SIGNAL TRANSDUCTION Membrane-associated periodic …Fig. 1. CB1 and NCAM1 are recruited to the MPS upon cognate ligand binding. (A) Two-color STORM images of bII-spectrin (green) and

SIGNAL TRANSDUCTION

Membrane-associated periodicskeleton is a signaling platform forRTK transactivation in neuronsRuobo Zhou1,2,3, Boran Han1,2,3, Chenglong Xia1,2,3, Xiaowei Zhuang1,2,3*

Actin, spectrin, and related molecules form a membrane-associated periodic skeleton(MPS) in neurons. The function of the MPS, however, remains poorly understood. Usingsuper-resolution imaging, we observed that G protein–coupled receptors (GPCRs),cell adhesion molecules (CAMs), receptor tyrosine kinases (RTKs), and related signalingmolecules were recruited to the MPS in response to extracellular stimuli, resulting incolocalization of these molecules and RTK transactivation by GPCRs and CAMs, givingrise to extracellular signal–regulated kinase (ERK) signaling. Disruption of the MPSprevented such molecular colocalizations and downstream ERK signaling. ERK signalingin turn caused calpain-dependent MPS degradation, providing a negative feedback thatmodulates signaling strength. These results reveal an important functional role of theMPS and establish it as a dynamically regulated platform for GPCR- and CAM-mediatedRTK signaling.

Signal transduction mediated by cell sur-face receptors requires precise coordi-nation of a cascade of molecular events.Receptor tyrosine kinases (RTKs) constitutea large class of such cell surface receptors

that are expressed across many cell types andperform a broad spectrum of cellular functions,including promotion of cell survival, regulationof cell division and differentiation, and modu-lation of cellular metabolism and cell-to-cellcommunication (1, 2). RTKs are activated in re-sponse to extracellular signals, initiating a num-ber of intracellular signal transduction cascadesto alter gene expression in cells (1–4). The kinaseactivity of RTKs can be activated either directlyby their cognate ligands or through transactiva-tion by other transmembrane proteins (1, 2, 4–7).Among the RTK transactivators are G protein–coupled receptors (GPCRs), the largest class of cellsurface receptors in eukaryotes, and cell adhesionmolecules (CAMs), the class of transmembraneproteins responsible for cell-cell interactions(1, 4–7). In neurons, RTK transactivation byGPCRs and CAMs, as well as direct activationof RTKs by their cognate ligands, plays impor-tant roles in regulating neurite outgrowth andaxon guidance, controlling neuronal migrationand repair, and modulating synaptogenesis andsynaptic transmission (3–8). However, it is largelyunknown howGPCRs, CAMs, RTKs, and relatedsignaling components are spatially organized atthe neuronal cell surface and how these mole-cules are brought together to enable RTK trans-activation and downstream signaling.

Recently, it has been shown that actin, spectrin,and their interacting molecules form amembrane-associated periodic skeleton (MPS) structure inthe axons and dendrites of neurons (9–12). Theneuronal MPS contains molecular componentshomologous to those of the erythrocytemembraneskeleton (13), but it adopts a distinct ultrastruc-ture: in neurites, actin filaments are assembledinto ring-like structures that are periodicallyspaced by spectrin tetramers, forming a quasi–one-dimensional lattice structure underneath theplasma membrane with a periodicity of ~190 nm(9). This structure is present in distinct typesof neurons and across diverse animal species(14, 15). The MPS can organize transmembraneproteins, such as ion channels and adhesionmolecules, into periodic distributions alongaxons (9, 11, 16–18), raising the possibility thatthis submembrane lattice structure may medi-ate membrane-associated signal transduction byregulating the distributions of related signalingproteins in space and time.To test this hypothesis, we applied stochastic

optical reconstruction microscopy (STORM)(19, 20), a super-resolution imaging method, toexamine the spatial distributions of two trans-membrane proteins that are known to trans-activate RTKs in neurons (5, 7, 21, 22): (i) thecannabinoid type 1 receptor (CB1), the mostabundant GPCR in the brain and a therapeutictarget for regulating appetite, pain, mood, andmemory, and for treating neurodegenerativediseases (23); and (ii) the neural cell adhesionmolecule 1 (NCAM1), an immunoglobulin super-family CAM important for neuronal migration,neurite outgrowth and fasciculation, and neu-ral circuit development (7). We used two-colorSTORM to investigate the spatial relationshipbetween the MPS and these membrane proteinsin cultured hippocampal neurons (Fig. 1). TheMPS was visualized through immunolabeling of

the C terminus of bII-spectrin, which is located atthe center of each spectrin tetramer connectingadjacent actin rings and is near the binding sitefor ankyrin, an adaptor protein that can connecttransmembrane proteins to the membrane skel-eton (13, 24).Before stimulation with exogenous ligands,

CB1 and NCAM1 exhibited a small degree of co-localization with the C terminus of bII-spectrin,i.e., the center of the spectrin tetramer, in axons(Fig. 1, A and C, left). We quantified the degreeof colocalization using one-dimensional (1D)cross-correlation analysis by projecting thesignals to the longitudinal axis of the axonand calculating the average 1D cross-correlationfunction between the two color channels overmany axon segments. The 1D cross-correlationamplitude, defined as the average amplitude ofthe peaks at ±190 nm (the period of the MPS),quantifies not only the colocalization betweenthe signalingmolecules and theMPS but also thedegree of periodicity of these signalingmolecules(Fig. 1, B and D, blue). The observed averagecross-correlation amplitudeswere>10-fold greaterthan the values derived from single-color–labeledneurons, indicating that the observed colocaliza-tion was not the result of cross-talk between thetwo color channels (fig. S1). Upon treatment withligands, a CB1 agonist WIN 55,212-2 (hereafter,WIN; inhibition constant Ki = 62 nM) (23) or aNCAM1 antibody (NCAM1 Ab) that binds to theextracellular domain to mimic homophilic orheterophilic binding ofNCAM(7), CB1 orNCAM1,respectively, displayed a substantially higherdegree of colocalization with the MPS (Fig. 1, Aand C, middle) with a three- to fourfold increasein the cross-correlation amplitudes (Fig. 1, B andD, red), and a significant reduction in the aver-age distance between CB1 or NCAM1 and theirnearest-neighbor spectrin tetramer centers (fig.S2). Quantitatively similar ligand-induced in-crease in colocalization between CB1 or NCAM1and the MPS was observed using different cell-fixation protocols (fig. S3). Such colocalizationwas abolished by treatment with the actin de-polymerizing drugs latrunculin A (LatA) andcytochalasin D (CytoD) (Fig. 1, A and C, right; Band D, yellow), which is known to disrupt theMPS structure (10, 12). Together, these resultsindicate ligand-induced recruitment of CB1 andNCAM1 to the MPS. Coimmunoprecipitationexperiments also showed increased interac-tion of CB1 and NCAM1 with the MPS upon lig-and treatment (fig. S4), further supporting thisnotion.Next, we tested whether the recruitment of

CB1 and NCAM1 to the MPS is important forthe downstream signaling. It has been shownthat, upon ligand binding, both CB1 andNCAM1can activate the Raf-MEK-ERK signaling cascadethroughRTK transactivation in neurons (Fig. 2A)(7, 22). We thus measured the level of phospho-rylated (activated) ERK (pERK) using an immuno-fluorescence assay (25) to quantify the signalingstrength. Upon treatment with either the CB1agonist WIN or the NCAM1 Ab, we observed atransient increase in pERK signal in neurons,

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1Howard Hughes Medical Institute, Harvard University,Cambridge, MA 02138, USA. 2Department of Chemistry andChemical Biology, Harvard University, Cambridge, MA 02138,USA. 3Department of Physics, Harvard University,Cambridge, MA 02138, USA.*Corresponding author. Email: [email protected]

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followed by a decay to basal levels (Fig. 2, Band C, blue), consistent with previous reports(22, 26). Similar ERK activation was observedregardless of whether the analysis was done foraxons only or for all neurites (fig. S5). Pretreatingthe neurons with the CB1-specific antagonistSR141716 (SR;Ki = 2 nM), which has little activityon CB2 (27–29), abolished the observed WIN-induced pERK signal increase in neurons (Fig.2C, green), as well as the WIN-induced increasein CB1 and MPS colocalization (Fig. 1B, green).Disruption of the MPS structure by the LatA/

CytoD treatment completely abolished the tran-sient, ligand-induced ERK activation (Fig. 2C,yellow). Similar results were obtained by bII-spectrin knockdown (Fig. 2, C, red, and D), whichis also known to disrupt the MPS structure(10, 12). The cell-surface expression levels of CB1and NCAM1 did not decrease in bII-spectrinknockdown neurons (fig. S6), excluding thepossibility that the knockdown effect on ERKactivation was the result of a decrease in thesurface expression of CB1 or NCAM1. SimilarbII-spectrin–dependent, ligand-induced ERK ac-tivation was also observed using Western blotanalysis (fig. S7). Together, these results suggestthat theMPS plays an important role in enablingthe CB1- and NCAM1-mediated ERK signaling.Next, we investigated mechanistically how

CB1- or NCAM1-mediated ERK signaling is fa-cilitated by the MPS. To this end, we first exam-ined which step along the signaling pathwayis affected by MPS disruption. Both CB1- andNCAM1-mediated RTK transactivations activateprotein kinase C (PKC), which in turn activatesthe ERK cascade in neurons (Fig. 2A) (5, 7, 22).We added PDBu, a direct PKC activator, to neu-rons and measured the resulting pERK signal.The PDBu-induced increase in pERK signal wasnot diminished by bII-spectrin knockdown (fig.S8A), indicating that theMPS did not act directlyon the Raf-MEK-ERK cascade downstream ofPKC. Previous studies (5, 7, 21, 22) have suggestedthat CB1 and NCAM1 can transactivate two RTKtypes in neurons, tropomyosin receptor kinase B(TrkB) and fibroblast growth factor receptors(FGFRs). Indeed, the addition of TrkB and FGFRinhibitors, and likewise the knockdown of TrkBand FGFR1, strongly suppressed the increase inpERK signal induced by WIN or NCAM1 Ab (fig.S8, B and C). These results indicate that the ERKsignaling in neurons induced by CB1 andNCAM1ligands was primarily through transactivation ofthe two RTKs, TrkB and FGFR. To test whetherMPS facilitates CB1- and NCAM1-mediated trans-activation of these two RTKs or events down-streamof TrkB and FGFR activation, we examinedwhether MPS disruption inhibits the TrkB andFGFR activation induced directly by their owncognate ligands, brain-derived neurotrophic fac-tor (BDNF) for TrkB and basic fibroblast growthfactor (bFGF) for FGFR. The pERK signal increaseinduced by BDNF or bFGF remained quantita-tively similar in bII-spectrin knockdown neuronsas compared with wild type neurons (fig. S8, Dand E), suggesting that the MPS does not actdownstream of these RTKs but likely affects

their transactivation by CB1 and NCAM1. Sup-porting this notion, using Western blot analy-sis, we observed activation (phosphorylation)of TrkB and FGFR upon addition of the CB1ligand WIN, as well as activation of FGFR by

the NCAM1 Ab treatment, both in a bII-spectrin–dependent manner (Fig. 2, E and F).Next, we used STORM to examine the spatial

relationship of the two RTKs, TrkB and FGFR1,to the MPS, as well as to the RTK transactivators


Fig. 1. CB1 and NCAM1 are recruited to the MPS upon cognate ligand binding. (A) Two-colorSTORM images of bII-spectrin (green) and CB1 (magenta) in the axons of untreated neurons(left, “−WIN”), neurons treated with the CB1 agonist WIN (middle, “+WIN”), and neurons pretreatedwith LatA and CytoD to disrupt the MPS before addition of WIN (right, “+WIN, +LatA/CytoD”).1D projection traces of bII-spectrin (green) and CB1 (magenta) signals along the axon are shownat the bottom. bII-spectrin was visualized by immunostaining with an antibody against theC terminus of bII-spectrin. CB1 was visualized by immunostaining with CB1 antibody. (B) Left:Average 1D cross-correlation functions between the distributions of CB1 and bII-spectrin from manyCB1-positive axon segments for the three conditions described in (A), as well as for neuronspretreated with the CB1 antagonist SR before addition of WIN (“+WIN, +SR”). Right: Average 1Dcross-correlation amplitudes, defined as the difference between the average of the peaks at ±190 nmand the average of the valleys at ±95 nm and ±285 nm of the average 1D cross-correlation functions.**P < 0.01; actual P values (from left to right): 4.4 × 10−3, 1.6 × 10−3, and 8.7 × 10−3 (unpairedStudent’s t test). (C and D) Same as (A) and (B), but for neurons treated with NCAM1 antibody(NCAM1 Ab) instead of WIN. Neurons were preincubated with NCAM1 Ab at 4°C to allow antibodybinding in both “−NCAM1 Ab” and “+NCAM1 Ab” conditions. NCAM1 Ab treatment (“+NCAM1 Ab”)was achieved by a temperature increase to stimulate signaling (see supplementary materials andmethods), whereas the temperature increase step was skipped in the “−NCAM1 Ab” condition toprevent signaling, as previously described (26). NCAM1 was visualized through immunostainingwith the NCAM1 antibody. **P < 0.01, ***P < 0.001; actual P values (from left to right): 2.1 × 10−3 and5.8 × 10−4 (unpaired Student’s t test). Data in bar graphs are mean ± SEM (n = 3 biologicalreplicates; 100 to 200 axonal regions were examined per condition). Scale bars: 1 mm.

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CB1 and NCAM1. Upon addition of WIN, bothRTKs displayed a substantial increase in colocal-ization with the MPS, which was reflected by athree- to fivefold increase in 1D cross-correlationamplitudes, and disruption of the MPS by LatA/CytoD treatment abolished these colocalizations(Fig. 3A). Co-imaging of CB1 with TrkB or FGFR1showed little colocalization between these mole-cules before addition ofWIN, whereas the ampli-tude and periodicity of the 1D cross-correlationfunctions increased dramatically after WIN ad-dition (Fig. 3B), suggesting ligand-induced co-localizations between CB1 and the two RTKs.MPS disruption by LatA/CytoD treatment orbII-spectrin knockdown completely eliminatedthis ligand-induced colocalization (Fig. 3B).One potential caveat of this 1D analysis is thatthe increase in the 1D cross-correlation mayonly be a reflection of the recruitment of bothCB1 and RTKs to the MPS, and it may not neces-sarily indicate an enhanced spatial proximity

between CB1 and RTKs themselves. Thus, weperformed 2D cross-correlation analysis betweenCB1 and the two RTKs (fig. S9A), as well asnearest-neighbor distance analysis betweenthese molecules (fig. S9B), to further probe theircolocalization. Both analyses showed that WINtreatment enhanced proximity between CB1 andthe two RTKs in an MPS-dependent manner.Similarly, NCAM1 Ab treatment also inducedcolocalization between NCAM1 and FGFR1 inan MPS-dependent manner (fig. S10). As furthersupport, coimmunoprecipitation experimentsshowed that the interaction between CB1 andthe two RTKs greatly increased upon WIN treat-ment, and likewise the interaction betweenNCAM1 and FGFR greatly increased uponNCAM1Ab treatment, both in an MPS-dependent man-ner (fig. S11).We further examined the spatial distributions

of Src-family tyrosine kinases, which are knownas important mediators of RTK transactivation

by GPCRs and CAMs (Fig. 2A) (4–7, 30). As ex-pected, preincubation with PP2, a specific Src-family kinase inhibitor, abolished the increasein pERK signal induced by CB1 and NCAM1 li-gands in neurons (fig. S12). Notably, Src, a knownmediator for GPCR-mediated RTK transactiva-tion (4, 6), also became substantially more co-localized with the MPS upon WIN treatment,and this colocalization was abolished by MPSdisruption (Fig. 3C). To test whether this recruit-ment of Src to the MPS depends on Src activity,we generated three Src mutants with differentlevels of kinase activities (SrcAct, SrcSH2eng, andSrcSH2-3eng) by introducing mutations that pro-mote the open (active) or closed (inactive) con-formation of Src (Fig. 3D) (31). The degree ofcolocalization between the MPS and these Srcmutants scaled with their kinase activity in theabsence of WIN, and the effect of WIN treat-ment on the Src-MPS colocalization was sub-stantially reduced for the constitutively active


Fig. 2. MPS disruption abolishes RTK transactivation and downstreamERK signaling. (A) Left: Diagrams showing direct RTK activation (top)and RTK transactivation (bottom). RTK can be either activated directlyby binding of their cognate ligands or transactivated by other trans-membrane proteins, such as CB1 (upon binding of CB1 ligand) andNCAM1 (upon binding of NCAM1 Ab). Right: Diagram showing the ERKsignaling cascade downstream of RTK. PLC, phospholipase C; MEK,mitogen-activated protein kinase kinase. (B) Top: Immunofluorescenceimages of pERK in wild-type (WT) untreated neurons (left) and WTneurons treated with WIN for 10 min (right). WIN treatment was initiatedby addition of WIN at 37°C. Bottom: Same as top, but for treatment withNCAM1 Ab. Neurons were preincubated with NCAM1 Ab at 4°C to allowantibody binding in both “−NCAM1 Ab” and “+NCAM1 Ab” conditions,and NCAM1 Ab treatment (“+NCAM1 Ab”) was then initiated by atemperature increase to 37°C, whereas this temperature increase step waseliminated in the “−NCAM1 Ab” condition to prevent signaling. Scale bar:

25 mm. (C) Time courses of ERK activation upon WIN addition (left) or uponNCAM1 Ab treatment (right) for WTneurons (blue), WTneurons pretreatedwith the CB1 antagonist SR (green, closed symbols: 1 mM SR; green, opensymbols: 100 nM SR), WTneurons pretreated with LatA and CytoD (yellow),and bII-spectrin knockdown (KD) neurons (red). bII-spectrin KD wasinduced by adenovirus expressing bII-spectrin shRNA (fig. S6A). Dataare mean ± SEM (n = 3 biological replicates; 20 to 30 imaged regionswere examined per condition). (D) Same as (B), but for bII-spectrin KDneurons instead of WT neurons. (E) Western blot analysis for phosphoryl-ated (activated) TrkB (pTrkB) and total TrkB in whole-cell lysates from WTneurons (top) and bII-spectrin KD neurons (bottom) before and 10 minafter WIN treatment. (F) Western blot analysis for phosphorylated(activated) FGFR (pFGFR) and total FGFR in whole-cell lysates from WTneurons (top) and bII-spectrin KD neurons (bottom) before and 10 minafter the initiation of WIN or NCAM1 Ab treatment. Western blots arerepresentative examples from two independent biological replicates.


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mutant SrcAct, exhibiting only a 48% (insteadof a three to fourfold) increase in 1D cross-correlation amplitude, and the WIN-inducedincrease was completely abolished for the twoinactive Src mutants (SrcSH2eng and SrcSH2-3eng)(Fig. 3D and fig. S13). Likewise, Fyn, the Src-family kinase mediating NCAM1-induced RTKtransactivation (30), also exhibited enhancedcolocalization with the MPS upon NCAM1 Abtreatment (fig. S14).The above results suggest that the MPS acts

as a signaling platform that brings CB1, NCAM1,RTKs, and Src-family kinases into proximity toenable RTK transactivation by CB1 and NCAM1.Next, we investigated whether RTK transacti-vation and the downstream ERK signaling inturn have any effect on the MPS. After WIN orNCAM1 Ab treatment, the MPS was degradedgradually over time (Fig. 4, A and B, and fig. S15,A and B). Preincubation with the CB1-antagonist

SR blocked the WIN-induced MPS degradation(Fig. 4, A and C). Preincubation with U0126,an inhibitor (median inhibitory concentrationIC50 = 60 to 70 nM) ofMEK, the kinase upstreamof ERK (Fig. 2A), also protected the MPS fromdegradation (Fig. 4, A and C, and fig. S15, A andC), indicating that the MPS degradation was aresult of the ERK signaling. Brain spectrin is thesubstrate of the calpain protease (32) and RTK-induced ERK signaling activates calpain-2 (33),raising the possibility that the observed MPSdegradationmay result from cleavage by calpain.Indeed, we found that inhibiting calpain ac-tivity with an inhibitor MDL-28170 (MDL; Ki =8 nM) or short hairpin RNA (shRNA) againstcalpain-2 prevented signaling-induced MPS deg-radation (Fig. 4, A and C, and fig. S15, A and C).With calpain or MEK activities inhibited andhence the MPS retained, the ligand-inducedcolocalization between signaling molecules and

the MPS was also maintained (fig. S16). Theseresults indicate that CB1- and NCAM1-medi-ated RTK transactivation turns on an ERK-dependent calpain pathway that degrades theMPS. This degradationwas reversible: theMPSstructure reassembled within a few hoursafter ligand removal (fig. S17).Because the MPS structure brings RTKs, RTK

transactivators, and Src-family kinases into prox-imity to facilitate RTK transactivation, we envi-sioned that theMPS degradation could provide anegative feedback to reduce the strength of ERKsignaling induced byRTK transactivation. Indeed,preventingMPSdegradation by the calpain inhib-itorMDLor calpain-2 knockdown increased ERKsignaling induced by both WIN and NCAM1 Ab(Fig. 4, D andE, and fig. S15, D andE), supportingthe existence of such a negative feedback loop.Ligand binding could also induce receptor

endocytosis, a process known to positively or


Fig. 3. The MPS functions as a signaling platform that brings RTKs,RTK transactivators, and Src kinases into proximity. (A) Left panels:Two-color STORM images of bII-spectrin (green) and TrkB (magenta)(top panels) and of bII-spectrin (green) and FGFR1 (magenta) (bottompanels) in CB1-positive axons of untreated neurons (left, “−WIN”), neuronstreated with WIN (middle, “+WIN”), and neurons pretreated with LatAand CytoD before WIN addition (right, “+WIN, +LatA/CytoD”). Rightpanels: Average 1D cross-correlation functions and 1D cross-correlationamplitudes between the distribution of bII-spectrin and the distributionsof RTKs (TrkB or FGFR1) from many CB1-positive axons for the threeconditions. **P < 0.01, *** P < 0.001; actual P values (from left to right):2.3 × 10−3, 5.6 × 10−4, 2.8 × 10−3, and 1.2 × 10−3 (unpaired Student’st test). (B) Similar to (A), but for co-imaging of CB1 instead of bII-spectrin,with the two RTKs. The results for the +WIN condition in bII-spectrin KDneurons are additionally shown in green (+WIN, bII-spectrin KD). **P < 0.01,***P < 0.001; actual P values (from left to right): 6.7 × 10−4, 7.3 × 10−4, 3.8 ×10−4, 5.2 × 10−4, 6.8 × 10−4, and 1.0 × 10−3 (unpaired Student’s t test).

(C) Similar to (A), but for co-imaging of Src with bII-spectrin. ***P < 0.001;actual P values (from left to right): 6.4 × 10−4, 2.2 × 10−4 (unpairedStudent’s t test). (D) Left: Diagram showing the intramolecular domainorganizations of the three Src variants. SrcAct is a constitutively activemutant, and gray dots in SrcAct indicate the sites modified to disrupt theauto-inhibiting intramolecular domain interactions. SrcSH2eng and SrcSH2-3eng

are inactive mutants, and red dots in these mutants indicate the sites modifiedto facilitate the auto-inhibiting intramolecular domain interactions. The red“P” represents the major phosphorylation site of activated Src. Right: Average1D cross-correlation amplitudes between the distributions of bII-spectrin andthe three Src mutants. *P < 0.1; n.s., not significant (P > 0.1); actual P values(from left to right): 1.1 × 10−2, 0.74, and 0.49 (unpaired Student’s t test). Data inbar graphs are mean ± SEM (n = 3 biological replicates; 100 to 200 axonalregions were examined per condition). bII-spectrin and CB1 were visualized asdescribed in Fig. 1; TrkB, FGFR1, and Src variants were visualized by moderateexpression of GFP-tagged TrkB, FGFR1, or Src variants through low-titerlentiviral transfection and detection through GFP antibody. Scale bars: 1 mm.


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negatively regulate various signaling pathways(34, 35). In addition to providing a platform forrecruiting signaling molecules, could the MPSinfluence the endocytosis of these molecules,which in turn also impacts ERK signaling? Toexamine whether the MPS affects CB1 endocyto-sis, we examined how the rate of ligand-inducedCB1 endocytosis changed under two MPS pertur-bation conditions: (i) bII-spectrin knockdown,which disrupts the MPS, and (ii) MDL treat-ment, which protects the MPS from signaling-induced degradation. bII-spectrin knockdown ledto a substantial increase in WIN-induced CB1endocytosis, whereas MDL treatment inhibitedWIN-inducedCB1 endocytosis (fig. S18), indicatingthat the MPS structure can repress endocytosis.To estimate how much this effect of MPS on

CB1 endocytosis would contribute to the observednegative feedback on signaling, we examinedthe ERK signaling in clathrin heavy chain (CHC)knockdownneurons, as CB1 endocytosis is knownto occur in a clathrin-dependent manner in neu-

rons (36). Although CHC knockdown inhibitedCB1 endocytosis at least as strongly as MDLtreatment did (fig. S18), it did not have an ap-preciable effect on ERK signaling induced byWIN (fig. S19), suggesting that the enhancementin ERK signaling observed under calpain inhi-bition (Fig. 4E) was not primarily the resultof inhibition of endocytosis. Hence, for CB1-mediated RTK transactivation, the negativefeedback caused by the signaling-induced MPSdegradation was likely a direct effect of theloss of the structural platform for signaling-molecule recruitment. Whether the same istrue for NCAM1-mediated RTK transactivationremains to be investigated.Taken together, our results suggest that the

MPS serves as a structural platform for bringingsignaling molecules, including GPCRs, CAMs,RTKs, and Src-family kinases, into proximity toenable GPCR- and CAM-mediated transactiva-tion of RTKs and the downstream ERK signaling(Fig. 4F). These signalingmolecules were recruited

to sites near the center of the spectrin tetramer,where the adaptor protein ankyrin binds. Bothspectrin and ankyrin are large scaffolding pro-teins containingmultiple domains, which couldprovide multiple binding sites for signaling mol-ecules and bring them into proximity to formsignaling complexes. It has been shown thatGPCR-signaling components, CAMs, and theSrc kinase can interact with specific moleculardomains of spectrin or ankyrin (13, 37, 38). It isalso possible that some of these signaling mol-ecules are first recruited to the MPS to increasetheir local concentration, which in turn facilitatesthe recruitment of other signaling moleculesthrough multivalent interactions. In support of thisview, optogenetically induced self-oligomerizationof the SH2 domain, a common protein domaininmany signalingmolecules, including Src andFyn kinases, has been shown to facilitate com-plex formation between RTKs and SH2, therebyactivating RTKs (39). Our results raise the in-teresting possibility that MPS may facilitate


Fig. 4. ERK signaling causes disassembly of the MPS structure,providing a negative feedback for signaling. (A) 3D STORM imagesof bII-spectrin in CB1-positive axons of untreated neurons, neurons treatedwith WIN for 1 hour in the absence and presence of SR (a CB1 antagonist),U0126 (a MEK inhibitor), MDL (a pan-calpain inhibitor), and calpain-2 KDneurons treated with WIN for 1 hour. Calpain-2 KD was induced by adenovirusexpressing calpain-2 shRNA (fig. S15A). Scale bars: 1 mm. Colored scale barindicates the z-coordinate information. (B) Average 1D auto-correlationamplitude of the bII-spectrin distribution, indicating the degree of theperiodicity in the MPS, calculated from many axon segments at differenttime points after addition of WIN. **P < 0.01, ***P < 0.001; actual P values(from left to right): 1.6 × 10−3 and 5.9 × 10−4 (unpaired Student’s t test).(C) Average 1D auto-correlation amplitudes for the six conditions described in(A). **P < 0.01, ***P < 0.001; actual P values (from left to right): 8.0 × 10−4,9.2 × 10−4, 6.3 × 10−4, and 3.5 × 10−3 (unpaired Student’s t test). Data in (B)and (C) are mean ± SEM (n = 3 biological replicates; 50 to 100 axonal regionswere examined per condition). (D) Immunofluorescence images of pERK inneurons pretreated with MDL, before (left) and after (right) WIN treatment.

Scale bar: 25 mm. (E) Time courses of ERK activation upon addition ofWIN for control neurons (blue), neurons pretreated with MDL (green), andcalpain-2 KD neurons (red). The curve for control neurons is reproducedfrom Fig. 2C. Data are mean ± SEM (n = 3 biological replicates; 20 to30 imaged regions were examined per condition). (F) Schematic showing theMPS functioning as a dynamically regulated platform to recruit signalingmolecules and enable RTK transactivation. Upon ligand binding to RTKtransactivators (CB1 and NCAM1), these transactivators, RTKs (TrkB andFGFR), and related Src-family tyrosine kinases (Src and Fyn) are recruited tothe MPS and brought into proximity of each other, enabling RTK trans-activation and downstream ERK signaling. ERK activation in turn inducesMPS degradation in a calpain-dependent manner, providing a negativefeedback loop to attenuate the strength of ERK signaling. MPS degradationalso leads to an increase in receptor endocytosis. Because the ligand-inducedincrease in the pERK signal was followed by a decay under both controlconditions and conditions where the MPS degradation was inhibited byinhibiting calpain activity (Fig. 4E and fig. S15E), other MPS-independentattenuation mechanisms may contribute to the observed pERK signal decay.


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multivalent-interaction–mediated phase sep-aration of signaling molecules. Our observa-tions of the recruitment of signaling moleculesto the MPS upon ligand stimulation suggest acritical role of the MPS in signaling. Indeed,disruption of the MPS abolished ligand-inducedRTK transactivation by CB1 or NCAM1 and thedownstream ERK signaling. Furthermore, wedemonstrated that the ERK signaling inducedreversible MPS degradation in a calpain-protease–dependent manner, which in turn caused anattenuation of signaling strength, providing anegative feedback loop (Fig. 4F). In addition,we observed that MPS can regulate endocytosis,potentially providing another mechanism for sig-naling regulation. Overall, our results demon-strate that the MPS functions as a dynamicallyregulated structural platform for GPCR- andCAM-mediated RTK transactivation and sig-naling, providing a mechanism for regulatingsignal transduction in neurons.

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ACKNOWLEDGMENTS

We thank H. Babcock and Y. Fu for helping with two-color STORMimaging setup construction and data analysis, K. Xu for providingthe software for processing the two-color STORM imaging data,and M. Rasband for providing the adenoviruses expressing shRNAagainst bII-spectrin. Funding: This work is supported in part bythe National Institutes of Health. R.Z. is an HHMI Fellow of the LifeSciences Research Foundation. X.Z. is a Howard Hughes MedicalInstitute investigator. Competing interests: The authors declareno competing interests. Author contributions: R.Z. and X.Z.designed the experiments. R.Z. and C.X. performed the experiments.R.Z. and B.H. performed data analysis. R.Z. and X.Z. wrotethe manuscript with input from B.H. and C.X. Data and materialsavailability: All data are available in the manuscript orsupplementary materials.

SUPPLEMENTARY MATERIALS

science.sciencemag.org/content/365/6456/929/suppl/DC1Materials and MethodsFigs. S1 to S19References (40–48)

8 January 2019; accepted 2 August 201910.1126/science.aaw5937



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neuronsMembrane-associated periodic skeleton is a signaling platform for RTK transactivation in

Ruobo Zhou, Boran Han, Chenglong Xia and Xiaowei Zhuang

DOI: 10.1126/science.aaw5937 (6456), 929-934.365Science

, this issue p. 929Sciencecoordinates signal transduction in neurons.downstream signaling in turn leads to degradation of the MPS. Thus, the MPS is a dynamically regulated platform thatpathways leads to transactivation of RTK, which initiates intracellular signaling. In a negative feedback loop, the spectrin, and related molecules in the axons and dendrites of neurons. The colocalization of signaling proteins in differentcolocalization of signaling proteins on the membrane-associated periodic skeleton (MPS) that is formed by actin,

used super-resolution imaging to visualizeet al.whose activation can depend on other signaling pathways. Zhou In neurons, many cellular processes are regulated by receptor tyrosine kinases (RTKs), cell surface receptors

A dynamic signaling scaffold

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REFERENCES

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SIGNAL TRANSDUCTION Membrane-associated periodic …Fig. 1. CB1 and NCAM1 are recruited to the MPS upon cognate ligand binding. (A) Two-color STORM images of bII-spectrin (green) and