Key determinants of the dual clamp/activator function of Complexin

Reviewer #1 (Public Review):

Summary:

Using chromaffin cells as a powerful model system for studying secretion, the authors study the regulatory role of complexin in secretion. Complexin is still enigmatic in its regulatory role, as it both provides inhibitory and facilitatory functions in release. The authors perform an extensive structure-function analysis of both the C- and N-terminal regions of complexin. There are several interesting findings that significantly advances our understanding of cpx/SNARe interactions in regulating release. C-terminal amphipathic helix interferes with SNARE complex assembly and thus clamps fusion. There are acidic residues in the C-term that may be seen as putative interaction partners for Synaptotagmin. The N-terminus of Complexin promoting role may be associated with an interaction with Syt1. In particular the putative interaction with Syt1 is of high interest and supported by quite strong functional and biochemical evidence. The experimental approaches are state of the art, and the results are of the highest quality and convincing throughout. They are adequate and intelligently discussed in the rich context of the standing literature. Whilst there are some concerns about whether the facilitatory actions of complexion have to be tightly linked to Syt1 interactions, the proposed model will significantly advance the field by providing new directions in future research.

Reviewer #2 (Public Review):

Summary:

Complexin (Cplx) is expressed at nearly all chemical synapses. Mammalian Cplx comes in four different paralogs which are differentially expressed in different neurons or secretory cell types, either selectively or in combination with one or two other Cplx isoforms. Cplx binds with high affinity to assembled SNARE complexes and promotes evoked synchronous release. Cplx is assumed to preclude premature SV fusion by preventing full SNARE assembly, thereby arresting subsequent SNARE-driven fusion ("fusion-clamp" theory). The protein has multiple domains, the functions of which are controversially discussed. Cplx's function has been studied in a variety of model organisms including mouse, fly, worm, and fish with seemingly conflicting results which led to partly contradicting conclusions.
Makee et al. study the function of mammalian Cplx2 in chromaffin cells by making use of Cplx2 ko mice to overexpress and functionally characterize mutant Cplx2 forms in cultured chromaffin cells. The main conclusion of the present study are:

The hydrophobic character of the amphipathic helix in Cplx's C-terminal domain is essential for inhibiting premature vesicle fusion at a [Ca2+]i of several hundreds of nM (pre-flash [Ca2+]i). The Cplx-mediated inhibition of fusion under these conditions does not rely on expression of either Syt1 or Syt7.

Slow-down of exocytosis by N-terminally truncated Cplx mutants in response to a [Ca2+]i of several µM (peak flash [Ca2+]i) occurs regardless of the presence or absence of Syt7 demonstrating that Cplx2 does not act as a switch favoring preferential assembly of the release machinery with Syt1,2 rather than the "slow" sensor Syt7.

Cplx's N-terminal domain is required for the Cplx2-mediated increase in the speed of exocytosis and faster onset of exocytosis which likely reflect an increased apparent Ca2+ sensitivity and faster Ca2+ binding of the release machinery.

Strengths:

The authors perform systematic truncation/mutational analyses of Cplx2. They analyze the impact of single and combined deficiencies for Cplx2 and Syt1 to establish interactions of both proteins.
State-of-the-art methods are employed: Vesicle exocytosis is assayed directly and with high resolution using capacitance measurements. Intracellular [Ca2+] is controlled by loading via the patch-pipette and by UV-light induced flash-photolysis of caged [Ca2+]. The achieved [Ca2+ ] is measured with Ca2+ -sensitive dyes.
The data is of high quality and the results are compelling.

Weaknesses:

With the exception of mammalian retinal ribbon synapses (and some earlier RNAi knock down studies which had off-target effects), there is little experimental evidence for a "fusion-clamp"-like function of Cplxs at mammalian synapses. At conventional mammalian synapses, genetic loss of Cplx (i.e. KO) consistently decreases AP-evoked release, and generally either also decreases spontaneous release rates or does not affect spontaneous release, which is inconsistent with a "fusion-clamp" theory. This is in stark contrast to invertebrate (D. m. and C. e.) synapses where genetic Cplx loss is generally associated with a strong upregulation of spontaneous release.

There are alternative scenarios explaining how Cplx may phenomenological "clamp" vesicle fusion rates without mechanistically assigning a "clamping" function to Cplx (Neher 2010, Neuron). In fact, changes in asynchronous release kinetics following conditioning AP trains observed at Cplx1 ko calyx of Held synapses do not favor a "fusion clamp" model (Chang et al., 2015, J.Neurosci.), while an alternative model, assigning Cplx the role of a "checkpoint" protein in SNARE assembly, quantitatively reproduces all experimental observations (Lopez et al., 2024, PNAS). It might be helpful for a reader to mention such alternative scenarios.

Author response:

The following is the authors’ response to the original reviews.

Response to reviewers (minor points):

We thank all reviewers for their very helpful suggestions and greatly appreciate their positive evaluation of our work.

Reviewer #1:

Ad 1) The reviewer states: Fig 5 While the data very nicely show that CPX and Syt1 have interdependent interactions in the chromaffin neurons, this seems to be not the case in neurons, where the loss of complexins and synaptotagmins have additive effects, suggesting independent mechanisms (eg Xue et al., 2010). This would be a good opportunity to discuss some possible differences between secretion in endocrine cells vs neurons.

We greatly appreciate the insightful suggestion by the reviewer. To accommodate the reviewer’s suggestion, we now discuss this issue on page 21, line 486-491: “In murine hippocampal neurons, loss of CpxI and Syt1 has additive effects on fast synchronous release, suggesting independent mechanisms (Xue et al., 2010). On the other hand, the same study also showed that Syt1 heterozygosity fails to reduce release probability in wild-type neurons, but does so in the absence of Cpx, again suggesting that Cpx and Syt1 may functionally interact in Ca2+-triggered release.”

Ad 2) The reviewer states: Fig 8 Shows an apparent shift in Ca sensitivity in N-terminal mutants suggesting a modification of Ca sensitivity of Syt1. Could there be also an alternative mechanism, that explains this phenotype which is based on a role of the n-term lowering the energy barrier for fusion, that in turn shifts corresponding fusion rates to take place at lower Ca saturation levels?

We fully agree with the reviewer. While our data indicate that Cpx and Syt1 act in a dependent manner in accelerating exocytosis, they do not provide decisive evidence that the NTD of CpxII directly modulates the Ca2+ affinity of Syt1, an issue that we discuss on page 23 , line 523529: ”The results favor a model wherein the CpxII NTD either directly regulates the biophysical properties of the Ca2+-sensor by increasing the apparent forward rate of Ca2+-binding or indirectly affects SytI-SNARE or SytI-membrane interactions, thereby, lowering the energy barrier of Ca2+triggered fusion.”

Reviewer #2:

Ad 1) The reviewer states: The authors provide a "chromaffin cell-centric" view of the function of mammalian Cplx in vesicle fusion. With the exception of mammalian renal ribbon synapses (and some earlier RNAi knockdown studies that had off-target effects), there is very little evidence for a "fusion-clamp"-like function of Cplxs in mammalian synapses. At conventional mammalian synapses, genetic loss of Cplx (i.e. KO) consistently decreases AP-evoked release, and generally either also decreases spontaneous release rates or does not affect spontaneous release, which is inconsistent with a "fusion-clamp" theory. This is in stark contrast to invertebrate (D. m. and C. e.) synapses where genetic Cplx loss is generally associated with strong upregulation of spontaneous release, providing support for Cplx acting as a "fusion-clamp".

We agree with the reviewer that it is difficult to reconcile contradictory findings regarding the role of Cpx in membrane fusion in vertebrates and invertebrates or between murine hippocampal neurons and neuroendocrine cells. On the other hand, we respectfully disagree with the statement of providing a "chromaffin cell-centric" view of the function of mammalian Cplx in vesicle fusion. In fact, a large number of model systems (in vitro and in vivo studies) support a scenario where complexin takes center stage in clamping of premature vesicle release. For example, in vitro analyses using a liposome fusion assay (Schaub et al., 2006, Nat Struct Mol Biol 13, 748; Schupp et al., 2016) or Hela cells that ectopically express “flipped” SNAREs on their cell surface (Giraudo et al., 2008, JBC 283, 21211) showed that complexin can inhibit the SNARE-driven fusion machinery. Likewise, several studies boosting complexin action by either genetic overexpression or peptide supplementation have provided evidence for the complexin clamp function in neuronal and nonneuronal cells (e.g. Itakura et al., 1999, BBRC 265, 691; Liu et al., 2007, Biochemistry 72, 439; Abderrahmani et al., 2004, J Cell Sci 117, 2239; Archer et al., 2002, JBC 277, 18249; Tang et al, 2006,

Cell 126, 1175; Vaithianathan et al., 2013, J Neurosci 33, 8216; Roggero et al., 2007, JBC, 282, 26335.)

In addition, chromaffin cells enable the investigation of secretion on the background of a well-defined intracellular calcium concentration. Indeed, CplxII knock-out in chromaffin cells demonstrated an enhanced tonic release which is evident at elevated levels of [Ca]i (>100nM), but absent at low resting [Ca]i (Dhara et al., 2014). Given this observation, it is tempting to speculate that variations in [Ca]i among the different preparations may contribute to the deviating expression of the complexin null phenotype in different preparations.

Ad 2) The reviewer states: The authors use a Semliki Forest virus-based approach to express mutant proteins in chromaffin cells. This strategy leads to a strong protein overexpression (~7-8 fold, Figure 3 Suppl. 1). Therefore, experimental findings under these conditions may not necessarily be identical to findings with normal protein expression levels.

As shown in Fig. 4, we use the secretion response of wt cells as a control so that we can assess the specificity and quality of the rescue approach in our experiments. In addition, the comparative analysis of the CpxII mutants was performed with respect to the equally overexpressed CpxII wt protein (Fig. 3 Suppl. 1), which we used as a control to determine the standard response under these conditions.

Ad 3) The reviewer states: Measurements of delta Cm in response to Ca2+ uncaging by ramping [Ca2+ ] from resting levels up to several µM over a me period of several seconds were used to establish changes in the release rate vs [Ca2+ ]i relationship. It is not clear to this reviewer if and how concurrently occurring vesicle endocytosis together with a possibly Ca2+-dependent kinetics of endocytosis may affect these measurements.

By infusing bovine chromaffin cells with 50µM free Ca2+, Smith and Betz have shown that the total capacitance increase is dominated by exocytosis and that significant endocytosis only sets in after 3 minutes (Smith and Betz, 1996, Nature, 380, 531). In the same line, we previously showed that mouse chromaffin cells (infused with 19µM free calcium over 2 minutes) responded with robust increase in membrane capacitance which strongly correlated with the number of simultaneously recorded amperometric events monitoring fusion of single vesicles (Dhara et al., 2014, Fig. 5B). Thus, capacitance alterations recorded under tonic intracellular Ca2+ increase in chromaffin cells are solely due to exocytosis and are not contaminated by significant endocytosis. As our Ca2+ ramp experiments were carried out for 6 seconds and the intracellular free [Ca]i did not exceed 19 µM the observed phenotypical differences between the experimental groups are most likely due to changes in exocytosis rather than endocytosis.

Ad 4) The reviewer states: It should be pointed out that an altered "apparent Ca2+ affinity" or "apparent Ca2+ binding rate" does not necessarily reflect changes at Ca2+-binding sites (e.g. Syt1).

We fully agree with the reviewer’s comment. As pointed out also in the response to reviewer 1, our experiments do not provide decisive evidence that the NTD of CpxII directly modulates the Ca2+ affinity of Syt1, an issue that we discuss on page 23 , line 523-529: ” The results favor a model wherein the CpxII NTD either directly regulates the biophysical properties of the Ca2+sensor by increasing the apparent forward rate of Ca2+-binding or indirectly affects SytI-SNARE or SytI-membrane interactions, thereby, lowering the energy barrier of Ca2+-triggered fusion.”

AD 5) There are alternative models on how Cplx may "clamp" vesicle fusion (see Bera et al. 2022, eLife) or how Cplx may achieve its regulation of transmitter release without mechanistically "clamping" fusion (Neher 2010, Neuron). Since the data presented here cannot rule out such alternative models (in this reviewer's opinion), the authors may want to mention and briefly discuss such alternative models.

The study by Bara et al reiterates the model proposed by the Rothman group which attributes the clamping function of Cpx to its accessory alpha helix by hindering the progressive SNARE complex assembly. We have explicitly stated this issue in the original version of the manuscript (page 19, line 425) “As the accessory helix of Cpx has been found to bind to membrane proximal cytoplasmic regions of SNAP-25 and SybII (Malsam et al., 2012; Bykhovskaia et al., 2013; Vasin et al., 2016), an attractive scenario could be that both domains of CpxII, the CTD and the accessory helix, synergistically cooperate to stall final SNARE assembly”. In this context, we will now cite also the study by Bera et al..

A related view of the function of complexin suggested that it may act as an allosteric adaptor for sytI (Neher 2010, Neuron). Here, rather than postulang independent "clamp" and "trigger" functions for the dual action of complexin, these were explained as facets of a simple allosteric mechanism by which complexin modulates the Ca2+ dependence of release. Yet, this interpretation appears to be difficult to reconcile with the observation of our and other laboratories, showing that the fusion-promoting and clamping effects are separable (e.g. Dhara et al., 2014; Lai et al., 2014; Makke et al., 2018; Bera et al., 2022).

Some parts of the Discussion are quite general and not specifically related to the results of the present study. The authors may want to consider shortening those parts.

Considering the contrary findings in the field of SNARE-regulating proteins, the authors hope that the reviewer will agree that it is necessary to discuss the new observations in a broader context, as also acknowledged by the first reviewer.

Last but not least, the presentation of the results could be improved to make the data more accessible to non-specialists, this concerns providing necessary background information, choice of colors, and labeling of diagrams.

Done

Recommendations for the authors:

Reviewer #2 (Recommendations For The Authors):

Regarding figures:

(1) Please use clearly distinct colors in diagrams. For example, in Figure 2 Suppl. 3, four different shades of red (or reddish) are used to color the traces and the respective bars. These different shades of red are difficult to discriminate. In Figure 5 Suppl. 1, the two greens are nearly indistinguishable.

Done

(2) RRP size and SRP size on the one hand, and SR rate on the other represent different quantities which are measured in different units. Please use a separate y-axis for the SR (a rate measured in fF/s) and do not combine with RRP and SRP (pool sizes measured in fF). This would also automatically alleviate the need for axis breaks in the plots of RRP size and SRP size. In general, please do not use axis breaks which make interpretation of data unnecessarily more complicated.

In order to clarify the display, we now define the different units together with the quantified parameter (e.g. RRP [fF], SRP [fF], SR [fF/s]) allowing us to omit a second axis in those subpanels.

(3) When plotting bar graphs showing mean tau_RRP, mean tau_SRP, and mean delay, please always use the correct y-axis labels, i.e. use "tau_RRP", "tau_SRP" and "delay" as y-axis labels as it was done for example in Figure 4D, and do not use "tau_RRP", "tau_SRP" and "delay" as x-axis labels as it was done for example in Figure 1D and many other figure panels.

We have standardized the figure display. Yet, we would prefer to keep our way subpanel labelling which states the parameter underneath the bar graph and thereby makes the results more accessible.

(4) Are the asterisks indicating statistical significance perhaps missing in Figure 4D, middle panel (tau_SRP)?

There was not a statistically significant difference (wt vs cpxIIko+CpxII EA, P=0.0826, Kruskal-Wallis with Dunn’ post hoc test).

(5) According to the Results section (pages 12 to 13), I assume that in Figures 6 and 7 the labels "+Cplx XYZ" are used by the authors to identify an overexpression of Cplx XYZ in a Cplx WT background. The legend text reads however " ... cells expressing either Cplx2 wt or the mutant ...", which would not be correct. Please check.

We have changed the formulations to “overexpression” accordingly.

(6) The x-axis unit in Figure 8C is likely "µM" and not "M".

Done.

(7) The abbreviations "CplxII LL-EE" and "CplxII LL-WW", and "CplxII LLEE" and "CplxII LLWW" are very similar but refer to different mutants. Could you please think of a more specific and unambiguous abbreviation? Perhaps "CplxII L124E-L128E"?

We have changed the abbreviations, accordingly (i.e. CpxII L124E-L128E).

Regarding the manuscript text:

Line 65: "prevents" instead of "impairs"?

done

Line 67: why "in vivo"?

We changed the formulation to ‘Several’

Line 83: "in addition to the clamping function ..." This is misleading. Many of the studies listed here did not provide evidence for enhanced spontaneous release following Cplx loss and often observed the opposite, reduced spontaneous release. The enhanced delayed release was observed by Strenzke et al 2009 J.Neurosci. and by Chang et al. 2015 J.Neurosci. (which the authors may want to cite). However, that enhanced delayed release occurred despite reduced spontaneous release indicating that it is not simply the result of a missing "fusion clamp".

To accommodate the reviewer’s suggestion, we have changed the formulation to “Independent of the clamping function of Cpx….”

Line 104: "speeds up exocytosis that is controlled by the forward rate of Ca2+ binding" This is difficult to understand without context.

We have now added the corresponding citations (Voets et al., 2001; Sorensen et al., 2003), which showed that exocytosis timing in chromaffin cells is largely determined by the kinetics of Ca2+-binding to SytI.

Line 116: "Cplx2 knock out ..." Please provide (here or earlier in the manuscript) information to the reader about which Cplx paralogs are expressed in chromaffin cells.

We now state on line 111 that “CpxII is the only Cpx isoform expressed in chromaffin cells (Cai et al., 2008)”

Line 118: "=~" either "=" or "~".

done

Line 120: "instead" seems superfluous.

done

Line 272: "calcium binding rates" should perhaps better read "apparent calcium binding rates".

done

Line 290: "enhancing SytI's Ca2+ affinity" should perhaps better be "enhancing the apparent Ca2+ affinity of the release machinery". Ca2+ binding kinetics is never directly assayed here.

We agree and have phrased the sentence accordingly.

Line 300: "Expression of Cplx ... in Syt1 R233Q ki cells, ..." Perhaps better "Overexpression of Cplx ... in Syt1 R233Q ki/Cplx2 wt cells, ..." for clarification?

done

Lines 313ff: What is assayed here is the apparent Ca2+ binding kinetics and apparent KD values of the release machinery. Ca2+ binding to Syt1 is never directly measured!

We agree and have changed the wording accordingly to “CpxII NTD supports the forward rate of calcium binding to SytI in accelerating exocytosis”

Line 347: "Complexin plays a dual role ..." This is partially misleading. It does so in chromaffin cells and D.m. and C.e. NMJs but not at conventional mammalian synapses.

We agree and have changed the formulation to “In many secretory systems, Complexin plays a dual role in the regulation of SNARE-mediated vesicle fusion”