+ elevations, and that the best fit to experimental data is achieved if about 15 Syt molecules are positioned along the surface of the vesicle, with three (on average) Syt molecules binding to PIP2 and Ca2+ before vesicle fusion is triggered. Thus, the study provides an explanatory framework for predicting the synaptotagmin copy number distribution per vesicle.
STRENGTHS
The proposed model occupies a "sweet spot" in terms of level of detail, going beyond widely used phenomenological models that are too simple to provide descriptive insight into the molecular interactions underlying observed Ca2+ dependence of vesicle fusion rate and latency, but staying away from highly detailed molecular models that cannot be fully constrained using existing experimental data, and which may not be needed to account for the main features of fast calciumtriggered exocytosis.
The advantage of the proposed model is that it holistically ties together within a single, relatively simple framework the main features of neurotransmitter release at central synapses, namely: (1) the high order (4th or 5th order) cooperativity in Ca2+ binding, despite the fact that only two Ca2+ ions bind to the relevant C2B domain of any single Syt molecule during fast calciumtriggered vesicle fusion; (2) the increase in Syt affinity to Ca2+ upon PIPs binding; (3) Syt copy number distribution per vesicle.
An appealing feature of the model is that it naturally leads to (and therefore explains in more depth) the phenomenological model of vesicle fusion put forward previously by Lou, Scheuss and Schneggenburger (2005), whereby vesicle fusion can proceed in several stages with increasing rate, which the proposed model explains in terms of the increase in fusion probability with increasing number of SytPIP2 "bridges" formed between the vesicle and synaptic membrane upon Syt binding to Ca2+ and PIP2. I think this model can also be viewed as a more detailed extension of the "excess binding site" model of Stephen D. Meriney and colleagues.
Another strength of this work is that it carefully examines and solves a fully stochastic model of vesicle fusion, rather than a simplified massaction representation that would not be adequate given the small copy number of Syt1 and SytPIP2 "bridges" proposed in this model. For the case of constant Ca2+ pulses, the Authors take advantage of the exact solution of the underlying Markov process, and perform stochastic simulation for a transient Ca2+ signal that mimics an incoming action potential.
Finally, parameter values were constrained very systematically, using an automated optimization algorithm that allows an unbiased estimate for modeling parameters achieving the best fit with experimental data. Quite appropriately, a derivativefree optimization approach was used, since optimization methods based on gradient descent would not perform well if the objective/cost function calculation involves numerical integration and is susceptible to other sources of numerical noise.
Given the relative simplicity of the model, it can readily be used in computational studies that rely on accurate representation of calciumtriggered vesicle fusion that goes beyond simple massaction schemes, but does not require molecularlevel precision and resolution in modeling the underlying processes.
Importantly, the simulation source code is included, allowing to reproduce the results of the model.
Finally, I would like to note that despite the technical nature of the model, the manuscript is written in an accessible way, and figure are well designed, making it easy to follow the logic of this work.
WEAKNESSES
Given the Authors' choice of modeling detail level, and the overall scope of the study, I do not detect significant weaknesses in the approach or results. The only potential weakness I can see is that the assumption of simultaneous binding of two calcium ions, rather than a sequence of two binding events, may affect the estimation of vesicle release latency examined in this study.
Of course, the simplified model proposed here cannot fully account for all molecular interactions involved in fast nonconstitutive vesicle fusion, and in particular, the interaction of synaptotagmin molecules with the SNARE machinery is only represented as the height of the barrier to vesicle fusion, but a full model may not be needed for a descriptive understanding of this phenomenon.
More minor suggestions for improvement are conveyed in a separate recommendation for the Authors.

Reviewer #2 (Public Review):
Vesicular synaptotagmins are the undisputed Ca2+ sensors for synchronous neurotransmitter release. However, despite intense study it remains unclear exactly how the binding of 5 Ca2+ ions to Synaptotagmin's two C2 domains leads to the observed Ca2+ dependence of vesicle fusion. Moreover, it is unclear how many Syts bind Ca2+ to initiate fusion. This is a fascinating question. For decades, the 45 power dependence of exocytosis on Ca2+ was attributed to cooperative binding of 5 Ca ions to Syt1. This study convincingly proposes an alternative scenario, where cooperative Ca/PIP2 binding and multiple Syt1 molecules can explain the Ca dependence of release. The authors make interesting and testable predictions about the molecular mechanism by which the C2B domain initiates fusion, and about the number of Syts …
Reviewer #2 (Public Review):
Vesicular synaptotagmins are the undisputed Ca2+ sensors for synchronous neurotransmitter release. However, despite intense study it remains unclear exactly how the binding of 5 Ca2+ ions to Synaptotagmin's two C2 domains leads to the observed Ca2+ dependence of vesicle fusion. Moreover, it is unclear how many Syts bind Ca2+ to initiate fusion. This is a fascinating question. For decades, the 45 power dependence of exocytosis on Ca2+ was attributed to cooperative binding of 5 Ca ions to Syt1. This study convincingly proposes an alternative scenario, where cooperative Ca/PIP2 binding and multiple Syt1 molecules can explain the Ca dependence of release. The authors make interesting and testable predictions about the molecular mechanism by which the C2B domain initiates fusion, and about the number of Syts required for synchronous release. To improve the readability and impact of this study, the authors should highlight what is novel about the predictions of their model, and put them in context with previous studies and interpretations.

Reviewer #3 (Public Review):
In this paper by Kobbersmed et al., the authors used mathematical analysis to investigate the role played by the interaction of Ca2+, PIP2, and synaptotagmin in shaping the kinetics of neurotransmitter release. Published data from in vitro analysis of the affinities of synaptotagmin1 for Ca2+ and PIP2 and the copy numbers of synaptotagmin molecules per vesicle are used in the model. Applying the model predictions to published data at a specific synapse (calyx of Held), the authors show that conformational stabilization of a crosslinking synaptotagmin/Ca2+/PIP2 complex plays a role in extending the dwell time needed to allow multiple crosslinking. These crosslinks are assumed to lower the energy barrier for vesicle fusion. An assumed strong cooperativity of Ca2+ and PIP2 binding to synaptotagmin can solve the …
Reviewer #3 (Public Review):
In this paper by Kobbersmed et al., the authors used mathematical analysis to investigate the role played by the interaction of Ca2+, PIP2, and synaptotagmin in shaping the kinetics of neurotransmitter release. Published data from in vitro analysis of the affinities of synaptotagmin1 for Ca2+ and PIP2 and the copy numbers of synaptotagmin molecules per vesicle are used in the model. Applying the model predictions to published data at a specific synapse (calyx of Held), the authors show that conformational stabilization of a crosslinking synaptotagmin/Ca2+/PIP2 complex plays a role in extending the dwell time needed to allow multiple crosslinking. These crosslinks are assumed to lower the energy barrier for vesicle fusion. An assumed strong cooperativity of Ca2+ and PIP2 binding to synaptotagmin can solve the problem that the Ca2+ affinity of synaptotagmin measured in vitro (Kd in the range of 200 µM) is much lower that the estimated Ca2+ affinity of the Ca2+ sensor for vesicle fusion measured at the calyx of Held (Kd in the range of 5 µM). A minimum of three crosslinked synaptogamins per vesicle was enough to account for physiological data obtained at the calyx of Held, nevertheless higher copy number of synaptogamins ensured a higher probability of interaction of synaptotagmin with Ca2+ and PIP2. The approach is innovative because the author aim to explain synaptic parameters with simulations of single synaptotagmin molecule. The main concern is that the validity of the conclusions is difficult to judge as the study relies only on modeling. Some of the assumptions of the modeling could be wrong and there is no attempt for experimental validation of the model.

