Molecular basis of interactions between CaMKII and α-actinin-2 that underlie dendritic spine enlargement
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eLife assessment
This fundamental study from Gold and colleagues substantially advances our understanding of the synaptic targeting of a major postsynaptic protein kinase, CaMKII, which is the basis for the persistence of excitatory synaptic strength in synaptic plasticity. The evidence supporting the claims of the authors is convincing, with cell biological, biochemical, as well as structural biological approaches. This work will be of interest to cell and computational biologists working on learning/memory.
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Abstract
Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is essential for long-term potentiation (LTP) of excitatory synapses that is linked to learning and memory. In this study, we focused on understanding how interactions between CaMKIIα and the actin-crosslinking protein α-actinin-2 underlie long-lasting changes in dendritic spine architecture. We found that association of the two proteins was unexpectedly elevated within 2 minutes of NMDA receptor stimulation that triggers structural LTP in primary hippocampal neurons. Furthermore, disruption of interactions between the two proteins prevented the accumulation of enlarged mushroom-type dendritic spines following NMDA receptor activation. α-Actinin-2 binds to the regulatory segment of CaMKII. Calorimetry experiments, and a crystal structure of α-actinin-2 EF hands 3 and 4 in complex with the CaMKII regulatory segment, indicate that the regulatory segment of autoinhibited CaMKII is not fully accessible to α-actinin-2. Pull-down experiments show that occupation of the CaMKII substrate-binding groove by GluN2B markedly increases α-actinin-2 access to the CaMKII regulatory segment. Furthermore, in situ labelling experiments are consistent with the notion that recruitment of CaMKII to NMDA receptors contributes to elevated interactions between the kinase and α-actinin-2 during structural LTP. Overall, our study provides new mechanistic insight into the molecular basis of structural LTP and reveals an added layer of sophistication to the function of CaMKII.
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Author Response
Reviewer #1 (Public Review):
The manuscript by Curtis et al. reports the interaction between CaMKII and alpha-actinin-2. The authors found that the interaction was elevated after NMDA receptor activation in dendritic spines. In addition, this study reveals NMDA receptor binding to CaMKII facilitates alpha-actinin-2 access to the CaMKII regulatory segment, indicating that the NMDA receptor is involved in this interaction. The authors identified the EF1-4 motifs mediated this interaction, and overexpression of this motif inhibited structural LTP. Moreover, biochemical measurements of affinities from various combination of protein fragments including autoinhibited CaMKII 1-315, regulatory segments of CaMKII, and the EFhand motif reveals that autoinhibited CaMKII has limited access to alpha-actinin-2. The authors also …
Author Response
Reviewer #1 (Public Review):
The manuscript by Curtis et al. reports the interaction between CaMKII and alpha-actinin-2. The authors found that the interaction was elevated after NMDA receptor activation in dendritic spines. In addition, this study reveals NMDA receptor binding to CaMKII facilitates alpha-actinin-2 access to the CaMKII regulatory segment, indicating that the NMDA receptor is involved in this interaction. The authors identified the EF1-4 motifs mediated this interaction, and overexpression of this motif inhibited structural LTP. Moreover, biochemical measurements of affinities from various combination of protein fragments including autoinhibited CaMKII 1-315, regulatory segments of CaMKII, and the EFhand motif reveals that autoinhibited CaMKII has limited access to alpha-actinin-2. The authors also solved the structure of the interaction, supporting their finding in neurons at the molecular level. The authors claim that the interaction between CaMKII and alpha-actinin-2 is essential for structural LTP through cooperative action by the NMDA receptor and actin cytoskeleton.
Overall, the experiments are well-designed and the results are largely convincing and well-interpreted. But some aspects of the experiments need to be clarified.
- Time resolution of the interaction analysis appears to be poor, as calcium elevation in a dendritic spine would be at milli-second order. What is the time window to interact alpha-actinin-2 with CaMKII during NMDA receptor activation or LTP?
We have performed additional time-course experiments to determine how quickly interactions between alpha-actinin-2 and CaMKII are elevated following NMDAR activation. The results of these experiments are shown in Figure 2A and Figure 2-Figure Supplement 1. We found that the change in association was established rapidly after NMDAR activation (t50% = 22±1 s, Figure 2A), which is consistent with proposed time-courses for CaMKII interactions following the induction of LTP (see Yasuda, Hayashi & Hell, Nat Rev Neuroscience, 2022, PMID 36056211). We have included additional text in the results (lines 138-147), methods (lines 609-611 & 650-652), and discussion (lines 426-427) sections explaining these experiments, and figure legends are provided for the new figures on lines 10061009 and lines 1096-1101.
- The authors analyzed the binding of CaMKII and alpha-actinin-2 with partial fragments. It remains to be unknown whether CaMKII can form a protein complex with GluN2B and alpha-actinin-2 in a single CaMKII protomer.
The reviewer is referring to experiments shown in figure 5, in which we found that a fragment of GluN2B (1260-1492) increases pull-down of full-length CaMKIIa with a fusion of GST to the EF3-4 region of a-actinin-2. This region of GluN2B contains a CaMKII phosphorylation sequence (positions 1290-1309) that occupies the substrate binding groove of the kinase domain (Stratton et al., Cell Reports, 2023, PMID 35830796). Therefore, the most logical explanation for the results of the pulldown experiment is that GluN2B increases a-actinin-2 access to the regulatory segment by binding to the substrate binding groove of the same CaMKII protomer. Nevertheless, we discuss the difficulty of conceptualising and investigating interactions between oligomeric proteins within the PSD on lines 451461.
- Besides synaptic localization, the effect of the interaction on the enzymatic activity of CaMKII is not known.
The Colbran laboratory has previously examined the effect of a-actinin-2 on CaMKII activity. Jalan-Sakrikar and colleagues (JBC, 2012, PMID 22427672) showed that a fragment of aactinin-2 corresponding to EF hands 3 and 4 is able to weakly activate CaMKII (~ 10 % compared to Ca2+/CaM) towards peptide substrates autocamtide-2 and GluN2B but not syntide-2 (see Figure 1B&C of this paper). An earlier study by Robison and colleagues (JBC, 2005, PMID 16172120) found that aactinin-2 antagonises Ca2+/CaM-dependent activation of unphosphorylated CaMKII towards autocamtide2, but does not affect the activity of pT286 auto-activated CaMKII (see Figure 4A of this paper). This work is referred to on lines 63-65 of the introduction.
- Although the authors quantify the effect of the EF-hand disruptor by measuring numbers of the dendritic spine by its shape, the specificity of the EF-hand disruptor needs to be clarified.
There are two known interaction partners for the EF hand region of a-actinin-2: CaMKII and Titin (Young et al., EMBO J, 1998, PMID 9501083; Atkinson et al., Nat Struct Biol, 2001, PMID 11573089). Titin is an extremely long sarcomeric protein that is expressed in striated muscle cells but not neurons. Therefore, the effects of the disruptor are highly likely to reflect disruption of interactions to CaMKII. We also performed control experiments with EF34 L854R that does not bind CaMKII effectively (Figure 3-figure supplement 1C). We have added a sentence to clarify the specificity of the EF-hand disruptor on lines 182-184, as follows: ” Furthermore, the only known interaction partner for the EF14 region of a-actinin-2 besides CaMKII is the muscle-specific protein titin (Young et al., 1998), so any effects of EF14 in neurons are likely to reflect destabilisation of native interactions between CaMKII and a-actinin-2”.
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eLife assessment
This fundamental study from Gold and colleagues substantially advances our understanding of the synaptic targeting of a major postsynaptic protein kinase, CaMKII, which is the basis for the persistence of excitatory synaptic strength in synaptic plasticity. The evidence supporting the claims of the authors is convincing, with cell biological, biochemical, as well as structural biological approaches. This work will be of interest to cell and computational biologists working on learning/memory.
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Reviewer #1 (Public Review):
The manuscript by Curtis et al. reports the interaction between CaMKII and alpha-actinin-2. The authors found that the interaction was elevated after NMDA receptor activation in dendritic spines. In addition, this study reveals NMDA receptor binding to CaMKII facilitates alpha-actinin-2 access to the CaMKII regulatory segment, indicating that the NMDA receptor is involved in this interaction. The authors identified the EF1-4 motifs mediated this interaction, and overexpression of this motif inhibited structural LTP. Moreover, biochemical measurements of affinities from various combination of protein fragments including autoinhibited CaMKII 1-315, regulatory segments of CaMKII, and the EF-hand motif reveals that autoinhibited CaMKII has limited access to alpha-actinin-2. The authors also solved the structure …
Reviewer #1 (Public Review):
The manuscript by Curtis et al. reports the interaction between CaMKII and alpha-actinin-2. The authors found that the interaction was elevated after NMDA receptor activation in dendritic spines. In addition, this study reveals NMDA receptor binding to CaMKII facilitates alpha-actinin-2 access to the CaMKII regulatory segment, indicating that the NMDA receptor is involved in this interaction. The authors identified the EF1-4 motifs mediated this interaction, and overexpression of this motif inhibited structural LTP. Moreover, biochemical measurements of affinities from various combination of protein fragments including autoinhibited CaMKII 1-315, regulatory segments of CaMKII, and the EF-hand motif reveals that autoinhibited CaMKII has limited access to alpha-actinin-2. The authors also solved the structure of the interaction, supporting their finding in neurons at the molecular level. The authors claim that the interaction between CaMKII and alpha-actinin-2 is essential for structural LTP through cooperative action by the NMDA receptor and actin cytoskeleton.
Overall, the experiments are well-designed and the results are largely convincing and well-interpreted. But some aspects of the experiments need to be clarified.
1. Time resolution of the interaction analysis appears to be poor, as calcium elevation in a dendritic spine would be at milli-second order. What is the time window to interact alpha-actinin-2 with CaMKII during NMDA receptor activation or LTP?
2. The authors analyzed the binding of CaMKII and alpha-actinin-2 with partial fragments. It remains to be unknown whether CaMKII can form a protein complex with GluN2B and alpha-actinin-2 in a single CaMKII protomer.
3. Besides synaptic localization, the effect of the interaction on the enzymatic activity of CaMKII is not known.
4. Although the authors quantify the effect of the EF-hand disruptor by measuring numbers of the dendritic spine by its shape, the specificity of the EF-hand disruptor needs to be clarified. -
Reviewer #2 (Public Review):
Gold and his colleagues first ectopically expressed aACTN2 constructs with various deletions and determine the spatial proximity to CaMKII by PLA. Chemical LTP induced by brief glycine application in hippocampal cultures strongly augmented the PLA puncta density in spines (postsynaptic sites). This interaction specifically depended on the 4 EF hands near the C-terminus of aACTN. At the same time expression of the 4 EF hands (plus the C-terminal PDZ ligand) impaired the formation of larger mushroom spines under unstimulated conditions and the increase in mushroom spines seen after chemLTP when compared to non-transfected conditions or transection of the EF hands with a point mutation (L854R) that disrupted binding to CaMKII.
To further define the interaction between aACTN and CaMKII the authors then solved a …
Reviewer #2 (Public Review):
Gold and his colleagues first ectopically expressed aACTN2 constructs with various deletions and determine the spatial proximity to CaMKII by PLA. Chemical LTP induced by brief glycine application in hippocampal cultures strongly augmented the PLA puncta density in spines (postsynaptic sites). This interaction specifically depended on the 4 EF hands near the C-terminus of aACTN. At the same time expression of the 4 EF hands (plus the C-terminal PDZ ligand) impaired the formation of larger mushroom spines under unstimulated conditions and the increase in mushroom spines seen after chemLTP when compared to non-transfected conditions or transection of the EF hands with a point mutation (L854R) that disrupted binding to CaMKII.
To further define the interaction between aACTN and CaMKII the authors then solved a crystal structure formed by the aACTN EF3/4 and regulatory segment of CaMKII. This structure confirmed the role of L854 in the interaction. It also explained earlier results that phosphorylation of threonine in position 306 but not of threonine 305 of the CaMKII regulatory domain impaired aACTN binding as T306 but not T305 is engaged in critical interactions. This contrasts with Ca/CaM binding to CaMKII, which engages both threonines and is blocked by the phosphorylation of either residue. Consistently, earlier structures of Ca/CaM with the CaMKII regulatory domains showed respective differences to the new aACTN-CaMKII structure.
Additional analysis of these data indicated that the association of the regulatory domain with the kinase domain occludes access to aACTN EF3/4. This is an important finding because it implies that only active CaMKII like T286 autophosphorylated CaMKII or bound to GluN2B would be able to effectively interact with aACTN in intact cells.
Finally, and remarkably, binding was augmented by a protein fragment of the GluN2B C-terminus that contains the binding site for CaMKII even when Ca/CaM was still present. This result suggests that with GluN2B present aACTN can bind to CaMKII even though in the absence of GluN2B Ca/CaM occludes this binding. This finding opens up new research directions.
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Reviewer #3 (Public Review):
This manuscript builds upon prior work showing that alpha-actinin-2 binds to the regulatory domain of the major postsynaptic protein kinase, CaMKII. The authors report the structure of a complex between the relevant domain in alpha-actinin-2 and a peptide based on the CaMKII regulatory domain. Data are presented indicating that the interaction of the NMDA receptor GluN2B subunit with the CaMKII catalytic domain stabilizes the complex with alpha-actinin-2. Furthermore, the authors present proximity ligation assay (PLA) data obtained in cultured neurons demonstrating that NMDA receptor activation strongly enhances the colocalization of CaMKII with alpha-actinin-2. Data obtained using mutated proteins indicate that this co-localization is mediated by the interaction characterized structurally.
Strengths:
Signifi…
Reviewer #3 (Public Review):
This manuscript builds upon prior work showing that alpha-actinin-2 binds to the regulatory domain of the major postsynaptic protein kinase, CaMKII. The authors report the structure of a complex between the relevant domain in alpha-actinin-2 and a peptide based on the CaMKII regulatory domain. Data are presented indicating that the interaction of the NMDA receptor GluN2B subunit with the CaMKII catalytic domain stabilizes the complex with alpha-actinin-2. Furthermore, the authors present proximity ligation assay (PLA) data obtained in cultured neurons demonstrating that NMDA receptor activation strongly enhances the colocalization of CaMKII with alpha-actinin-2. Data obtained using mutated proteins indicate that this co-localization is mediated by the interaction characterized structurally.
Strengths:
Significant strengths of this work are:
1. The high-quality structures of the complex that are reported.
2. Integration of these findings with the much better-studied complex of CaMKII and GluN2B.
3. The convincing PLA analyses show that NMDA receptor activation increases CaMKII colocalization with alpha-actinin-2.
4. The careful comparisons of data from these new studies with data reported in previous publications.Weaknesses:
Despite the significant strengths of the work, there are some gaps/weaknesses.
1. Although there is abundant published evidence that activated CaMKII colocalizes with NMDA receptors, the evidence for the involvement of GluN2B in the CaMKII-alpha-actinin-2 complex in neurons is lacking.
2. The evidence supporting a role for the EF1 and EF2 domains of alpha-actinin-2 in binding to CaMKII is not very convincing.
3. CaMKII autophosphorylation at multiple sites plays an important role in regulating the subcellular localization of CaMKII, but the role of autophosphorylation is not explored here.Taken to together the manuscript describes novel data that provide a significant extension to prior work, and the data convincingly, but perhaps only partially, support an interesting proposed model for the control of CaMKII targeting in spines.
This more sophisticated delineation of the mechanisms underlying CaMKII targeting synapses will be of interest to the broader field of investigators studying the molecular basis for the regulation of excitatory synaptic transmission, learning, and memory.
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