Myosin motors at neuronal synapses

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Synaptic plasticity is believed to be the cellular basisof learning and memory 1 and relies on at least two keymechanisms: the remodelling of synaptic circuits by theformation of new synapses and/or the elimination of oldsynapses; and the selective strengthening and weakeningof subsets of existing synapses2. Strengthening and weak-ening of individual synapses involves adaptive changesat presynaptic active zones that control vesicle fusion andneurotransmitter release probability 3. Moreover, post-synaptic dendritic spines undergo activity-controlledgrowth or shrinkage4 with dynamic regulation of syn-aptic neurotransmitter receptor numbers through eithersurface membrane diffusion or active cytoskeleton-based transport5.

Synaptic function, synaptic plasticity and spine mor-phology depend on the actin cytoskeleton as well as otherfactors6–9. Actin filaments are abundant near synapses(FIG. 1a) and consist of two helically intertwined strandsof polymerized actin monomers. The actin filament has

inherent structural polarity with a barbed plus end anda pointed minus end. Both the dendritic spine head andthe spine neck that connects the spine head to the den-drite shaft contain a mix of linear and branched actin fila-ments of non-uniform orientation8,10 (FIG. 1a). Althoughthe precise functional organization of actin filaments inspines remains unknown9, there is evidence that, in spineheads, the fast-growing barbed ends of the filamentsare oriented predominantly towards the surface8,10–12. Inspine necks, actin filaments oriented with their barbedends towards the distal end of the spine seem to domi-nate, although barbed ends have also been observed at thebase of the spine, suggesting that spine necks can contain

actin filaments of mixed polarity 8,10,11. As at the leadingedge of migrating fibroblasts, actin filaments in dendriticspines are highly dynamic — they undergo continuouspolymerization and depolymerization8. Reorganizationand enhanced polymerization of the actin cytoskel-eton occurs within seconds of the induction of long-termpotentiation (LTP) and is required for the spine growththat accompanies LTP6–9,12,13. The actin cytoskeleton alsosupports submembraneous receptor trafficking and influ-ences the diffusion rate of receptors within the plasmamembrane6,14. Presynaptically, opposing roles for actinfilaments have been observed. Actin appears either tofacilitate the docking of vesicles of the readily releasablepool or to act as a barrier that prevents the fusion ofthese vesicles with the plasma membrane, and theseactions might depend on synaptic activity 6. At leastsome aspects of synaptic vesicle recycling also dependon actin filaments6.

Myosins are mechanoenzymes that interact with

actin filaments and hydrolyse ATP to generate move-ment and force15–25 (BOX 1). This enables myosins to pro-pel the sliding of actin filaments, to produce tension onactin filaments and to walk along these filaments. As aresult, myosins can regulate the structure and dynam-ics of the actin cytoskeleton and affect the localizationand transport of cellular components. The differentmyosins are grouped into classes on the basis of theirmotor domains. There are 35 known classes of myo-sin, and humans have 40 myosin genes that fall into 13classes (I, II, III, V, VI, VII, IX, X, XV, XVI, XVIII, XIXand XXXV; see the CyMoBase website)26. Here, we pro-

vide an overview of how neuronal myosins participate in

Active zones

These are presynaptic

specializations at which

docking, priming and fusion of

synaptic vesicles occur. They

organize neurotransmitter

release and are crucial forpresynaptic plasticity.

Dendritic spines

Small, actin-rich protrusions

from a neuronal dendrite.

Spines are postsynaptic

compartments that receive

input from presynaptic

terminals.

Long-term potentiation

(LTP). An activity-dependent,

long-lasting increase in the

strength of a neuronal synapse.

Myosin motors at neuronal synapses:drivers of membrane transport andactin dynamicsMatthias Kneussel and Wolfgang Wagner

Abstract | Myosins are a large family of actin-based cytoskeletal motors that use energy

derived from ATP hydrolysis to generate movement and force. Myosins of classes II, V and VI

have specific pre- and postsynaptic roles that are required for synapse function. They alsofacilitate several forms of synaptic plasticity. Interestingly, the myosins of these classes differ

markedly in important aspects of their molecular mechanisms of function. Accordingly, their

major roles at synapses are diverse and include the regulation of actin cytoskeleton dynamics

in dendritic spines and powering of synaptic cargo transport.

University Medical Center

Hamburg-Eppendorf,

Center for Molecular

Neurobiology (ZMNH)

Falkenried 94, 20251

Hamburg, Germany.

e-mails: wolfgang.wagner@

zmnh.uni-hamburg.de;

[email protected]

hamburg.de

doi:10.1038/nrn3445

Published online 13 March

2013

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© 2013 Macmillan Publishers Limited. All rights reserved

http://www.cymobase.org/http://www.cymobase.org/http://www.cymobase.org/


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Low myosin–actinaffinity

High myosin–actinaffinity

Power stroke

Pi

Lever armdisplacement

ADP.Pi

+

ADP

ATPATP hydrolysislever arm recocking

+

++

ADP.Pi

+

ATP

ADP

1

2

3

4

Myosin motor domain

A conserved, globular domain

that binds actin, hydrolyses

ATP and produces force. It is

also known as the myosin head.

RAB GTPases

A family of small GTPases that

are anchored in diverse

membranes via geranylgeranyl

moieties. When bound to GTP,

RAB GTPases bind effector

proteins to recruit them to

membranes.

Lever arm

A rod-like element attached to

the myosin motor domain that

acts as a lever that amplifies the

conformational changes

generated in the motor domain.

Both the coiled-coil region and the globular taildomain (GTD) of vertebrate class V myosin are involvedin cargo binding (FIG. 2b). To attach to its different cargoes,the myosin uses organelle-specific receptors that often

comprise RAB GTPases that are inserted in the organellemembrane by geranylgeranyl moieties20,41 (FIG. 3b). Whenmyosin V is not bound to cargo, it folds into an inactiveconformation in which the GTD interacts with the motordomain and inhibits its ATPase activity 42,43, preventingfutile ATP hydrolysis by cargo-free myosin. Binding ofa cargo receptor to the GTD leads to myosin V unfold-ing, thereby activating the myosin’s ATPase activity 44.Similarly, Ca2+ unfolds myosin V, but it also induces theloss of calmodulin light chains from the lever arm. This lossseverely compromises the myosin’s ability to walk alongactin, probably because the myosin’s lever arm becomesfloppy in the absence of bound calmodulin light chains45.

Class VI myosin. Myosin VI is the only known myosinthat walks towards the actin filament’s pointed end21,22,46.This reversal of direction is due to a distinctive inserttermed ‘reverse gear’ that forms part of the myosin VIlever arm (FIG. 2b) and repositions the lever comparedwith other myosins21,22. The reverse gear insert and thelever arm sequence that follows bind one calmodulinlight chain each21,22. The C-terminal end of the myo-sin VI heavy chain features a globular cargo-bindingdomain (CBD) that allows the myosin to associatewith diverse membranes using different linker proteinstermed cargo adaptors23 (FIG. 2b). Alternative splic-ing can add extra residues at two sites in the CBD 47.Unlike class II and class V myosins, purified myosin VIshows very little heavy chain dimerization48–50. However,binding of cargo adaptors links together two CBDs andinduces myosin VI heavy chain dimerization51,52 (FIG. 3c).Dimerized myosin VI senses tension and behaves differ-ently depending on the load that acts on it. Under a lowload, myosin VI moves processively towards the pointedend of an actin filament (BOX 2), whereas under a high

load, the myosin acts as a cytoskeletal anchor that stablylinks cargo to actin21,22,53.

Myosin VI acts at several stages of membrane traf-ficking. It is involved in clathrin-mediated endocytosisand becomes recruited to endocytic sites through itsadaptor disabled homologue 2 (DAB2)23,47,54,55 (FIG. 3c).Myosin VI also associates with uncoated endocytic

vesicles (UEVs) through GIPC1 and is important forthe motility of these vesicles56–58. By binding optineurin,myosin VI becomes recruited to the Golgi apparatus59.Both myosin VI and optineurin are needed for normalGolgi morphology 59, for vesicle delivery to the leadingedge60, for vesicle fusion with the plasma membrane 61 and for the delivery of endosomal cargo to autophago-somes62. Endocytic trafficking of a number of surfacemolecules including the cystic fibrosis transmembraneconductance regulator (CFTR) depends on myosin VI63.Myosin VI also promotes E-cadherin-based cell-to-cellcontacts64,65 and the maintenance of actin-based protru-sions of inner ear hair cells termed stereocilia66,67.

Little is known about whether specific cellular tasksrequire myosin VI to dimerize, and whether they requirethe myosin to act as a load-induced anchor or processivetransporter. In hair cells, the myosin might maintain stere-ocilia structure by functioning as an anchor that generatestension between the plasma membrane and the underly-ing actin cytoskeleton22,66. By contrast, when associated

with UEVs, the myosin might act as a processive trans-porter21,58. Further investigations are needed to defineprecisely how myosin VI performs its different tasks.

The mammalian genome contains only onemyosin VI heavy chain gene ( MYO6 ), and no othermammalian myosin contains the reverse gear insert.Thus, myosin VI might be the only reverse directionmyosin in mice and humans. It is therefore surprising that MYO6 -null mutations are not lethal67,68. Instead, owingto inner ear hair cell defects, mice such as Snell’s waltzermice that carry MYO6 mutations develop deafness andshow a prominent circling behaviour indicating vestibulardysfunction67,69 (BOX 3).

Box 1 | The conserved actomyosin mechanochemical cycle

The myosin motor domain (head) is the site of ATP hydrolysis and is responsible for actin

binding. Typically, the presence of actin activates the myosin’s ATPase activity. During

the actomyosin mechanochemical cycle, ATP hydrolysis is tightly coupled to

conformational changes in the motor domain that lead to the power stroke (a swing of

the lever arm). The lever arm is a rod-like element attached to the motor domain that

amplifies the conformational changes generated in the motor domain. The magnitude

of lever arm displacement depends on the length of the lever arm and takes placetowards the barbed plus end of the actin filament in the case of myosin II and myosin V

(shown) and towards the pointed minus end in case of myosin VI (not shown). Usually,

calmodulin light chains or calmodulin-related light chains associate with and stabilize

the lever. When ATP (1) or the hydrolysis products ADP and Pi (2) are bound to the motor

(shown in blue), the myosin has low affinity for its actin track (shown in dark red). With Pi

release, the myosin transitions into a state of high actin affinity and produces the power

stroke (3). ADP is released (4) and, with subsequent binding of ATP, the motor transitions

back into the state of weak actin affinity (1). The motor ‘recocks’ its lever arm, allowing

another ATP hydrolysis cycle to take place. The coupling of ATP hydrolysis with changes

in actin affinity and swinging of the lever arm appears to be conserved in all myosins.

However, different myosins such as myosin II, myosin V and myosin VI have undergone

kinetic and structural adaptations that allow them to fulfil different functions, such as

crosslinking of actin filaments, transport of cargo, anchoring of organelles to the actin

cytoskeleton or sensing of mechanical tension. Adaptations include variations in the

motor domain and the lever arm, differences in the ability of myosin heavy chains to di-or multimerize and distinct capacities of myosins to interact with cargo via their tail

domains15,24,25,155,156.

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a

b

Motor(head)

Lever(neck)

Tail

Proximal Distal

Myosin II Homodimer of

heavy chains

Myosin VIHeavy chain

monomer

Myosin VHomodimer of

heavy chains

Regulatory light chain

Essential light chain

N C

Calmodulinlight chain

Loops

Globular taildomain

DYNLL2

Cargo-binding

Cargo-bindingdomain

Reversegear

Cargo-binding,dimerization

Calmodulinlight chain

Postsynaptic density(PSD). An electron-dense

submembrane compartment in

dendritic spines. It is directly

opposed to the active zone and

harbours neurotransmitter

receptors, scaffold proteins

and signalling molecules.

Blebbistatin

An inhibitor that blocks the

ATPase activity of myosin II by

slowing down phosphate

release, thereby locking the

myosin motor domain in a

state of low actin affinity.

Synaptic roles of myosin II

All three isoforms of non-muscle myosin II (IIa, IIb andIIc) are found in the brain, with non-muscle myosin IIband myosin IIc being predominantly expressed in neuralcells, and non-muscle myosin IIa being mainly expressedin the vasculature70–74. Alternative splicing of MYH10 and MYH14 pre-mRNAs in the brain creates variantsof non-muscle myosin IIb and myosin IIc with distinctenzymatic properties75–79. Non-muscle myosin IIb hasbeen detected at both pre- and postsynaptic sites80–83.In mature hippocampal neurons, the myosin is foundin spines (as well as in other sites), where it localizes tothe spine neck and proximal spine head but also over-laps with the postsynaptic density (PSD) scaffoldingprotein PSD95 (REFS 10,82,83) (FIG. 1b). All three non-mus-cle myosin II isoforms co-fractionate with the PSD83–86.

Myosin II and dendritic spine morphology. Severalindependent studies have established that non-musclemyosin IIb regulates the morphology and dynamicsof dendritic spines of cultured hippocampal neurons.

The cell-permeable compound blebbistatin inhibitsmyosin II (including non-muscle and sarcomeric iso-forms) but not myosin I, myosin V or myosin X31,87,88;however, blebbistatin has also been found to influencemyosin II-independent processes89. Acute exposure ofhippocampal cultures to blebbistatin leads to transfor-mation of existing mushroom-headed spines into longer,more filopodia-like spines83. Moreover, both blebbistatinexposure and knockdown of non-muscle myosin IIb byshort hairpin RNA (shRNA) result in increased rates ofspine protrusion and retraction82 and in an increase inthe number of spines with protrusions emerging fromtheir heads83,90. Non-muscle myosin IIb is also requiredfor the maturation of spine heads into mushroom-likeheads after NMDA receptor (NMDAR) activation82.Finally, the morphology, size and subspine localizationof the PSD depend on non-muscle myosin IIb82.

Non-muscle myosin IIb can translocate actin fila-ments, but it can also crosslink and maintain tension onthem78,91. A MYH10 mutation (R709C) that separatesthese two functions provided insight into how the myo-sin acts in cells. The R907C mutation disrupts the ATPaseactivity of non-muscle myosin IIb and the myosin’sability to translocate actin, while leaving its capacity tocrosslink actin filaments intact30. Notably, this mutationand the homologous mutation in non-muscle myosin IIa(the MYH9 R702C mutation) lead to severe defects in

mice and humans, respectively 74,92 (BOX 3). Nevertheless,the MYH10 R709C mutation does not abolish themyosin’s ability to drive actomyosin ring contraction,indicating that the ability of non-muscle myosin IIb tomaintain tension by actin crosslinking is sufficient forcytokinesis30. Similarly, actin filament translocationby non-muscle myosin IIa does not seem to be neces-sary for focal adhesion maturation93. By contrast, actinfilament translocation mediated by non-muscle myo-sin IIb, and not solely the myosin-mediated crosslink-ing of actin filaments, seems to be required for spinemorphology, as overexpression of wild-type but not ofmutant R709C non-muscle myosin IIb accelerates spine

Figure 2 | Domain structure of synaptic myosins.

a | Representation of the myosin heavy chain primary

structure. The myosin heavy chain has an N-terminal motor

(head) domain that binds actin filaments, hydrolyses ATP

and generates force (BOX 1). The head is followed by a neck

region, which differs in length and contains binding sites forcalmodulin light chains or calmodulin-related light chains

and acts as a lever arm. The myosin tail comprises a

proximal region that often contains a dimerizing coiled-coil

sequence and a distal region, which can be globular or

non-helical. b | Molecular organization of myosin II, myosin

V and myosin VI18–23. Myosin II comprises two heavy chains

that homodimerize via an extended coiled-coil domain

(purple). Each heavy chain binds two calmodulin-related

light chains (green) — one essential light chain and one

regulatory light chain. The C terminus holds a short

non-helical region (red). Myosin V comprises a homodimer

of heavy chains, each of which has six IQ-motifs that bind to

calmodulin light chains or calmodulin-related light chains

(green). The myosin V heavy chains dimerize via a

coiled-coil region (purple) that is interrupted by loops. Inneuronal myosin Va, the dimeric light chain dynein light

chain 2 (DYNLL2; light blue) binds in the coiled-coil region.

Both the coiled-coil region and the globular tail domain

(red) are involved in cargo binding. Purified myosin VI is

predominantly monomeric and consists of a heavy chain

with two associated calmodulin light chains (green). The

more N-terminal calmodulin-binding site is located in the

‘reverse gear’ insert (dark blue). This insert allows myosin VI

to move towards the pointed end of an actin filament.

C-terminal to the second calmodulin-binding site is a

region comprising a three-helix bundle and a single α-helixdomain (purple). The cargo-binding domain (red) dimerizes

upon binding to cargo adaptors.

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+

RE

UEV

Bipolar myosin II filament

+

+

+

Myosin IIActin filament

contraction andcrosslinking

Myosin VOrganelle tethering

and transport

Myosin VIOrganelle anchoring

and motility, actinfilament

organization

a

b

+

DAB2 CCP

c

ER

+

+

Myosin Vb

RAB11-FIP2

RAB11

Myosin Va

Unknown organellereceptor

36 nm step size

+

TRKB

GIPC1

GluA1βSAP97

+

AMPA receptor

A tetrameric glutamate

receptor that mediates fast,

excitatory synaptic

transmission and that is

composed of diverse

combinations of the subunits

GluA1–GluA4.

maturation in culture82. Moreover, non-muscle myosinIIb-mediated spine maturation is probably regulated byRLC phosphorylation82. For example, increased amountsof diphosphorylated RLC are detected in spines afterNMDAR stimulation. Conversely, inhibition of theupstream kinase ROCK leads a decrease in diphospho-rylated RLC and an increase in spine length. This defectis reversed by the expression of an RLC mutant thatmimics the diphosphorylated form82. Thus, activation

of non-muscle myosin IIb by RLC phosphorylation andthe myosin’s ability to contract actin filaments seem to becrucial for myosin-driven formation and maintenance ofmature hippocampal spines82.

An enzymatically inactive splice isoform of non-muscle myosin IIb has a specific role in cerebellarPurkinje neurons76. Insertion of extra residues (spe-cifically, the B2 insert) by alternative splicing near theactin-binding region of MYH10 creates a non-musclemyosin IIb isoform with diminished actin-activatedATPase activity even when the RLCs are phosphoryl-ated75,79. This isoform does not translocate actin fila-ments in vitro, although it can bind actin75. Therefore,minifilaments consisting of this isoform might functionby crosslinking actin filaments and exerting tension75.Non-muscle myosin IIb containing the B2 insert is pre-dominantly expressed in Purkinje neurons79. Mice thatlack this isoform show reduced density of dendriticspines on Purkinje neuron dendrites, misorientationand decreased branching of Purkinje neuron dendritesand ectopic localization of Purkinje neuron cell bodies76.

Consistent with defective cerebellar function, these micehave motor coordination deficits76.

The mRNA of one of the sarcomeric myosin II heavychains, MYH7B, is also expressed in hippocampal neu-rons, albeit at very low levels90. A green fluorescent pro-tein-tagged version of this sarcomeric myosin distributesequally to the dendritic spines and to the dendrites ofcultured hippocampal neurons90. Its role appears to bedistinct from that of non-muscle myosin IIb90. For exam-ple, MYH7B knockdown primarily affects a subpopula-tion of spines and leads to more irregularly shaped spineheads with increased spine head area, whereas MYH10 knockdown leads to protrusions of increased length.Moreover, simultaneous knockdown of MYH10 and MYH7B has an additive effect on spines, suggesting thatthese myosins function in parallel to determine dendriticspine morphology 90.

Postsynaptic myosin II, synaptic transmission and LTPmaintenance. Consistent with the idea that myosin II isimportant for postsynaptic function, short-term blebbi-statin treatment causes clusters of the AMPA receptor (AMPAR) subunit GluA1 to become larger but to decreasein number, and MYH7B knockdown causes smaller andless intense GluA1 surface clusters83,90. Importantly,interfering with postsynaptic myosin II activity reducesexcitatory synaptic transmission. Blebbistatin (50 μM)

introduced through the recording pipette into postsyn-aptic CA1 neurons in acute hippocampal slices causesdepression of AMPAR-mediated excitatory postsynapticcurrents (EPSCs) that are evoked by Schaffer collateralstimulation83. Similarly, in cultured hippocampal neurons, MYH10 knockdown predominantly decreases the fre-quency of miniature EPSCs (mEPSCs), whereas MYH7B knockdown reduces their amplitude90, again suggestingthat the roles of non-muscle myosin IIb and sarcomericmyosin differ. Treatment with blebbistatin reduces boththe amplitude and the frequency of mEPSCs83, which isconsistent with the idea that blebbistatin can inhibit bothmyosins.

Figure 3 | Mechanisms of function of synaptic myosins. a | Myosin II molecules

associate in an antiparallel fashion to form bipolar ‘thick filaments’ (sarcomeric myosin II)

or ‘minifilaments’ (non-muscle myosin II). Translocation (indicated by arrows) of the

myosins in bipolar assemblies towards the barbed plus ends of antiparallel actin

filaments leads to contraction of the actin filament array. The ability of non-muscle

myosin IIb to drive actin translocation appears to be important for dendritic spine

morphogenesis but is dispensable for cytokinesis (see text). b | Myosin V can associate

with different organelles and moves towards the barbed end of an actin filament(indicated by the arrow). Organellar cargoes in dendritic spines include recycling

endosomes (REs) for myosin Vb and the endoplasmic reticulum (ER) for myosin Va.

Organelle-specific receptors interact with the myosin’s globular tail domain (for

example, RAB11 and RAB11-FIP2) or with the coiled-coil region (purple) to recruit

myosin V to an organelle. c | Myosin VI dimerizes upon binding of cargo adaptors to its

cargo-binding domain (red). Parts of the proximal tail (purple) might contribute to heavy

chain dimerization. Dimerized myosin VI walks processively towards the pointed minus

end of actin (indicated by arrows). Myosin VI associates with distinct cargoes by using

specific receptors; for example, GIPC1, a cargo adaptor that interacts with the

brain-derived neurotrophic factor–receptor tyrosine kinase TRKB (tropomyosin-related

kinase B) complex. GIPC1 bridges myosin VI to uncoated endocytic vesicles (UEVs) in

non-neuronal cells. Thus, myosin VI might be linked to UEVs via GIPC1 and TRKB. Theβ-splice isoform of the postsynaptic scaffolding protein synapse-associated protein 97 (βSAP97) binds myosin VI and the AMPA receptor subunit GluA1. Myosin VI also interacts

with the clathrin adaptor disabled homologue 2 (DAB2), which can mediate the myosin’sassociation with clathrin-coated pits (CCPs).

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In addition to its role in spine morphology andbasal synapse function76,82,83,90, non-muscle myosin IIbis required postsynaptically for maintaining synapticplasticity 94. To analyse synaptic plasticity, CA1 neuronsof live rats were transduced with recombinant adeno-associated virus (rAAV) particles carrying MYH10 shRNA constructs. In acute hippocampal slices fromthese rats, the maintenance, but not the initial induc-tion, of LTP was disrupted at Schaffer collateral–CA1synapses94. Inhibition of myosin II function by bath-application of 10 μM blebbistatin had an equivalenteffect without altering basal synaptic transmission,dendritic spine morphology, paired-pulse facilitation

or the frequency of mEPSCs94. This is in apparent con-flict with previous findings that convincingly linkednon-muscle myosin IIb to basal synaptic function andspine morphology 76,82,83,90,95. However, whereas 10 μMblebbistatin is sufficient to block LTP maintenance94,five- to tenfold higher concentrations of blebbistatinwere used to reveal the depression of basal transmis-sion and defects in spine morphology 82,83. Althoughother experimental differences (such as intracellular 83

versus field recordings94 and knockdown in dissociatedculture90 versus in live animals94) could contribute tothis discrepancy, it is likely that maintenance of synap-tic plasticity is more sensitive to myosin II inhibition

than spine morphology or basal transmission but thatnon-muscle myosin IIb is important for both basal andactivity-induced processes.

Insight into how non-muscle myosin IIb maintains hip-pocampal LTP has been obtained94. The myosin is activatedby RLC phosphorylation during LTP induction through asignalling cascade involving NMDARs and ROCK (FIG. 4).FRAP (fluorescence recovery after photobleaching)experiments using cultured hippocampal neurons indi-cate that non-muscle myosin IIb promotes actin filamentturnover in spines during neuronal activity. Furthermore,in hippocampal slices from MYH10-knockdown rats,the appearance of newly polymerized, activity-inducedactin filaments at synapses subjected to LTP inductionwas disrupted. Both blebbistatin and the actin polymeri-zation inhibitor latrunculin A, when applied 30 secondsafter LTP induction, disrupted the appearance of activity-induced actin filaments and LTP maintenance. Finally,application of jasplakinolide, a potent activator of actinpolymerization, rendered myosin II activity dispensablefor LTP maintenance. Together, these findings indicate that

non-muscle myosin IIb promotes the de novo polymeriza-tion of actin filaments shortly after LTP induction and thatthe myosin-driven formation of new filaments is neededfor the stabilization of synaptic plasticity 94 (FIG. 4).

How does non-muscle myosin IIb promote the for-mation of new actin filaments, and how is its activityintertwined with other major actin regulatory proteinsthat are found in spines8,9? In protrusive structures suchas the neuronal growth cone, actin filaments grow by theaddition of subunits at the filaments’ barbed ends nearthe plasma membrane. Together with simultaneous dis-assembly of the filaments in a zone more distant from theplasma membrane, this gives rise to the retrograde flowof actin subunits. Non-muscle myosin II in the zone ofactin disassembly is thought to contribute to retrogradeflow by exerting force on the actin meshwork and by pro-moting the disassembly and recycling of actin bundles19,28.A similar situation might exist in dendritic spines, whereactin polymerization takes place close to the surface nearthe spine tip11,12, whereas non-muscle myosin IIb local-izes to the proximal part of the spine head10,82,83. There, themyosin might drive actin filament disassembly by exertingcontractile force, thereby helping to generate the supply ofmonomeric actin subunits that is needed for new filamentgrowth. Pushing force exerted by a growing actin mesh-work on the spine plasma membrane might be essentialfor spine head growth during LTP. Notably, the myosin

might drive basal spine morphology and dynamics by thesame mechanism.

To affect structural and functional plasticity, non-muscle myosin II must cooperate tightly with otherimportant drivers of actin filament dynamics in spines.For example, the actin-related protein 2/3 (ARP2/3) com-plex, which nucleates new actin filaments that branchoff from pre-existing ones, is concentrated in a specificzone underneath the spine surface and is important forspine morphology 8–11. As in neuronal growth cones96,non-muscle myosin II and the ARP2/3 complex mightcooperatively regulate actin dynamics in spines.Furthermore, the actin-severing factor cofilin is important

Box 2 | Mechanochemical properties of synaptic myosins

Stepping

The myosin’s step size depends on its lever arm length36,37 and is the distance by which

the power stroke propels the myosin forward. The myosin V lever is three times longer

than that of myosin II. Accordingly, the step size of myosin II is ~7 nm165, whereas

myosin V takes ~36-nm steps39. Myosin V steps in a hand-over-hand fashion, whereby

the trailing head detaches from actin and, by virtue of the leading head’s power

stroke, is propelled towards the barbed end to become the new leading head20,166

.Dimerized myosin VI moves processively in the opposite direction by taking ~36-nm

hand-over-hand steps and smaller, inchworm-like steps21,22,46,167. Myosin VI’s large step

size is surprising given its apparently short lever (comprising just two

calmodulin-binding sites). However, C-terminal to the calmodulin-binding sites, an

extendable three-helix bundle and a single α-helix (SAH) domain are found. Theextended three-helix bundle or both the bundle and the SAH domain might

lengthen the lever, thereby enabling myosin VI to take 36-nm steps21,22.

Processivity

Myosins use different strategies in order to move processively: that is, to take many

steps before dissociating from their track. A feature that helps two-headed myosin V

to move processively is its motor’s high duty ratio; that is, a head spends a large

fraction of time in an ATP hydrolysis cycle state with high affinity for actin. This is the

consequence of ADP release being the rate-limiting step in myosin V’s ATP hydrolysis

cycle and decreases the chance that both myosin V heads simultaneously detach from

actin20,156. Another feature that promotes myosin V processivity is coordination of thetwo heads’ ATP hydrolysis cycles during stepping38. This coordination involves

intramolecular strain that is generated when both heads are attached to actin and that

differentially affects trailing and leading head168. In the case of myosin VI, slow ADP

release and slow ATP binding to the nucleotide-free head generate a high-duty ratio

motor21,22. Heavy chain dimerization and thus intramolecular strain appears to be

dispensable for processive, myosin VI-mediated transport169,170. By contrast,

sarcomeric skeletal myosin II is a low-duty ratio motor that spends most of its time in a

low actin affinity state (rate-limiting Pi release)15. The presence of large numbers of

heads in sarcomeric myosin II filaments ensures that sufficient heads are strongly

attached to actin. Non-muscle myosin IIb shows an intermediate-duty ratio that is

increased by subjecting the myosin to load, enabling non-muscle myosin IIb

assemblies to efficiently function in tension maintenance91.

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for actin dynamics and morphology of spines11 and mightalso regulate the binding of myosin II to actin filaments97.Interestingly, NMDAR stimulation leads to phosphoryla-tion of both cofilin and the RLC of non-muscle myosin II94,suggesting that synaptic plasticity involves coordinatedcontrol of the actin regulatory machinery. Finally, RHOGTPases and their effectors control spine actin dynamicsand spine morphology in part by regulating actomyo-sin contractility 98. For example, PAK3, an effector of theRHO GTPase RAC1, regulates spine morphology andsynapse formation by affecting the phosphorylation stateof the non-muscle myosin II RLC through MLCK99. RHO

GTPases might act also upstream of non-muscle myosinIIb during LTP stabilization. Importantly, several genesinvolved in RHO GTPase signalling, including PAK3, arelinked to mental retardation associated with abnormalspine morphology and/or density 98.

Myosin II and memory. Is non-muscle myosin IIb alsoimportant for learning and memory? CA1 neuronsin live rats were transduced with rAAV particles car-rying MYH10 shRNA constructs, and the rats under-went single-trial contextual fear conditioning 30 dayslater94. Compared with controls, these rats showedimpaired freezing behaviour during a 24-hour long-term

memory (LTM) test. During training, however, bothgroups acquired the context–shock association normallyand had comparable levels of exploratory activity, indicat-ing that non-muscle myosin IIb does not regulate learningbut is selectively involved in stabilizing acquired contex-tual associations for LTM storage. Infusions of blebbistatininto CA1 produced similar results and also showed thatmyosin inhibition did not affect the acquisition of novelconditioned stimulus–unconditioned stimulus associa-tions but did impair the consolidation of LTMs. Consistentwith the LTP analysis, pretreatment with jasplakinolide toactivate actin polymerization prevented blebbistatin from

disrupting LTM consolidation94. This indicates that non-muscle myosin IIb motor activity drives actin dynamicsthat are essential for contextual memory consolidation.Similarly, blebbistatin infusion experiments suggest thatmyosin II activity is required in the lateral amygdala forfear memory consolidation100.

Myosin II and synaptic vesicle motility. Myosin II hasalso been implicated in regulating neurotransmitterrelease at presynaptic nerve terminals80,95,101–103. Injectionof an antibody against non-muscle myosin IIb or of adominant-negative myosin tail fragment into culturedsuperior cervical ganglion neurons (SCGNs) attenuated

Box 3 | Nervous system phenotypes caused by mutations in mammalian myosin genes

Non-muscle myosin II

MYH9.On the basis of Taiwanese blood samples, fine mapping of human chromosome 22q12 suggested that the myosin

heavy chain 9 (MYH9) gene conferred vulnerability for specific subtypes of schizophrenia171. However, a follow-up study

in a Japanese population failed to confirm increased susceptibility to schizophrenic disorders172. Humans with MYH9

point mutations, such as the R702C mutation, suffer from macrothrombocytopenia and can develop deafness92.

MYH10. In mice, the R709C mutation causes an abnormal migration of pontine neurons, cerebellar granule cells and

facial neurons74

; deletion of alternatively spliced exon B1 in mice causes an abnormal migration of facial neurons; anddeletion of the alternatively spliced exon B2 results in abnormal development of cerebellar Purkinje neurons76.

Myosin V

MYO5A. In humans, mutations in the gene encoding myosin Va heavy chain, MYO5A, cause Griscelli syndrome type I,

which is characterized by neurologic impairment and severe nervous system dysfunction173. Patients show abnormal eye

movement, are mentally retarded and develop seizures174,175. Murine mutations such as dilute-lethal and flailer, which are

characterized by loss of function of myosin Va, produce neurological defects, including ataxia, cerebellar Purkinje neuron

loss, poor myelination, opisthotonus and convulsive limb movement34,126,127,176–178. Similarly, the rat dilute-opisthotonus

autosomal recessive mutation in Myo5a causes ataxia, opisthotonus and convulsive limb movement128. AMYO5A gene

mutation in horse causes lavender foal syndrome, a lethal disease with severe neurological abnormalities including

tetanic-like seizures, opisthotonus, stiff or paddling leg movements and nystagmus179.

MYO5B.A single-nucleotide polymorphism in the gene encoding myosin Vb heavy chain, MYO5B, was found in a

whole-genome association study of bipolar disorder (also known as manic depressive disorder), which is characterized by

profound mood symptoms that include episodes of mania, hypomania and depression180. MYO5B mutations cause

microvillus inclusion disease

181

, but direct effects of the mutation on nervous system function have not been reported.Transgenic mice expressing Myo5b-Y119G are conditional mutants in which the mobility of myosin Vb can be inhibited

through application of the non-hydrolysable ADP analogue N6-2-phenylethyl-ADP (PE-ADP). Functional inactivation of

myosin Vb through PE-ADP markedly attenuates long-term potentiation in hippocampal CA1 neurons115.

Myosin VI

MYO6.Mutations in the gene encoding myosin VI heavy chain, MYO6, are associated with deafness and vestibular

dysfunction in the Snell’s waltzer (sv ) mutant mouse and in humans67,182. Homozygous mouse mutants (sv /sv ) show

synaptic abnormalities in the hippocampal CA1 region, which is characterized by fewer synapses and shorter dendritic

spines than the CA1 region in controls140. Myosin VI to has been linked to neurodegenerative disease, as it modifies

tau-induced neurodegeneration in Drosophila melanogaster 161, and the fibrillary inclusions of brains from patients with

tauopathies such as Alzheimer’s disease and frontotemporal dementia with Parkinsonism-17 contain myosin VI160.

Furthermore, mutations in optineurin and the RNA-binding protein TLS (also known as FUS) — two myosin VI-bindingpartners — lead to amyotrophic lateral sclerosis183,184. Finally, Snell’s waltzer mice develop profound astrogliosis, whichis consistent with neurodegeneration in the absence of myosin VI140.

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DAG

SV

SV

SV

Folded

mGluRPlasmamembrane supply

AMPARsurface

removal

P

Directed movementof evoked vesicle

Evokedvesicle

Spontaneous

vesicle TRKB

BDNF

Synaptic release

NMDAR

x

x xx

xxx

Exocytosis

Endocytosis Endocytosis

?

Ca2+

Ca2+

Ca2+

LTDROCK

MLCK

RLC

?

P L C

IP3

RE

RE

Actin turnovernew filaments

Spine head growth

LTP establishment

Recycling

Lysosomaldegradation

PSD

Glutamate

Presynapse

Postsynapse

PKC

ExocytosisEndocytosis

Actin filament

PICK1

AMPAR

Myosin IIb(non-muscle)

MYH7B

Myosin Va

Myosin Vb

Myosin VI

IP3R

ER

UEV

RE

LTP maintenance

BDNF-dependentLTP

Cargo adaptor

Recycling

Cargo-bound

Actin filament

contraction

Clathrin

?

?

AMPARsurfacedelivery

acetylcholine release at their presynaptic terminals80,101.More recently, myosin II has been proposed to supportsynaptic transmission during periods of sustained neu-ronal activity by promoting synaptic vesicle motility 95.Live imaging of cultured hippocampal neurons provided

direct evidence that myosin II is required for synaptic vesicle motility during evoked activity 95. The vesicleswere labelled while being generated by endocytosiseither during evoked synaptic activity (‘evoked vesicles’)or during spontaneous activity (‘spontaneous vesicles’).

Figure 4 | Integrated model of the function of synaptic myosins. Schematic of a presynaptic terminal contacting a

dendritic spine. Arrows represent cellular processes that are mediated by, or are upstream of, the respective myosins.

Presynaptic functions of myosins include: promotion of directed movement of synaptic vesicles (SVs) generated by

endocytosis after evoked glutamate release by myosin II; and promotion of synaptic recycling of vesicles and induction of

brain-derived neurotrophic factor (BDNF)-dependent long-term potentiation (LTP) by myosin VI. Postsynaptically,

non-muscle myosin IIb and myosin Vb are activated by an NMDA receptor (NMDAR)-dependent Ca2+ influx. Non-muscle

myosin IIb promotes the turnover of actin filaments in spines, thereby contributing to spine head growth and the

maintenance of LTP. Myosin Vb trafficks AMPA receptor (AMPAR) subunit GluA1-carrying recycling endosomes (REs) into

spines and thereby contributes to spine head growth and AMPAR surface delivery during LTP establishment. In Purkinje

neurons, myosin Va transports the endoplasmic reticulum (ER) into dendritic spines, allowing local Ca

2+

release that isnecessary for long-term depression (LTD). Increased endocytosis of AMPARs after induction with AMPA or insulin depends

on myosin VI. Possible roles for myosin VI include promoting clathrin-mediated AMPAR endocytosis or driving the motility

of endocytic AMPAR carriers. DAG, diacylglycerol; IP3, inositol 1,4,5-triphosphate; IP

3R, IP

3 receptor; mGluR, metabotropic

glutamate receptor; MLCK, myosin light chain kinase; MYH7B, myosin II heavy chain 7B; PICK1, PRKCA-binding protein;

PKC, protein kinase C; PLC, phospholipase C; PSD, postsynaptic density; RLC, regulatory light chain; ROCK,

RHO-associated, coiled-coil-containing kinase; TRKB, tropomyosin-related kinase B; UEV, uncoated endocytic vesicle.

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Recycling endosomes

Membrane compartments via

which endocytosed membrane

receptors are recycled back to

the plasma membrane.

Long-term depression

(LTD). A long-lasting reduction

in the strength of a neuronal

synapse following certain

stimuli.

The motile behaviour of these two types of vesicles dif-fered markedly: compared with the spontaneous vesicles,the evoked vesicles showed increased directionality andmoved faster (~150 nm per second versus ~90 nm persecond) and, consequently, passed through a larger areawithin 20 seconds95. Exposure to blebbistatin or to theMLCK inhibitor ML-9 reduced speed and directional-ity of the evoked vesicles, indicating that myosin II isneeded for the increased motility of synaptic vesicles thatare formed during periods of neuronal activity 95 (FIG. 4).Moreover, the exposure of acute hippocampal slices to100 μM blebbistatin reduced EPSC amplitudes in CA1neurons during high-frequency stimulation of Schaffercollaterals when neurotransmission depends on theresupply of synaptic vesicles95. Additional evidence thatnon-muscle myosin II is important presynaptically comesfrom the Drosophila melanogaster neuromuscular junc-tion (NMJ), where the myosin is required for synaptic

vesicle motility and synaptic transmission102,103. It remainsunclear precisely how non-muscle myosin II, which pro-motes actin organization and dynamics19, drives vesicle

motility. Interestingly, myosin Va associates with synap-tic vesicles104 (FIG. 1b), and myosin V-dependent organelletransport depends on actin filament dynamics in mel-anophores105. Therefore, we propose that non-musclemyosin II might indirectly promote directional motilityof vesicles at synaptic terminals by affecting the dynamicsof actin filaments along which processive myosins move.

Synaptic roles of myosin VTwo of the three class V myosins (myosin Va andmyosin Vb) are found in the brain35,106–109. Myosin Vbshows relatively strong expression in the hippocam-pus107, whereas myosin Va is most strongly expressed inother brain regions108. Both myosins are present in den-dritic spines and co-fractionate with the PSD85,107,110,111 (FIG. 1b). The splice isoform of myosin Va found in thebrain binds an additional, dimeric light chain calleddynein light chain 2 (DYNLL2)112,113 (FIG. 2b). Moreover,there are two myosin Vb splice isoforms (with andwithout exon D) in the brain. Exon D encodes extraresidues in the myosin’s coiled-coil region, permittingthe myosin to associate with RAB10 (REF. 114).

Myosin V and hippocampal LTP induction. To investigatethe role of myosin Vb in LTP induction, mice were engi-neered to express mutant MYO5B ( MYO5B-Y119G) inwhich ATPase activity can be conditionally blocked by the

non-hydrolysable ADP-analogue N6-2-phenylethyl-ADP(PE-ADP)115,116. PE-ADP disrupted LTP in slices fromthe transgenic mice but did not affect NMDAR currents.PE-ADP arrests the myosin in a state with high affinityfor its actin track. Thus, the ability of myosin Vb to movealong actin filaments rather than simply to bind to actinseems to be required for LTP induction115.

How is myosin Vb involved in the induction of syn-aptic plasticity? LTP establishment requires a supply ofGluA1 AMPAR subunits from recycling endosomes to theplasma membrane and, consequently, to the synapse117,118.Myosin Vb associates with recycling endosomes and reg-ulates trafficking through the recycling pathway 20,116,119,120.

In cultured hippocampal neurons, dominant-negativemyosin Vb constructs disrupt the trafficking of GluA1(but not GluA2) to the plasma membrane and reducethe frequency of mEPSCs, which is consistent with theidea that myosin regulates the delivery of GluA1 tosynapses107. Within dendritic spines, myosin Vb andrecycling endosomes colocalize and show correlatedmovement115. Stimulation with glycine to induce LTPthrough NMDAR activation leads to the recruitmentof myosin Vb to recycling endosomes that are found inthe dendritic shaft and that are only weakly decoratedby the myosin under basal conditions. Subsequently,these recycling endosomes move into spines115 (FIG. 4).Interaction between myosin Vb and its recycling endo-some organelle receptor, which comprises RAB11 andRAB11-FIP2 (FIG. 3b), is required for the recruitment ofthe myosin to recycling endosomes, for the traffickingof endosomes into spines and for the burst of exocyto-sis and spine growth that follows glycine application115.Thus, during LTP induction, myosin Vb interacts withGluA1-containing recycling endosomes in the dendritic

shaft to drive their delivery into spines. There, the recy-cling endosomes fuse with the plasma membrane, lead-ing to surface insertion of GluA1 AMPAR subunits andto spine surface growth that accompanies LTP115 (FIG. 4).

The recruitment of myosin Vb to recycling endosomesseems to be regulated by Ca2+, which promotes the myo-sin’s transition from a folded to an open conformation115.After LTP-inducing, glycine-mediated NMDAR activa-tion, the cytosolic Ca2+ concentration in spines increases.This increase is necessary to recruit myosin Vb to recy-cling endosomes in the dendritic shaft115. Ca2+ is thoughtto affect the myosin in two ways45,115. First, Ca2+-inducedunfolding of myosin Vb promotes the GTD’s interactionwith the receptor on recycling endosomes115. In supportof this, constitutively open myosin Vb shows increasedassociation with recycling endosomes in the absence ofglycine stimulation and enhances transport of recyclingendosomes into spines115. Second, increased spine Ca2+ might reduce the ability of myosin Vb to move alongactin115, possibly by inducing the loss of calmodulin lightchains from the lever arm45. This could contribute to theaccumulation of myosin Vb at the base of the spine, wherethe GluA1-carrying recycling endosomes wait. As Ca2+ rises only locally within the spines after NMDAR activa-tion, myosin Vb at the spine base is proposed to recover itsability to walk along actin filaments, allowing the myosinto drive recycling endosomes into spines115 (FIG. 4).

It remains unclear whether myosin Va is also importantfor hippocampal synaptic plasticity 121. Although MYO5A mutations lead to severe neurological abnormalities (BOX 3),synaptic function including LTP and long-term depression (LTD) at Schaffer collateral–CA1 synapses was found tobe normal in slices from adolescent dilute-lethal micethat lack myosin Va122. However, the absence of defectsat these synapses might be explained by compensationthrough myosin Vb122. Notably, similar to myosin Vb107,myosin Va was found to associate with RAB11 and GluA1(REFS 114,121). To test whether myosin Va is important forthe delivery of GluA1 to spines during synaptic plastic-ity, LTP was mimicked in cultured hippocampal neurons

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Endoplasmic reticulum

A cellular organelle that forms

an interconnected network of

tubules and cisternae and that

is crucial for protein secretion,

membrane protein translation

and lipid synthesis.

Furthermore, it serves as an

intracellular Ca2+

store involvedin regulating cytosolic Ca2+

concentration.

Opisthotonus

A state of severe

hyperextension and spasticity

caused by spasm of the axial

muscles along the spinal

column.

Astrogliosis

A proliferation of astrocytes

that can be a consequence of

neurodegenerative processes

or epilepsy.

by expressing constitutively active Ca2+/calmodulin-dependent protein kinase II (tCaMKII), which leads toan increased accumulation of AMPAR subunits in spines.A dominant-negative construct consisting of the GTD ofmyosin Va abolished tCaMKII-induced GluA1 accumula-tion121. However, the dominant-negative effect might bedue to inhibition of myosin Vb, as the myosin Va GTDinhibits both myosin Va and myosin Vb115,123. Moreover,unlike myosin Vb, myosin Va does not show corre-lated movement with recycling endosomes in spines115.Nevertheless, acute interference with myosin Va by smallinterfering RNA knockdown blocks LTP at Schaffer col-lateral–CA1 synapses and disrupts the synaptic GluA1accumulation that has been induced by overexpression oftCaMKII or PSD95 (REF. 121) . As myosin Va associates withseveral postsynaptic proteins that are important for den-dritic spine morphology and/or synapse function in addi-tion to RAB11 and GluA1 (TABLE 1), we believe t hat furtherinvestigation of the mechanism (or mechanisms) by whichmyosin Va acts at hippocampal synapses is warranted.

Myosin Va and cerebellar LTD. Dilute-lethal mice showa striking organelle positioning defect in the cerebellum.Normally, the dendritic spines of cerebellar Purkinjeneurons contain smooth endoplasmic reticulum tubulesthat are connected to the rest of the cytoplasm-wideendoplasmic reticulum network 124,125. However, thereis no endoplasmic reticulum in the spines of Purkinjeneurons from rodents with Myo5a mutations, such asdilute-lethal mice126, flailer mice127 and dilute-opistho-tonus rats128, although it is conserved in their dendrites.The induction of LTD at synapses between parallel fibresand Purkinje neurons involves activation of postsynap-tic metabotropic glutamate receptor 1 (mGluR1), whichinduces Ca2+ release from the endoplasmic reticulumwithin Purkinje neuron spines129; this local Ca2+ tran-sient is absent from dilute-lethal spines129,130. This defectis thought to result in the observed disruption of paral-lel fibre–Purkinje neuron LTD in Myo5a mutants, as theLTD depends on mGluR1-downstream signalling thatculminates in protein kinase C-dependent stimulationof GluA2 endocytosis129,131 (FIG. 4). Parallel fibre–Purkinjeneuron LTD might contribute to cerebellum-dependentmotor learning132.

There is relatively high expression of myosin Va inPurkinje neurons106,108, and in vitro experiments indicatethat the myosin functions cell-autonomously in these neu-rons to target the endoplasmic reticulum to spines130. The

mechanochemical properties of myosin Va suggest thatit can function as a point-to-point organelle transporterthat carries cargo along actin filaments20. However, in

vertebrate cells, class V myosins were found to positionorganelles by capturing them after their delivery by micro-tubule-based transport and by dynamically tethering themto the actin cytoskeleton20,24,116,133. Nevertheless, analysisof endoplasmic reticulum dynamics in cultured Purkinjeneurons showed that myosin Va is required for the move-ment of the endoplasmic reticulum into spines and notsimply for tethering the organelle there130. Moreover,the myosin associates with the tip of the motile endo-plasmic reticulum during its movement into spine-like

protrusions110,130 (FIG. 3b). In addition, the replacement ofwild-type myosin Va with slow-walking versions of themyosin leads to a corresponding reduction in the maxi-mum velocity of endoplasmic reticulum movement intospines130. Together, these data show that myosin Va acts asan organelle transporter that moves endoplasmic reticu-lum compartments along actin filaments into Purkinjeneuron spines130. In addition to defective LTD at parallelfibre–Purkinje neuron synapses, dilute-lethal mice showsevere ataxia (BOX 3), which is consistent with cerebel-lar malfunction. As disruption of LTD alone does notcause a detectable motor coordination or motor learn-ing phenotype134, we propose that myosin Va or myosinVa-mediated transport of the endoplasmic reticulum hasadditional physiological significance in Purkinje neurons.

Other synaptic functions of myosin Va. Myosin Va alsofunctions at other synapses. For example, at the verte-brate NMJ, myosin Va regulates postsynaptic acetylcho-line receptor trafficking to promote NMJ plasticity andmaintenance135,136. Myosin Va is also required for synaptic

transmission at photoreceptor synapses137. Furthermore,myosin Va is present on synaptic vesicles (FIG. 1b) andpromotes retrograde long-range movements of vesiclescontaining synaptic vesicle protein 2 in SCGN axons104,138.However, myosin Va is not generally required for synaptictransmission because disruption of its function does notimpair neurotransmitter release at hippocampal, cerebel-lar and SCGN synapses80,121,122,129. Finally, myosin Va hasbeen implicated in maturation, transport and exocyto-sis of secretory granules and large dense core vesicles(LDCVs) in neuroendocrine cells and hippocampalneurons, respectively 139. LDCVs carry neuropeptides andhormones that, upon release from cells, affect processessuch as synaptic plasticity. Therefore, we speculate thatmyosin Va might also influence synaptic function in theCNS through its role as an LDCV-associated motor.

Synaptic roles of myosin VIMyosin VI is widely expressed in the adult mouse brain140,and exposure to traumatic stress temporarily increases itsexpression in the hippocampus141. Myosin VI is foundthroughout hippocampal neurons, including in den-dritic spines (FIG. 1b), and it partially colocalizes andco-fractionates with the PSD86,140. The loss of myosin VIexpression in Snell’s waltzer mice leads to reductions insynapse numbers and spine length in the CA1 region ofthe hippocampus140. Several studies using cultured hip-

pocampal neurons confirmed the requirement of myosinVI for normal synapse numbers and revealed, for exam-ple, that Snell’s waltzer neurons have fewer presynapticactive boutons140,142,143. In addition, the brains of thesemice show widespread astrogliosis140.

Myosin VI and BDNF-dependent synaptic plasticity. Snell’s waltzer mice have a defect in brain-derived neu-rotrophic factor (BDNF)-dependent neurotransmissionand synaptic plasticity in the hippocampus143. BDNF is asecreted neurotrophin that affects synaptic transmissionand plasticity by acting on both pre- and postsynaptic tar-get cells144. BDNF binds to the transmembrane receptor

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tyrosine kinase TRKB (tropomyosin-related kinase B; alsoknown as NTRK2) to activate multiple downstream sig-nalling cascades145. TRKB forms a complex with myosinVI and GIPC1 (REF. 143). GIPC1 apparently acts as a cargoadaptor that bridges the myosin to the cytosolic juxtam-embrane region of TRKB143,146 (FIG. 3c). Like TRKB, GIPC1is present at pre- and postsynaptic sites, and immunoelec-tron microscopy shows that GIPC1 associates with smallpresynaptic vesicles143.

Mice deficient in myosin VI or GIPC1 share severalsynaptic phenotypes, including a reduction in basal syn-aptic transmission at Schaffer collateral–CA1 synapses143.Defects in enhanced paired-pulse facilitation and reducedpost-tetanic potentiation indicate that both proteins areimportant for presynaptic short-term plasticity. Consistentwith the idea that myosin VI and GIPC1 mediate thedownstream effects of BDNF–TRKB signalling at syn-apses, various BDNF-dependent phenomena are disruptedin the absence of myosin VI and GIPC1. These includethe BDNF-dependent increase of EPSC amplitudes andthe BDNF–TRKB-mediated enhancement of glutamate

release from presynaptic terminals143. Finally, the BDNF-dependent facilitation of LTP at Schaffer collateral–CA1synapses at postnatal days 12–13 (but not LTP in adultmice) requires myosin VI and GIPC1 (REF. 143). Thus,myosin VI and GIPC1 act on the presynaptic side to pro-mote BDNF–TRKB-dependent neurotransmitter releaseand synaptic plasticity, possibly by promoting synaptic

vesicle recycling143. Further investigations are neededto identify the mechanism by which myosin VI acts inBDNF–TRKB signalling. It is possible that the myosinfunctions as an organelle motor that locally promotesexo- or endocytic trafficking of TRKB-containing vesiclesat synaptic terminals, thereby affecting TRKB signallingat the synapse (FIG. 4).

There are other instances in which myosin VI affectspresynaptic function. Besides stereocilia maintenance,myosin VI is required in inner ear hair cells for ribbon syn-apse maturation and Ca2+-dependent exocytosis at thesesynapses147,148. At the D. melanogaster NMJ, myosin VI isneeded for synaptic vesicle localization, synaptic transmis-sion and short-term plasticity 149.

Myosin VI and postsy naptic receptor traf ficking. Postsynaptically, myosin VI is involved in regulating thetrafficking of AMPARs140,142,150. The myosin’s CBD bindsthe β-splice isoform of synapse-associated protein 97(SAP97; also known as DLG1) (FIG. 3c), which is a scaffold-

ing protein that interacts with the AMPAR subunit GluA1and regulates its surface expression150,151. A complex ofmyosin VI, AMPAR and SAP97 co-immunoprecipitatesfrom brain tissue140. In cultured hippocampal neurons,exposure to AMPA or insulin stimulates AMPAR endo-cytosis. This upregulation of endocytosis is abolished inSnell’s waltzer neurons, indicating that it requires myo-sin VI140 (FIG. 4). Using a dominant-negative myosin VItail construct to interfere with the myosin’s functionin cultured hippocampal neurons, another study sug-gested that myosin VI is required for the trafficking ofAMPARs to the surface and the synapse142. Several keyquestions regarding the function of myosin VI in AMPAR

trafficking remain open. For example, does myosin VIdrive AMPAR trafficking directly by acting at the postsyn-aptic side or indirectly by promoting glutamate release? Itis unclear which AMPAR trafficking event (endocytosis,recycling or exocytosis) is directly mediated by myosin VIand whether the myosin functions as a processive trans-porter or an anchor. Given the mixed orientation of actinfilaments in spine necks, it is also unknown whether myo-sin VI (in its capacity as an organelle motor) moves bothinto and out of the spines. Whether myosin VI-mediatedAMPAR trafficking is relevant for synaptic plasticity andfor learning and memory is unknown.

In addition to AMPAR trafficking, myosin VI is alsoimportant for the trafficking of inhibitory type A GABAreceptors (GABA

ARs)14,152,153, which mainly localize to syn-

apses on dendritic shafts. In brain lysate and hippocampalneurons from Snell’s waltzer mice, the cell surface levelsof GABA

ARs containing the α1 subunit were markedly

increased, indicating that myosin VI is involved in actin-dependent receptor endocytosis152. Myosin VI and theGABA

AR α1 subunit bind to the trafficking factor mus-

kelin, which also takes part in dynein-mediated GABAARtransport downstream from the sorting endosome andparticipates in melanosome trafficking152. Interactions ofmuskelin154 with myosin VI and dynein transport com-plexes might help to shuttle myosin VI cargoes from actinfilaments to microtubule tracks.

Future directionsUnravelling the functions of myosins in neurons isimportant for understanding molecular mechanismsat pre- and postsynaptic sites that regulate short- andlong-term plasticity and influence learning and mem-ory. Important challenges lie ahead if we are to obtain acomplete picture of how synapse function, learning andmemory and possibly neurodegeneration are affected bythe function and dysfunction of myosins.

The wealth of knowledge about the bio- and mechano-chemical properties of myosin II, myosin V and myosinVI18–22,155,156 (BOXES 1,2), and about the structure of thecargo-binding tails of class V and class VI myosins32,51,157 will be fundamental for further dissecting the synapticroles of myosins because it enables investigators to probea myosin’s mechanism of function by introducing pointmutations with known effects on the myosin’s function.For example, testing whether synaptic plasticity is affectedby mutations with known effects on the myosin’s motormechanism, the myosin’s binding to specific cargo adaptors

or the myosin’s regulation will lead to deeper insights intohow a myosin drives synaptic plasticity. In the case of theorganelle motors myosin V and myosin VI, understand-ing their synaptic function will depend on identifying thefull set of cargoes with which the myosins interact to pro-mote synapse function. Several proteins involved in syn-apse function that interact with those myosins are known(TABLE 1). It will be important to investigate the relevanceof these interactions of the myosins for synaptic plasticity.Moreover, alternative splicing in the brain generates myo-sin isoforms that differ in their mechanochemical prop-erties or their cargo-binding tails47,75,77–79,114. However, it islargely unknown whether and how the synaptic functions

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of the distinct myosin splice isoforms differ. Last, in vitro data provide hints on how myosin V and myosin VI coop-erate despite moving in opposite directions on actin158,159.It will be interesting to see whether such cooperation alsoexists at synapses, for example, during endosomal traf-

ficking and recycling of AMPARs. Moreover, the questionarises whether class II myosin-driven actin dynamics regu-late myosin V- and myosin VI-based transport of synapticcargoes at the pre- and/or postsynaptic side.

Another major challenge concerns the consequencesof synaptic, myosin-mediated processes for cognition andanimal behaviour. With few exceptions94,100, the impor-tance of myosin function for learning and memory hasnot yet been examined. Moreover, the potential link ofmyosin VI to neurodegenerative disease160,161 (BOX 3) deserves investigation. Approaches such as the genera-tion of mutant mice with point mutations that disruptspecific aspects of myosin function74,115 might be needed

to advance research in this area. In addition, there is aneed to further dissect the roles of myosins in the plastic-ity of neuronal circuits in adults as opposed to their rolesin nervous system development. Towards this end, theability to inhibit specific myosins in an acute fashion in

live adult animals, perhaps using the recently discoveredmyosin VI inhibitor162 or shRNA-mediated knockdown

via viral transduction94, will continue to be pivotal.In conclusion, we need a more complete picture of the

different myosins that directly affect synapse function.Whereas the field is relatively advanced regarding thesynaptic roles of myosin II, myosin V and myosin VI, thismight just represent the tip of the iceberg. Various othermyosins are expressed in neurons, some of which havebeen shown by proteomic studies to be components of thePSD (for example, myosin Id163,164 and myosin XVIIIa84).Whether these myosins are involved in synaptic transmis-sion or plasticity has not yet been investigated.

Table 1 | Synapse and/or spine proteins that interact with myosin V or myosin VI

Myosin Interactionpartner

Comment Refs

Myosin V Drebrin Spine protein that modulates myosin V–actin interaction 185,186

CaMKII Myosin V activates CaMKII; CaMKII is a kinase crucial for long-termpotentation (LTP) and translocates into spines during LTP

187,188

GKAP Postsynaptic density scaffolding molecule; co-precipitates with myosin Va;synaptic clustering of GKAP is reduced by myosin Va heavy chain (MYO5A)knockdown

111,189

GluA1 Long-tailed AMPA receptor subunit 107,121

PTEN Activated by myosin V; PTEN is a phosphatase that regulates NMDAreceptor-dependent long-term depression at postsynaptic terminals

123,190

RAB10 Binds myosin V splice isoforms containing exon D 114

RAB11 Part of myosin Vb organelle receptor on recycling endosome; also binds tomyosin Va

114,115,121

RAB11-FIP2 Part of myosin Vb organelle receptor on recycling endosome 115

RILPL2 Binds to myosin Va; regulates hippocampal spine morphology 191

TLS Facilitates RNA targeting to spines by coupling mRNA to myosin Va;regulates spine morphology;TLSmutation causes amyotrophic lateral

sclerosis

184,192,193

TRIM2 andTRIM3

Required for efficient transferrin receptor recycling and for regulation ofdendritic spine morphology

194–197

Myosin VI βSAP97 Binds both AMPA receptor subunit GluA1 and myosin VI 140,150

AP2 Clathrin adaptor required for clathrin-mediated endocytosis of AMPAreceptors

140

GIPC1 Putative linker between TRKB and myosin VI; promotes BDNF-dependentLTP together with myosin VI

143

Muskelin Localizes to inhibitory synapses; required for normal hippocampal networkoscillations

152

Optineurin Binds and inhibits metabotropic glutamate receptor; promotes membranetrafficking events together with myosin VI; optineurin mutation causesamyotrophic lateral sclerosis

59–62,183,198

Otoferlin Required together with myosin VI for inner ear hair cell ribbon synapsematuration and function

147,148

TLS Regulates spine morphology;TLS mutation causes amyotrophic lateralsclerosis

184,193,199

AP2, adaptor protein 2 complex; BDNF, brain-derived neurotrophic factor; CaMKII, Ca2+/calmodulin-dependent proteinkinase II; GKAP, G kinase-anchoring protein; PTEN, phosphatase and tensin homologue; RILPL2; RILP-like protein 2; βSAP97,β-splice isoform of synapse-associated protein 97; TRIM, tripartite motif-containing protein; TRKB, tropomyosin-related kinase B.

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1. Yuste, R. & Bonhoeffer, T. Morphological changes in

dendritic spines associated with long-term synaptic

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