Articles
https://doi.org/10.1038/s41593-021-00843-3

CaMKII activation persistently segregatespostsynaptic proteins via liquid phase separation
Tomohisa Hosokawa1,2,3,8, Pin-Wu Liu 1,8, Qixu Cai 4, Joana S. Ferreira
Florian Levet 5, Corey Butler5, Jean-Baptiste Sibarita5, Daniel Choquet
Eric Hosy 5, Mingjie Zhang 4,7 ✉ and Yasunori Hayashi 1 ✉,5, Laurent Groc5,

5,6Transient information input to the brain leads to persistent changes in synaptic circuits, contributing to the formation of memory engrams. Pre- and postsynaptic structures undergo coordinated functional and structural changes during this process, but
how such changes are achieved by their component molecules remains largely unknown. We found that activated CaMKII, acentral player of synaptic plasticity, undergoes liquid–liquid phase separation with the NMDA-type glutamate receptor subunit GluN2B. Due to CaMKII autophosphorylation, the condensate stably persists even after Ca2+ is removed. The selective
binding of activated CaMKII with GluN2B cosegregates AMPA receptors and the synaptic adhesion molecule neuroligin intoa phase-in-phase assembly. In this way, Ca2+-induced liquid–liquid phase separation of CaMKII has the potential to act as anactivity-dependent mechanism to crosslink postsynaptic proteins, which may serve as a platform for synaptic reorganization
associated with synaptic plasticity.

I

nside a central excitatory synapse, various molecules are segregated into functional nanodomains to accomplish the intricate
regulation of synaptic transmission and plasticity. In the presynaptic compartment, the readily releasable pool of vesicles is concentrated at specialized nanodomains referred to as active zones. On
the postsynaptic membrane, different classes of glutamate receptor form discrete nanodomains1–4. These pre- and postsynaptic
nanodomains are matched with each other, forming trans-synaptic
nanocolumns or nanomodules that ensure an efficient transmission
between pre- and postsynaptic structures1,2,5,6.
However, how such nanodomains are formed and regulated byneuronal activity in the absence of any demarcating membranous
structures has not been fully elucidated. Recently, liquid–liquid
phase separation (LLPS) of biological macromolecules was foundto play a critical role in regulating the assembly and segregation ofmolecules in various intracellular structures7,8. In this regard, Ca2+/
calmodulin-dependent protein kinase II (CaMKII)—a highly abundant protein kinase in the postsynaptic density (PSD)—has idealfeatures to undergo LLPS7,8. Ca2+/calmodulin binding to CaMKII
opens up a binding pocket called the T-site, which is occupied by
the autoinhibitory domain encompassing threonine (T) 286 in inactive kinase, and forms a stable complex, at micromolar affinity, withvarious synaptic proteins, such as the carboxyl tail of NMDA-typeglutamate receptor (NMDAR) subunit GluN2B (Supplementary
Fig. 1) and RacGEF protein Tiam1 (refs. 9,10). Once bound, it persists
even when the cellular Ca2+ concentration decreases9,10. Finally, thedodecameric structure of CaMKII11 allows multivalent interactions.
Given this, we explored whether CaMKII has the ability toundergo LLPS with PSD proteins and, if it does, how it can affectsynaptic protein distribution and function. We found that Ca2+ activation of CaMKII results in persistent LLPS with PSD proteins ina manner requiring T286 autophosphorylation. CaMKII then segregates two subtypes of glutamate receptor, AMPAR and NMDAR,
through the formation of phase-in-phase, which was recapitulated
in neurons as shown by super-resolution microscopy. Neuroligin-1(NLGN1), a neuronal adhesion molecule, which clusters presynaptic neurexin and other active zone proteins, segregates together
with AMPAR. From these observations, we propose that the persistent LLPS of activated CaMKII serves as an activity-dependent
mechanism to crosslink proteins beneath postsynaptic membrane.
This may also align the AMPAR nanodomain with the presynaptictransmitter release site, thereby serving as a previously unknown
mechanism of synaptic plasticity.

Results

CaMKII undergoes LLPS with GluN2B carboxyl tail. To test theidea that CaMKII can undergo LLPS with its T-site binding partner,
we combined purified CaMKII with the carboxyl tail of GluN2B, aprototypical T-site binding protein (residues 1226–1482, GluN2Bc).
GluN2Bc was fused with dimeric near-infrared fluorescent protein
eqFP670 to label it and to mimic the subunit stoichiometry of the
GluN2B subunit in the endogenous NMDAR complex. We used alow-speed centrifugation assay to assess macromolecular complex
formation12–14. The cytoplasmic concentration of CaMKII in thesynapse is estimated to be 20–80 µM as a monomer15. Here, we used
10 µM of CaMKII as this was the practical limit of the preparation.
Generally, proteins form condensates more readily at higher concentration. Therefore, we are towards the more conservative sidein making this conclusion. On the other hand, GluN2B is a membrane protein and it is difficult to define its concentration/density.
Also, association with the membrane limits the diffusion and stability of GluN2B, which can effectively increase the valency of the

Department of Pharmacology, Kyoto University Graduate School of Medicine, Kyoto, Japan. 2RIKEN Brain Science Institute, Saitama, Japan. 3Departmentof Molecular Biology, Division of Biological Science, Nagoya University Graduate School of Science, Nagoya, Japan. 4Division of Life Science, Hong Kong
University of Science and Technology, Kowloon, Hong Kong, China. 5Université de Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297,
Bordeaux, France. 6Bordeaux Imaging Center, Bordeaux, France. 7School of Life Sciences, Southern University of Science and Technology, Shenzhen, China.
8
These authors contributed equally: Tomohisa Hosokawa, Pin-Wu Liu. ✉e-mail: mzhang@ust.hk; yhayashi-tky@umin.ac.jp
1

Nature Neuroscience | www.nature.com/natureneuroscienceArticlesinteraction. Therefore, we tentatively used GluN2B at the same concentration as CaMKII. When CaMKII, GluN2Bc and calmodulin
were mixed in the absence of Ca2+, the proteins stayed in the supernatant (Fig. 1a,b). However, upon addition of Ca2+, most CaMKII
moved to the pellet with GluN2Bc, indicating that Ca2+ stimulationof CaMKII induces the formation of a macromolecular complex
with GluN2Bc. Differential interference contrast (DIC) and fluorescent microscopy showed no condensate in the absence of Ca2+ (Fig.
1c). However, the addition of Ca2+ induced formation of protein
condensates containing CaMKII and GluN2Bc, consistent with thesedimentation assay12,14. Upon point photobleaching in a single condensate, both CaMKII and GluN2Bc fluorescence recovered after
photobleaching (Fig. 1d and Supplementary Fig. 2). Once formed,
the condensates were stable, and we could observe two droplets
fusing together to form a larger droplet (Fig. 1e). These observations indicate that the condensate retained liquid-like properties.
GluN2Bc without CaMKII, or with CaMKII with eqFP670 fusion
tag only, did not pellet or form condensates, indicating that both
CaMKII and GluN2Bc are required (Supplementary Fig. 3a–d).
The carboxyl tails of AMPA receptor subunits GluA1 and GluA2
did not form condensates with CaMKII (Supplementary Fig. 3e).
Together, our results indicate that Ca2+/calmodulin can trigger formation of protein condensates containing CaMKII and GluN2Bc bya LLPS-mediated mechanism.
Autophosphorylation of CaMKII is required for persistence ofprotein condensate. CaMKII and GluN2Bc protein condensates
persisted even after the addition of ethylene glycol tetra-acetic acid
(EGTA), a Ca2+-chelator (Fig. 1a–c). In contrast, in the absence ofATP, condensates could form but dissolved upon addition of EGTA
(Fig. 1b,f and Supplementary Fig. 4a). This indicates that the kinase
reaction is involved in the persistence of condensates after Ca2+
dissipates. All experiments described hereafter were performed in
the presence of ATP unless otherwise indicated. Consistent withthe experiment in the absence of ATP, a kinase null CaMKII K42R
mutant formed condensates in the presence of Ca2+ but failed topersist after the addition of EGTA (Fig. 1g). Mutation of the autophosphorylation site at T286, a site required for the constitutive
activation of CaMKII beyond the period of elevated Ca2+ concentration, to alanine (T286A) replicated the results of the kinase nullmutant (Fig. 1h). These results indicate that autophosphorylation at
T286 is not critical for the initial formation of condensates by Ca2+
but is required for the persistent maintenance of the condensates in
the absence of Ca2+.
We next tested if the multivalent interaction between CaMKII and
GluN2Bc is required for the formation of condensates. Consistent
with the requirement of multivalency of CaMKII, a catalytically
active but monomeric CaMKII mutant 1–314 lacking the association domain failed to form condensates (Supplementary Fig. 4b).
To prevent the specific interaction between CaMKII and GluN2Bc,
we turned to a model of binding between CaMKII and GluN2B (ref.
16). The model shows the interaction between a hydrophobic pocket
made by I205 and F98 of CaMKII with L1298 of GluN2B as wellas electrostatic interactions between E139 of CaMKII and R1300 of
GluN2B. A CaMKII T-site mutant I205K failed to form condensates(Supplementary Fig. 4c). Also, GluN2Bc mutants that cannot interact with CaMKII, L1298A/R1300Q (LR/AQ) and R1300Q/S1303D(RS/QD) (refs.9,17) failed to form condensates (Supplementary Fig.
4d,e). These results indicate that multivalent interactions via thosehydrophobic and electrostatic interactions between the CaMKII
T-site and GluN2Bc are required for the formation of condensates.
To obtain temporal information on the formation and dispersion of condensates, we measured the turbidity of the protein mixture (Supplementary Fig. 5) (ref. 18). The turbidity of the CaMKII/
GluN2Bc sample increased within 30 s of the addition of Ca2+, andremained after adding EGTA. On the other hand, the turbidity of

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the T286A/GluN2Bc sample increased similarly to the wildtype
CaMKII sample but decreased to baseline amounts within 30 s of
EGTA treatment.
Segregation of AMPAR and NMDAR by a CaMKII LLPS-mediatedmechanism. We then added further components of the PSD toexamine if CaMKII can form condensates with other main PSD
proteins as well. We tested the carboxyl tail of Stargazin (STGc), an
auxiliary subunit of AMPAR critical for determining its synaptic distribution, as a proxy of the AMPA receptor, and PSD-95, which caninteract with both GluN2Bc and STGc through PDZ domains13,19,20(Supplementary Fig. 1). STGc was fused with a tetrameric fluorescent protein DsRed2 to mimic the stoichiometry of the endogenous
AMPAR complex. When CaMKII, calmodulin, PSD-95, GluN2Bc
and STGc were combined, PSD-95, GluN2Bc and STGc formed
homogenous condensates while CaMKII remained in the more
dilute phase in the absence of Ca2+ (Fig. 2a–c)20. However, whenCa2+ was added, CaMKII partitioned into the condensates, which
persisted after the addition of EGTA. Intriguingly, we found segregation of proteins in the condensate. CaMKII and GluN2Bc came tothe periphery and surrounded PSD-95 and STGc, which formed aphase-in-phase organization (Fig. 2c). Z-axis reconstruction showedthat CaMKII and GluN2Bc entirely covered PSD-95 and STGc
(Fig. 2d). While STGc was exclusively enriched in the inner phase,
PSD-95 was partitioned in the peripheral phase as well (Fig. 2e).
Conversely, both CaMKII and GluN2Bc were also partly partitioned
in the inner phase. Again, consistent with liquid-like properties, weobserved condensates fusing with each other (Fig. 2f).
Formation of the phase-in-phase organization requires
CaMKII. Without CaMKII, Ca2+ failed to induce phase-in-phaseassembly (Supplementary Fig. 6a). In the presence of CaMKII,
phase-in-phase assembly could be induced in the absence ofATP (Supplementary Fig. 6b). However, after addition of EGTA,
CaMKII moved to the more dilute phase and the condensates
became homogenous (Supplementary Fig. 4b). Essentially the same
results were obtained by using the CaMKII K42R (Supplementary
Fig. 6c) and T286A (Supplementary Fig. 7a) mutants in the presence of ATP. These results indicate that neither kinase activity nor
T286 phosphorylation is required for phase-in-phase assembly
formation although GluN2B, Stg and PSD-95 are all known to bephosphorylated by CaMKII (ref. 9,19,20). However, for the persistent phase-in-phase organization after the decrease in Ca2+ concentration, T286 autophosphorylation is crucial. CaMKII I205K
and 1–314 mutants did not induce segregation (Supplementary
Fig. 7b,c). Together, these results indicate that the segregation of
GluN2Bc and STGc requires multivalent binding at the CaMKII
T-site and GluN2Bc, but not the phosphorylation of any of thecomponents. However, the persistent segregation after Ca2+ receding requires CaMKII T286 phosphorylation.
T-site interaction peptide can dissolve protein condensates.
Different synaptic input patterns can induce bidirectional synaptic plasticity. We then wondered if there is any way to reverse
the protein condensates. We turned to Camk2n1, a small endogenous CaMKII inhibitor protein that interacts with the T-site of
CaMKII and is upregulated during memory processes21. Infusion of
Camk2n1 to protein condensates resulted in collapse of the condensates (Fig. 3a,b and Supplementary Videos 1 and 2). In condensatescomposed of CaMKII/GluN2Bc/PSD-95/STGc, the surrounding
CaMKII/GluN2Bc phase collapsed, while the PSD-95/STGc in the
inner phase was more resistant, consistent with the fact that PSD95/STGc by themselves form condensates20 (Fig. 3b). To confirm
that Camk2n1 disrupts the phase by competing with the T-site,
we used CN21, a peptide derived from the minimum T-site binding region of Camk2n1 (ref. 22). CN21, but not a scrambled peptide, collapsed the condensates formed by CaMKII and GluN2Bc
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Fig. 1 | CaMKII and GluN2Bc form LLPS condensates. a, Low-speed sedimentation assay. CaMKII (10 µM) and GluN2Bc (10 µM) were mixed in thepresence of 0.5 mM EGTA, 10 µM calmodulin, 5 mM MgCl2 and 2.5 mM ATP (−). Then, 2 mM CaCl2 (Ca2+) was added, followed by 2.5 mM EGTA
(Ca2+ → EGTA). The supernatant (S) and pellet (P) after centrifugation at 10,000g were subjected to SDS–PAGE and Coomassie brilliant blue staining.
A slight upward shift of GluN2Bc is likely due to phosphorylation by CaMKII. The asterisk indicates a degradation product of GluN2Bc. Calmodulin is
unobservable in the image due to its small size. An uncropped image of the gel is shown in Supplementary Fig. 13. b, Quantification of a (mean ± s.e.m.;n = 4 samples). c, DIC and confocal microscopic images of the protein mixture in a. CaMKII and GluN2Bc formed a condensate only in the presence ofCa2+. Once formed, the condensate persisted even after the addition of EGTA. Scale bar, 10 µm. d, FRAP after photobleaching of a single point inside acondensate (indicated by white dot) of CaMKII–GluN2Bc in the presence of Ca2+. Note that these are two separate experiments. Scale bar, 1 μm. See
Supplementary Fig. 2 for quantification. e, A fusion event of condensates. Scale bar, 1 µm. f, Same experiment as in c in the absence of Mg2+–ATP. Ca2+
triggers the formation of the condensate in the absence of Mg2+–ATP. However, if EGTA is added, the condensate dispersed. Scale bar, 10 µm. g,h, Sameexperiment as in c using K42R (g) and T286A (h) mutants of CaMKII. Only Ca2+ and Ca2+ → EGTA conditions are shown. In both cases, the condensate
formed in the presence of Ca2+ but did not persist after addition of EGTA. Combined, the results indicate that T286 phosphorylation is crucial for
persistence of the condensate. Scale bar, 10 µm.
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Fig. 2 | Segregation of AMPAR and NMDAR in protein condensate by active CaMKII. a, Sedimentation assay of 10 µM PSD-95, 2.5 µM GluN2Bc, 7.5 µM
STGc, 10 µM CaMKII and 10 µM calmodulin in the presence of Mg2+-ATP. The upward band shift and reduction in staining of PSD-95, GluN2Bc and STGc
is likely due to phosphorylation by CaMKII. An uncropped image of the gel is shown in Supplementary Fig. 13. b, Quantification of a (mean ± s.e.m.; n = 3
samples). c, Images of the protein mixture in a. The two right-most columns are higher magnifications of the dashed rectangles in the STGc channel. Scalebars, 10 µm and 1 µm for low- and high-magnification images. d, Magnification and Z projection of single condensates. Scale bar, 1 µm. e, Line scanning ofd in each color channel. f, Observation of a condensate fusion event. Scale bar, 1 µm. When stimulated with Ca2+, PSD-95/STGc formed phase-in-phase
while GluN2Bc/CaMKII formed a surrounding phase. This persisted even after addition of EGTA.

(Fig. 3c). Although CN21 is a CaMKII inhibitor, in this case, itdoes not affect existing phosphorylation as there is no phosphatase.
These results indicate that the LLPS mediated by CaMKII can bereversed by Camk2n1 competition with GluN2Bc.

Tiam1 behaves as a Ca2+-dependent client for CaMKII condensate. We then tested the possibility that other synaptic T-site proteins might serve as a client for the CaMKII/GluN2Bc condensate.
We previously found that persistent binding between CaMKIINature Neuroscience | www.nature.com/natureneuroscienceArticles

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Fig. 3 | Dispersion of protein condensates by competing T-site interaction. a, Time-lapse imaging of CaMKII–GluN2Bc condensates (Ca2+ → EGTA
condition) during infusion of 50 µM Camk2n1. The arrow shows the direction of infusion. See Supplementary Video 1. Scale bars, 10 µm and 1 µm for lowand high-magnification images, respectively. Note the complete dispersal of the condensate. b, Same experiment as in a using the condensates of CaMKII,
GluN2Bc, PSD-95 and STGc. Due to the limited number of color channels available, PSD-95 was not imaged. See Supplementary Video 2. Note that thephase-in-phase of PSD-95/STGc was resistant to Camk2n1 application, indicating that these two proteins formed condensates by themselves. c, Effect of
5 µM scrambled and CN21 peptides for CaMKII/GluN2Bc condensates in Ca2+ → EGTA condition. CN21 replicated the effect of Camk2n1. d, Effect of 5 µM
Tiam1 peptides for CaMKII/GluN2Bc condensates in Ca2+ → EGTA condition. Tiam1 peptide was taken up by the condensate without much affecting the
LLPS. Scale bars, 10 µm and 1 µm for low- and high-magnification images, respectively.

and Tiam1, a RacGEF protein, after long-term potentiation (LTP)
induction results in a reciprocally activating kinase-effector complex (RAKEC), which supports persistent Rac activity and theenlargement of dendritic spines10. We therefore tested if fluorescently labeled Tiam1 peptide corresponding to the CaMKII-binding
domain (1544-DSHASRMAQLKKQAALSGINGG-1565) can betaken up by the protein condensate (Fig. 3d). We found that peptide
was taken up by CaMKII/GluN2Bc condensates formed by the addition of Ca2+. Once taken up, the peptide remained even after Ca2+
was chelated. This indicates that the protein condensate formed by
CaMKII can serve as a mechanism to trap synaptic T-site binding
proteins in an activity-dependent fashion.
Disruption of CaMKII T-site interaction decreases segregation
between AMPAR and NMDAR. We then tested if CaMKII plays
a role in segregating AMPAR and NMDAR in neurons by using
direct stochastic optical reconstruction microscopy (dSTORM)3,4.
We immunolabeled the endogenous AMPAR subunit GluA2 andNMDAR subunit GluN1 by using antibodies against their extracellular domains and then analyzed the overlap of synaptic nanodomains between the two receptor subtypes. In control neurons treatedwith cell-permeable peptide tat-scrambled (SCR), we observed that
AMPAR and NMDAR formed distinct nanodomains (Fig. 4a). In
neurons treated with tat-CN21 (CN21), the overlap was increased
Nature Neuroscience | www.nature.com/natureneurosciencesignificantly compared with neurons treated with SCR, consistent with the idea that the segregation of AMPAR and NMDAR isdependent on CaMKII-mediated phase-in-phase assembly formation (Fig. 4b,c). The reason the proteins did not totally diffuse away
upon CN21 treatment, unlike in the LLPS experiment, is likely due
to the presence of several other mechanisms that still anchor the
receptors at the synapse. We found no change in the area of nanodomain, the number of localization or the density of localization in
CN21-treated neurons compared with neurons treated with SCR(Supplementary Fig. 8a–c).
NLGN cosegregates together with AMPAR. The trans-synaptic
nanocolumn composed of presynaptic active zone and postsynaptic glutamate receptor is refined by neuronal activity, which canenhance the efficiency of synaptic transmission2,5,23,24. We wondered whether CaMKII-mediated segregation of postsynaptic
proteins can communicate with the presynaptic terminal. We thus
turned to examining the role of neuroligin-1 (NLGN), a neuronaladhesion molecule. NLGN interacts with presynaptic neurexin
through its N-terminal extracellular domain, while the intracellular C-terminus interacts with the third PDZ domain (PDZ3) of
PSD-95 (refs. 23,25–27) (Supplementary Fig. 1). We fused the amino
terminus of the intracellular carboxyl tail of NLGN (NLGNc) tothe carboxyl terminus of dimeric Kusabira Green, a fluorescentArticles

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domainFig. 4 | Reduction of synaptic glutamate receptor segregation by competing T-site interaction. a, Top row: low-magnification epifluorescence image ofa dendrite from a hippocampal neuron in dissociate culture treated with 20 µM tat-scrambled peptide for 30 min and immunolabeled with anti-GluA2(AMPAR, green) and anti-GluN1 (NMDAR, magenta). Scale bar, 10 µm. Second row: high-magnification image of a single synapse (in white squares in the
low magnification image). Bottom two rows: dSTORM and thresholded images of the same region. Scale bar, 0.5 µm. b, Images of a synapse treated with
20 µM tat-CN21 for 30 min. c, Proportion of AMPAR nanodomains overlapping with NMDAR nanodomains (left, P = 0.0098) and of NMDAR nanodomains
overlapping with AMPAR nanodomains (right, P = 0.0019) in tat-scrambled (SCR) or tat-CN21 (CN21) treated neurons. There was significantly more
overlap in two receptor nanodomains in neurons treated with tat-CN21 than in those treated with tat-scrambled peptide. The dataset was obtained from118 (SCR) and 116 (CN21) spines, from a total of 10 neurons for each treatment group. All neurons were processed in parallel using the same staining,acquisition and analysis parameters in blind fashion. The statistical significance of the results was assessed by two-sided Mann–Whitney U test. **P < 0.01.
The center line, the top and bottom of the box, and the whiskers of the blot indicate median, 25th–75th percentile and 5th–95th percentile, respectively.

protein, and tested if it could form condensates. NLGNc alone (notshown) or together with PSD-95 did not form condensates (Fig.
5a). Only when we added GluN2Bc or GluN2Bc and STGc, did
NLGNc participate in condensates (Fig. 5a). Deletion of the PDZ
domain binding motif of NLGNc (NLGNc-Δ7) excluded it fromthe condensates (Supplementary Fig. 9). These results indicate that
NLGNc participates in PSD-95 condensates as a ‘client’ throughits PDZ-binding motif. When we added CaMKII, before additionof Ca2+, proteins other than CaMKII formed homogenous condensates (Fig. 5b with unlabeled CaMKII, Fig. 5c with unlabeled
PSD-95). Upon stimulation with Ca2+, NLGNc moved to the inner
phase together with STGc/PSD-95, whereas GluN2Bc and CaMKII
segregated to the outer phase (Fig. 5b,c and Supplementary Fig.
10). These results indicate that NLGN is partitioned together with
AMPAR and forms a phase distinct from NMDAR, which mightserve as a mechanism to position AMPAR beneath the presynaptic
active zone. To test if the segregation of NMDAR and NLGN1 inneurons also depends on CaMKII, we treated the neurons in dissociated culture with tat-CN21 or tat-scrambled peptides, thensurface-labeled and observed them by dSTORM (Supplementary
Fig. 11). In neurons treated with tat-Scrambled peptide, NMDAR
and NLGN1 were segregated from each other. In contrast, in neurons treated with tat-CN21, the segregation between them became
significantly less.

Discussion

In this study, we showed that CaMKII can undergo LLPS withmain PSD proteins, most notably GluN2B, through its multivalent interaction conferred by its dodecameric structure. This viewis consistent with several properties of synaptic CaMKII, such as
constant exchange at rest as shown by fluorescence recovery after
photobleaching (FRAP) analysis28, and rapid translocation to thesynapse upon LTP induction in a manner requiring the interactionof CaMKII T-site with GluN2B carboxyl tail17,29,30. The formation of
LLPS by CaMKII and GluN2B as well as Shank3 was reported as aseparate article31.
The initial formation of protein condensates was triggered by
Ca2+ and was independent of kinase activity. Once formed, thecondensate persisted even after the decrease in Ca2+ concentration. This persistence requires CaMKII T286 autophosphorylation,
which maintains CaMKII in an open conformation and exposes the
T-site32, thereby allowing the binding of GluN2B. In its absence, theautoinhibitory domain docks at the T-site11 and outcompetes binding with GluN2B. We speculate this is why T286 phosphorylationis required for the persistence of protein condensates. The size ofprotein condensate in the LLPS in vitro experiment is much larger
than in a synapse. One reason for this difference may be due to theavailability of the component molecules. In vitro, the amount ofproteins is enormous compared to the situation in neurons, which
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a

DIC

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Ca2+→
EGTA

Fig. 5 | Neuroligin-1 segregates into the STGc/PSD-95 phase by CaMKII. a, Images of protein mixtures (as indicated). PSD-95 (10 µM) was mixed
with 10 µM NLGNc (upper) and a further 10 µM GluN2Bc (middle) and 10 µM STGc (lower). No Ca2+ was added. NLGNc plus PSD-95 in the absence of
GluN2Bc did not form condensate. This indicates that NLGNc participates in the condensates formed by PSD-95 and GluN2Bc as a ‘client’. b, Condensatesof 10 µM PSD-95, 2.5 µM GluN2Bc, 7.5 µM STGc, 1 µM NLGNc and unlabeled CaMKII in −, Ca2+ and Ca2+ → EGTA conditions. The right column shows ahigher magnification image of condensates indicated by dashed rectangles in the NLGNc channel. Due to the limited number of color channels available,
we used unlabeled CaMKII and labeled PSD-95. c, Same experiment as in b but using labeled CaMKII and unlabeled PSD-95. Scale bars, 10 µm and 1 µm
for low- and high-magnification images, respectively. NLGNc/STGc segregate from CaMKII/GluN2Bc to form phase-in-phase while CaMKII and GluN2Bc
are in the surrounding phase in the presence of Ca2+. Once formed, the phase-in-phase organization remained, even after EGTA was added.

are surrounded by plasma membrane and have only a limited
amount of proteins. Membrane proteins (receptors and cell adhesion molecules) are especially spatially limited because they exist
two dimensionally at the synapse. Nevertheless, the result obtainedin vitro shows that it is possible for CaMKII to undergo LLPS withits binding partners at the synapse.
Nature Neuroscience | www.nature.com/natureneuroscience

In condensates, CaMKII segregated AMPAR and NMDAR into
different compartments. Super-resolution imaging of the native
AMPAR and NMDAR provided in vivo evidence that CaMKII
segregates these two subtypes of glutamate receptor into different nanodomains (Supplementary Fig. 12a). AMPAR partitions
together with NLGN and can form a link with the presynaptic active

Articleszone. This mechanism may enrich AMPAR beneath the transmitter release site. AMPAR has a low affinity to glutamate comparedwith NMDAR and is normally not saturated with glutamate at
the synaptic cleft33–35. Indeed, super-resolution imaging studies
showed the alignment of pre- and postsynaptic markers, termed
synaptic nanomodules or nanocolumns, is refined as a result ofneuronal activation5,6. The segregation of AMPARs under the transmitter release site can increase the efficacy of synaptic transmission (Supplementary Fig. 12b)24. Furthermore, cluster formationof NLGN induces clustering of presynaptic neurexin, which thenrecruits further axonal vesicular release machinery and eventuallyforms an active zone36. Therefore, the postsynaptic clustering of
NLGN may serve as a retrograde mechanism to increase presynaptic release probability (Supplementary Fig. 12b)27. Combining
these processes, we propose that postsynaptic activation of CaMKII
and resultant formation of LLPS can serve as a new modulatory
mechanism of synaptic transmission. Consistent with this idea, theactivation of postsynaptic NMDAR accumulates more active zone
proteins over the postsynaptic PSD-95 cluster, thereby forming atrans-synaptically matched nanocolumn of release machinery andreceptor complex at the synapse5.
GluN2B is a minor component of PSD proteins37 and the
CaMKII T-site can interact with other proteins, such as Tiam1,
GJD2/connexin 36, LRRC7/densin-180, Camk2n1 and L-type Ca2+
channel. Therefore, it is possible that CaMKII forms condensateswith these proteins as well, although GluN2B is the most important partner for CaMKII17. Through this mechanism, CaMKII canserve as a postsynaptic Ca2+-dependent hub, which underlies theactivity-dependent transport and crosslinking of several postsynaptic client proteins observed during LTP via the LLPS-mediatedmechanism30,38. This reasonably explains the dodecameric structure
and abundance of CaMKII.
In conclusion, we found a new property of CaMKII in undergoing LLPS in Ca2+-dependent manner and segregating different synaptic proteins. In the future, it will be crucial to test whether thismechanism works in situ and to determine the relative contribution of this versus other proposed mechanisms of synaptic plasticity
mediated by CaMKII, such as AMPAR phosphorylation, insertionand translocation39–41.

Online content

Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of
author contributions and competing interests; and statements ofdata and code availability are available at https://doi.org/10.1038/
s41593-021-00843-3.
Received: 9 June 2020; Accepted: 22 March 2021;
Published: xx xx xxxx

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Articles
Methods

Guidelines. All recombinant DNA and animal experiments were carried out
in accordance with the institutional guidelines of Kyoto University, Hong Kong
University of Science and Technology, University of Bordeaux and CNRS.
DNA constructs and protein purification. Rat CaMKII wildtype and mutants,
fluorescent proteins fused with Spy-catcher, Spy-tag fused with the intracellularcarboxyl tails of GluN2Bc (mouse, amino acids (a.a.) 1226–1482), STGc (mouse,a.a. 203–323), NLGNc (mouse, a.a. 719–843), GluA1 (rat, a.a. 827–907), andGluA2 (rat, a.a. 834–883) at their amino-termini were inserted into the vector
pSUMO (ref. 42). Amino acid residues were numbered with the initiation
methionine as 1. PSD-95 and calmodulin were inserted into the p32m3c vector
as previously described13.
All proteins were expressed in Escherichia coli strain BL21 DE3 RIL andpurified by affinity column using Nickel–NTA beads (Nacalai Tesque), gelfiltration column HiLoad 26/600 Superdex 200 pg (GE Healthcare) and anion
exchange column HiTrap Q HP (GE Healthcare). All tags for purification were cut
and removed. The I205K mutant of CaMKII was tagged with green fluorescentprotein (GFP) due to the difficulty of expressing and purifying untagged protein.
Fluorescent protein tagged Spy-catcher and Spy-tag tagged receptor C-tails42
were mixed with excess molar ratio of monomer C-tails and incubated for 2 h
at room temperature to covalently conjugate with each other. Extra monomer
C-tails were removed by further gel filtration. PSD-95 and CaMKII was labeled
by iFluor 405 succinimidyl ester or iFluor 488 succinimidyl ester (AAT Bioquest)
as previously described13. Labeled protein was mixed with unlabeled protein at
1:100. Protein concentration is expressed as monomer concentration throughout
the study.
Formation and observation of LLPS condensates. Proteins were mixed in a buffer
containing 50 mM Tris–HCl pH 7.5, 100 mM NaCl, 1 mM Tris(2-carboxyethyl)
phosphine (TCEP), 0.5 mM EGTA and 10 µM calmodulin in the presence of
5 mM MgCl2 and 2.5 mM ATP (− condition). MgCl2 and ATP were not added inFig. 1f and Supplementary Fig. 6b. CaCl2 (2 mM) was added to activate CaMKII(Ca2+ condition) and, 10 s later, 2.5 mM EGTA was further added to chelate Ca2+
(Ca2+ → EGTA condition) to mimic a transient Ca2+ signal.
The sedimentation assay was carried out as previously described12–14. The
protein sample in a low protein binding tube (WATSON) was centrifuged at
10,000g for 1 min. Pellet and supernatant was denatured by SDS loading buffer andadjusted to the same volume. Samples (5 µl) were loaded onto SDS–polyacrylamide
gel electrophoresis (SDS–PAGE) and visualized using Coomassie brilliant blue dye.
For confocal microscope imaging, a sample chamber was made between acoverslip (12 mm round coverslip, MATSUNAMI) and a slide glass (FRC-04,
MATSUNAMI) separated by double-sided adhesive paper tape as a spacer. Protein
mixture (5 µl) was injected into this space and the condensates were allowed
to settle to the bottom of coverslip for 5 min. Observation was by a confocal
microscopy system (FLUOVIEW FV1200, Olympus). The observation area wasdetermined randomly. Images of each color channel were obtained with excitation
wavelength and bandpass filters as follows: 405 nm for iFluor-405-tagged PSD-95
or CaMKII, 488 nm for iFluor-488-tagged CaMKII or Kusabira Green-tagged
NLGNc, 546 nm for DsRed2-tagged STGc and 647 nm for eqFP670-tagged
GluN2Bc and E2-Crimson-tagged GluA1c and GluA2c. Tiam1 peptide (mouse, a.a.
1540–1560) was labeled with fluorescein by N-hydroxysuccinimide (NHS)-ester at
the amino terminus.
Turbidity assay. CaMKII (10 µM) and GluN2Bc (10 µM) were mixed in buffer
containing 50 mM Tris–HCl pH 7.5, 100 mM NaCl, 1 mM Tris(2-carboxyethyl)
phosphine (TCEP), 0.5 mM EGTA and 10 µM calmodulin in the presence of 5 mM
MgCl2 and 2.5 mM ATP. The turbidity of the protein sample was measured as the
optical density at 420 nm by nanodrop ND-1000 (Thermo Fisher). The baseline
was defined as zero, and the turbidity was measured every 30 s for 4 min. CaCl2(2 µM) was added at between 1 and 1.5 min, and 2.5 mM EGTA was further added
between 2.5 and 3 min.
Cell culture, drug treatment and immunostaining. Banker-type cultures ofhippocampal neurons were prepared from embryonic day 18 (E18) Sprague–
Dawley rats of both sexes at a density of 200,000 cells per dish as described4,43.
The neurons at 16 days in vitro were treated with the CaMKII inhibitor
peptide CN21 fused with the cell-penetrating peptide TAT (TAT-CN21;YGRKKRRQRRRKRPPKLGQIGRSKRVVIEDDR) or a scrambled CN21 with TAT(TAT-scrambled; YGRKKRRQRRRVKEPRIDGKPVRLRGQKSDRI) (20 µM) for
30 min. After treatment, the neurons were surface-immunolabeled for endogenous
glutamate receptor labeling: GluA2 (anti-GluA2, clone 14B11, 0.0033 µg µl−1.
IgG2b; provided by E. Gouaux) and GluN1 (anti-GluN1, clone 10B11, 0.002 µg µl−1.
IgG1; provided by E. Gouaux) at 37 °C for 15 min (refs. 4,43). Neuroligin-1 waslabeled with biotin by coexpressing acceptor peptide (AP)-tagged neuroligin-1
and a biotin ligase BirA (ref. 44). The cells were surface labeled by incubating withmonovalent streptavidin coupled to Alexa 647 for 10 min. After three washes, thecells were fixed with 4% paraformaldehyde (Sigma-Aldrich, catalog no. P6148)/4%
sucrose (Sigma-Aldrich, catalog no. 0389) in phosphate buffered saline (PBS) at

NATure NeurosCIenCeroom temperature for 10 min and treated with blocking solution (1.5% bovine
serum albumin (Sigma-Aldrich, catalog no. A3059)/0.1% fish skin gelatin/0.1%
Triton X-100 in PBS/NH4Cl) at room temperature for 1 h. Cells were thenincubated with secondary antibodies, goat anti-mouse IgG2b Alexa 647 (Thermo
Scientific, catalog no. 21242. 0.002 µg µl−1) and goat anti-mouse IgG1 Alexa 532(Thermo Scientific, and coupling done at IINS. 0.002 µg µl−1) at room temperaturefor 1 h. Following three washes, a second fixation was performed and cells werethen imaged.
dSTORM imaging. dSTORM imaging was performed on a LEICA DMi8
microscope equipped with a Leica HCX PL APO ×160 1.43 NA oil immersion
TIRF objective and fiber-coupled laser launch (532 nm and 642 nm) (Roper
Scientific). Single fluorophores were detected with an EMCCD camera (Evolve
Photometrics). Samples were mounted on a Ludin chamber (Life Imaging Services)and 600 µl dSTORM pyranose switching buffer45 was added. An extra coverslip wasplaced on top to minimize buffer evaporation and oxygen exchanges with ambient
air. Before dSTORM imaging, a diffraction limited image of the target region(512 × 512 pixels, 1 pixel = 100 nm) was taken under wide-field epifluorescenceillumination. Image acquisition was steered by MetaMorph software (Molecular
Devices) with a 30 ms exposure time, 20,000 frames per color. The 642 nm
and 532 nm lasers were used sequentially. Multicolor fluorescent microspheres(Tetraspeck, Invitrogen, catalog no. T7279) were used as fiducial markers fornanometer scale lateral drift correction and dual color registration.
Nanodomain analyses. To analyze AMPAR and NMDAR nanodomains, intensity
super-resolution images with a pixel size of 25 nm were reconstructed during theacquisition using WaveTracer software operating as a plugin of MetaMorph46.
Lateral drifts were corrected automatically from the localizations of fluorescentfiduciary markers absorbed into the coverslip. Single-molecule localizations of
Alexa 532 and Alexa 647 were aligned postacquisition with PALMTracer software
using a third-order polynomial transform to correct for chromatic aberrations on
the whole field of view. SR-Tesseler and Coloc-Tesseler tessellation-based analysis
software47,48 were used to quantify the nanodomains and the colocalization of
AMPAR and NMDAR, respectively. The segmentation of AMPAR and NMDAR
nanodomains was performed separately. Single-molecule localizations wereused to compute the Voronoï tessellation, from which the first rank order local
density map was computed. Clusters were segmented automatically using athreshold of twice the average local density of the whole dataset, with a minimum
localizations number of five and a minimum area of 2 × 104 nm2. Next, theclusters’ nanodomains were segmented by applying a threshold of one times theaverage density in each cluster, with a minimum localizations number of 25, a
minimum area 0.02 (AMPAR) or 0.01 (NMDAR) × 104 nm2. The colocalization
between AMPAR and NMDAR was computed from the overlapping nanodomains
area in selected regions of interest (ROI). ROIs were identified from merged
epifluorescence images of AMPAR and NMDAR.
NLGN1 and NMDAR double-stained images were analyzed similarly but,because they hardly overlapped, we measured the distance from NMDAR
localization to the nearest NLGN1 localization with a cut-off of 500 nm. Statisticalsignificance was tested by Kolmogorov–Smirnov test; α was set at 0.05.
Statistics and reproducibility. No animals/datapoints were excluded. No statisticalmethods were used to predetermine sample sizes but our sample sizes are similar
to those reported in previous publications4,43. We used GraphPad Prism Software
v.8.3.1 to perform statistical analysis. The normality and equal variances ofdatasets were tested, and suitable statistical tests were chosen according to thedatasets. Two-sided Mann–Whitney U was used for Fig. 4 and Supplementary
Fig. 8 and Kolmogorov–Smirnov test was used for Supplementary Fig. 11. The
reproducibility of all micrographs shown in this paper was confirmed in more thanfour independent experiments.
Reporting Summary. Further information on research design is available in the
Nature Research Reporting Summary linked to this article.

Data availability

All relevant data of this manuscript are available from the corresponding author
upon reasonable request. Protein sequences are available from NCBI (https://www.
ncbi.nlm.nih.gov/protein/).

References

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Neurosci. 33, 13204–13224 (2013).
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45. Beghin, A. et al. Localization-based super-resolution imaging meets
high-content screening. Nat. Methods 14, 1184–1190 (2017).
46. Kechkar, A., Nair, D., Heilemann, M., Choquet, D. & Sibarita, J. B. Real-time
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48. Levet, F. et al. A tessellation-based colocalization analysis approach forsingle-molecule localization microscopy. Nat. Commun. 10, 2379 (2019).

Acknowledgements

We thank R. A. Nicoll, J. W. Hell and T. A. Blanpied for comments on the manuscript,
E. Gouaux, O. Thoumine, M. Sainlos, M. Rosendale and Bordeaux Imaging Center forthe reagents and assistance and L. Yu, A. Z. Weitemier and E. Agnello for editing. This
work was supported by RIKEN Presidents Fund, SPIRITS 2019 of Kyoto University,
Grant-in-Aid for Scientific Research JP20240032, JP22110006, JP16H01292, JP18H04733
and JP18H05434 from the MEXT, Japan, JST, CREST JPMJCR20E4, Japan, Programme
Exploration France from Ambassade de France au Japon, The Uehara Memorial
Foundation, The Naito Foundation, Research Foundation for Opto-Science andTechnology, Novartis Foundation, The Takeda Science Foundation and Japan Foundation
for Applied Enzymology to Y.H., The Takeda Science Foundation and Grants-in-Aid for
Scientific Research JP17K14947, JP18KK0421 and JP19K06885 from the MEXT, Japan
to T.H., grants from the Simons Foundation (Award ID 510178) and Research Grant

Nature Neuroscience | www.nature.com/natureneuroscience

Council of Hong Kong (AoE-M09-12 and C6004-17G) to M.Z., HFSP Research Grant(RGP0020/2019) jointly to Y.H. and M.Z, CRCNS-NIH-ANR AMPAR-T fellowship toE.H. and The National Center for Scientific Research (CNRS), Agence Nationale de laRecherche (DynHippo) to L.G. and J.F.

Author contributions

T.H. and P.-W.L. conducted and managed all experiments. Y.H. managed the overall
project. Q.C. and M.Z. participated in LLPS experiments. J.S.F., F.L., C.B., J.-B.S., D.C.,
L.G. and E.H. participated in super-resolution microscopy.

Competing interests

Y.H. is partly supported by Fujitsu Laboratories and Dwango.

Additional information

Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41593-021-00843-3.
Correspondence and requests for materials should be addressed to M.Z. or Y.H.
Peer review information Nature Neuroscience thanks Thomas Biederer, Dragomir
Milovanovic and the other, anonymous, reviewer(s) for their contribution to the peer
review of this work.
Reprints and permissions information is available at www.nature.com/reprints.

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Data collection

Describe the data collection procedure, including who recorded the data and how.

Timing and spatial scale Indicate the start and stop dates of data collection, noting the frequency and periodicity of sampling and providing a rationale forthese choices. If there is a gap between collection periods, state the dates for each sample cohort. Specify the spatial scale from which
the data are taken

Data exclusions

If no data were excluded from the analyses, state so OR if data were excluded, describe the exclusions and the rationale behind them,
indicating whether exclusion criteria were pre-established.

Reproducibility

Describe the measures taken to verify the reproducibility of experimental findings. For each experiment, note whether any attempts torepeat the experiment failed OR state that all attempts to repeat the experiment were successful.

Randomization

Describe how samples/organisms/participants were allocated into groups. If allocation was not random, describe how covariates were
controlled. If this is not relevant to your study, explain why.

Blinding

Describe the extent of blinding used during data acquisition and analysis. If blinding was not possible, describe why OR explain why
blinding was not relevant to your study.
Yes

Did the study involve field work?

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Sampling strategy

No

Field work, collection and transport
Field conditions

Describe the study conditions for field work, providing relevant parameters (e.g. temperature, rainfall).

Location

State the location of the sampling or experiment, providing relevant parameters (e.g. latitude and longitude, elevation, water depth).

Access & import/export Describe the efforts you have made to access habitats and to collect and import/export your samples in a responsible manner and incompliance with local, national and international laws, noting any permits that were obtained (give the name of the issuing authority,
the date of issue, and any identifying information).

DisturbanceDescribe any disturbance caused by the study and how it was minimized.

Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material,
system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.

Materials & experimental systems

Methods

n/a Involved in the study

n/a Involved in the study

Antibodies

ChIP-seq

Eukaryotic cell lines

Flow cytometry

Palaeontology and archaeology

MRI-based neuroimaging

Animals and other organisms
Human research participants
Clinical data
Dual use research of concern

Antibodies
Antibodies usedanti-GluA2, clone 14B11, 0.0033 µg/µl. IgG2b. From Dr. Eric Gouaux, anti-GluN1, clone 10B11, 0.002 µg/µl. IgG1. From Dr. Gouaux

Validation

Anti-GluA2 antibodies used in this study gave no staining in GluA2 knockout mice(see also Giannone et al., Biophysical journal 99, 1303-1310).
Specificity of anti-GluN1 was confirmed by FSEC traces in Lu et al., Science 2017 Mar 24; 355(6331)
April 2020

Eukaryotic cell lines
Policy information about cell lines
Cell line source(s)State the source of each cell line used.

Authentication

Describe the authentication procedures for each cell line used OR declare that none of the cell lines used were authenticated.

3

Confirm that all cell lines tested negative for mycoplasma contamination OR describe the results of the testing formycoplasma contamination OR declare that the cell lines were not tested for mycoplasma contamination.

Commonly misidentified lines

Name any commonly misidentified cell lines used in the study and provide a rationale for their use.

(See ICLAC register)

Palaeontology and Archaeology
Specimen provenance

Provide provenance information for specimens and describe permits that were obtained for the work (including the name of theissuing authority, the date of issue, and any identifying information).

Specimen deposition

Indicate where the specimens have been deposited to permit free access by other researchers.

Dating methods

If new dates are provided, describe how they were obtained (e.g. collection, storage, sample pretreatment and measurement), where
they were obtained (i.e. lab name), the calibration program and the protocol for quality assurance OR state that no new dates areprovided.

Tick this box to confirm that the raw and calibrated dates are available in the paper or in Supplementary Information.
Ethics oversightnature research | reporting summary

Mycoplasma contamination

Identify the organization(s) that approved or provided guidance on the study protocol, OR state that no ethical approval or guidance
was required and explain why not.

Note that full information on the approval of the study protocol must also be provided in the manuscript.

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal researchLaboratory animals

For laboratory animals, report species, strain, sex and age OR state that the study did not involve laboratory animals.

Wild animals

Provide details on animals observed in or captured in the field; report species, sex and age where possible. Describe how animals werecaught and transported and what happened to captive animals after the study (if killed, explain why and describe method; if released,
say where and when) OR state that the study did not involve wild animals.

Field-collected samples

For laboratory work with field-collected samples, describe all relevant parameters such as housing, maintenance, temperature,
photoperiod and end-of-experiment protocol OR state that the study did not involve samples collected from the field.

Ethics oversight

Identify the organization(s) that approved or provided guidance on the study protocol, OR state that no ethical approval or guidance
was required and explain why not.

Note that full information on the approval of the study protocol must also be provided in the manuscript.

Human research participants
Policy information about studies involving human research participants
Population characteristics

Describe the covariate-relevant population characteristics of the human research participants (e.g. age, gender, genotypic
information, past and current diagnosis and treatment categories). If you filled out the behavioural & social sciences study
design questions and have nothing to add here, write "See above."

Recruitment

Describe how participants were recruited. Outline any potential self-selection bias or other biases that may be present andhow these are likely to impact results.

Ethics oversight

Identify the organization(s) that approved the study protocol.

Note that full information on the approval of the study protocol must also be provided in the manuscript.

Clinical data
Policy information about clinical studies
All manuscripts should comply with the ICMJE guidelines for publication of clinical research and a completed CONSORT checklist must be included with all submissions.

Clinical trial registration Provide the trial registration number from ClinicalTrials.gov or an equivalent agency.
Note where the full trial protocol can be accessed OR if not available, explain why.

Data collection

Describe the settings and locales of data collection, noting the time periods of recruitment and data collection.

Outcomes

Describe how you pre-defined primary and secondary outcome measures and how you assessed these measures.

April 2020

Study protocol

4Policy information about dual use research of concern

Hazards
Could the accidental, deliberate or reckless misuse of agents or technologies generated in the work, or the application of information presented
in the manuscript, pose a threat to:
No YesPublic healthNational security
Crops and/or livestock
Ecosystems
Any other significant area

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Dual use research of concern

Experiments of concern
Does the work involve any of these experiments of concern:
No YesDemonstrate how to render a vaccine ineffective
Confer resistance to therapeutically useful antibiotics or antiviral agents
Enhance the virulence of a pathogen or render a nonpathogen virulent
Increase transmissibility of a pathogen
Alter the host range of a pathogen
Enable evasion of diagnostic/detection modalities
Enable the weaponization of a biological agent or toxin
Any other potentially harmful combination of experiments and agents

ChIP-seq
Data deposition
Confirm that both raw and final processed data have been deposited in a public database such as GEO.
Confirm that you have deposited or provided access to graph files (e.g. BED files) for the called peaks.

May remain private before publication.

For "Initial submission" or "Revised version" documents, provide reviewer access links. For your "Final submission" document,
provide a link to the deposited data.

Files in database submission

Provide a list of all files available in the database submission.

Genome browser session

Provide a link to an anonymized genome browser session for "Initial submission" and "Revised version" documents only, toenable peer review. Write "no longer applicable" for "Final submission" documents.

Data access links(e.g. UCSC)

Methodology
Replicates

Describe the experimental replicates, specifying number, type and replicate agreement.

Sequencing depth

Describe the sequencing depth for each experiment, providing the total number of reads, uniquely mapped reads, length of reads andwhether they were paired- or single-end.

Antibodies

Describe the antibodies used for the ChIP-seq experiments; as applicable, provide supplier name, catalog number, clone name, and lot
number.

used.

Data quality

Describe the methods used to ensure data quality in full detail, including how many peaks are at FDR 5% and above 5-fold enrichment.

SoftwareDescribe the software used to collect and analyze the ChIP-seq data. For custom code that has been deposited into a community
repository, provide accession details.

April 2020

Peak calling parameters Specify the command line program and parameters used for read mapping and peak calling, including the ChIP, control and index files

5PlotsConfirm that:
The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation

Describe the sample preparation, detailing the biological source of the cells and any tissue processing steps used.

Instrument

Identify the instrument used for data collection, specifying make and model number.

SoftwareDescribe the software used to collect and analyze the flow cytometry data. For custom code that has been deposited into acommunity repository, provide accession details.

Cell population abundance

Describe the abundance of the relevant cell populations within post-sort fractions, providing details on the purity of thesamples and how it was determined.

Gating strategy

Describe the gating strategy used for all relevant experiments, specifying the preliminary FSC/SSC gates of the starting cell
population, indicating where boundaries between "positive" and "negative" staining cell populations are defined.

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Flow Cytometry

Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.

Magnetic resonance imaging
Experimental design
Design type

Indicate task or resting state; event-related or block design.

Design specifications

Specify the number of blocks, trials or experimental units per session and/or subject, and specify the length of each trialor block (if trials are blocked) and interval between trials.

Behavioral performance measures

State number and/or type of variables recorded (e.g. correct button press, response time) and what statistics were used
to establish that the subjects were performing the task as expected (e.g. mean, range, and/or standard deviation acrosssubjects).

Acquisition
Imaging type(s)Specify: functional, structural, diffusion, perfusion.

Field strength

Specify in Tesla

Sequence & imaging parameters

Specify the pulse sequence type (gradient echo, spin echo, etc.), imaging type (EPI, spiral, etc.), field of view, matrix size,
slice thickness, orientation and TE/TR/flip angle.

Area of acquisition

State whether a whole brain scan was used OR define the area of acquisition, describing how the region was determined.

Diffusion MRI

Used

Not used

Preprocessing
Provide detail on software version and revision number and on specific parameters (model/functions, brain extraction,
segmentation, smoothing kernel size, etc.).

Normalization

If data were normalized/standardized, describe the approach(es): specify linear or non-linear and define image types used fortransformation OR indicate that data were not normalized and explain rationale for lack of normalization.

Normalization template

Describe the template used for normalization/transformation, specifying subject space or group standardized space (e.g.
original Talairach, MNI305, ICBM152) OR indicate that the data were not normalized.

Noise and artifact removal

Describe your procedure(s) for artifact and structured noise removal, specifying motion parameters, tissue signals andphysiological signals (heart rate, respiration).

April 2020

Preprocessing software6Volume censoring

Define your software and/or method and criteria for volume censoring, and state the extent of such censoring.

Model type and settingsSpecify type (mass univariate, multivariate, RSA, predictive, etc.) and describe essential details of the model at the first andsecond levels (e.g. fixed, random or mixed effects; drift or auto-correlation).

Effect(s) tested

Define precise effect in terms of the task or stimulus conditions instead of psychological concepts and indicate whether
ANOVA or factorial designs were used.

Specify type of analysis:
Statistic type for inference

Whole brain

ROI-based

BothSpecify voxel-wise or cluster-wise and report all relevant parameters for cluster-wise methods.

(See Eklund et al. 2016)

Correction

Describe the type of correction and how it is obtained for multiple comparisons (e.g. FWE, FDR, permutation or Monte Carlo).

Models & analysis

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Statistical modeling & inference

n/a Involved in the study
Functional and/or effective connectivity
Graph analysis
Multivariate modeling or predictive analysis

Functional and/or effective connectivity

Report the measures of dependence used and the model details (e.g. Pearson correlation, partial correlation,
mutual information).

Graph analysis

Report the dependent variable and connectivity measure, specifying weighted graph or binarized graph,subject- or group-level, and the global and/or node summaries used (e.g. clustering coefficient, efficiency,etc.).

Multivariate modeling and predictive analysis Specify independent variables, features extraction and dimension reduction, model, training and evaluation
metrics.

April 20207