Actin Filaments and Microtubules in Dendritic Spines

JOURNAL OF NEUROCHEMISTRY | 2013 | 126 | 155–164 doi: 10.1111/jnc.12313

*Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine, Maebashi, Japan †Laboratory of Cell and Neuronal Dynamics, Department of Biology, Faculty of Sciences, Universidad de Chile, Chile

Abstract occurring at the nascent excitatory postsynaptic site, and Dendritic spines are small protrusions emerging from their plays a pivotal role in spine formation as well as small parent dendrites, and their morphological changes are GTPases. It has been recently reported that microtubules involved in synaptic plasticity. These tiny structures are transiently appear in dendritic spines in correlation with composed of thousands of different proteins belonging to synaptic activity. Interestingly, it is suggested that microtubule several subfamilies such as membrane receptors, scaffold dynamics might couple with actin dynamics. In this review, we proteins, signal transduction proteins, and cytoskeletal pro- will summarize the contribution of both actin filaments and teins. Actin filaments in dendritic spines consist of double helix microtubules to the formation and regulation of dendritic of actin protomers decorated with drebrin and ADF/cofilin, and spines, and further discuss the role of cytoskeletal deregula- the balance of the two is closely related to the actin dynamics, tion in neurological disorders. which may govern morphological and functional synaptic Keywords: actin, dendritic spine, microtubules, synaptic plasticity. During development, the accumulation of drebrin- plasticity. binding type actin filaments is one of the initial events J. Neurochem. (2013) 126, 155–164.

Dendritic spines are small protrusions emerging from their mature neurons have dendritic spines capable of morpho- parent dendrites, and contain postsynaptic structures such as logical plasticity depending on synaptic activity (Fig. 2e postsynaptic density and actin filaments and, under certain and f). How does a neuron form dendritic spines? When a circumstances, microtubules. Dendritic spines observed in presynaptic terminal comes into contact with a filopodium, fixed brain tissue and cultured neurons show various shapes a cluster of actin filaments appears at the contact site in (Fig. 1a–c) and are generally classified into three types: the the filopodium (Fig. 2c and d). Eventually, the cluster- thin type having a slender neck and a small head, the containing processes change into mature dendritic spines mushroom type having a short neck and a relatively large (Takahashi et al. 2003). In this mini-review, we will head, and the stubby type having no neck. All these delineate the basic cytoskeletal elements in dendritic categories reflect a continuum rather than separate classes spines and discuss the role of cytoskeletons in the (Rochefort and Konnerth 2012). And in living neurons, spine dendritic spine formation, synaptic plasticity, and neuro- shapes easily interchange among the above three types. In logical disorders. other words, spine morphologies are snapshots of dynamic morphological changes. In fact, dendritic spines dynamically Received April 10, 2013; revised manuscript received May 13, 2013; change their morphology in response to synaptic transmis- accepted May 13, 2013. sion, which happens to be the structural basis of synaptic Address correspondence and reprint requests to Dr. Tomoaki Shirao, plasticity. Department of Neurobiology and Behavior, Gunma University Graduate Although at early developmental stages neurons form School of Medicine, Maebashi 371-8511, Japan. fi E-mails: [email protected]; [email protected] many thin lopodia on their dendrites (Fig. 2a and b), they Abbreviations used: AD, Alzheimer’s disease; ASD, autism spectrum are just similar to those emerging from non-neuronal cells disorders; DB-actin, drebrin-binding actin; LTP, long-term potentiation; and do not show any morphological plasticity. Only MAPs, microtubule-associated proteins.

(a) (d)

(b) PresynapƟc terminal (e) SynapƟc vesicles

Glutamate receptors Spine PSD

Microtubules MAP1B ?

AcƟn ﬁlaments MAP2 ADF/coﬁlin DendriƟc shaŌ

Fig. 1 (a) DiI labeling of a 21-day-in vitro hippocampal neuron. (b) with anti-drebrin antibody (M2F6). Blue shows microtubule-associated Enlarged images of the areas indicated by the square in (a). (c) Golgi proteins immunostained with anti-MAPs anti-serum. Note that yellow staining image of hippocampal neuron of the adult rat brain. (d) Triple signals indicate co-localization of drebrin and F-actin in dendritic spines. (e) staining images of a 21-day-in vitro hippocampal neuron. Red shows actin Schematic presentation of actin ﬁlaments, microtubules and postsynaptic ﬁlament stained with phalloidin. Green shows drebrin immunostained density (PSD). Scale bars are 20 lm in (a) and 2 lmin(b).

(a) (b) (c)(d) (e) (f)

Dynamic actin pool Stable actin pool PSD structure Presynaptic terminal

Drebrin

PSD95 ADF/cofilin

Cortical actin in dendritic shaft

Fig. 2 Schematic representation of dendritic spine formation from filament clusters are formed, postsynaptic density (PSD)-95 and dendritic filopodia. (a) and (b) are filopodia. A filopodium at early glutamate receptors are accumulated into these clusters in (d). (e) and developmental stages of a neuron is similar to that emerging from a (f) are mature spines. When DB-actin filament is predominant in the non-neuronal cell. When a presynaptic terminal contacts a filopodium, spine, the spine morphology is stable. Long-term potentiation (LTP) drebrin is accumulated at nascent contact sites in (b), (c) and (d) are signals might form mushroom-type spines in (e). When ADF/cofilin- immature spines. Drebrin-binding actin filaments form stable actin pool binding-actin filaments predominate, the spine morphology is unstable at postsynaptic site in (c). Once drebrin-binding actin (DB-actin) in (f).

Another possible mechanism is the capping of the barbed end Cytoskeletal structures in dendritic spines of an actin filament, which results in the inhibition of actin Overview of actin filaments polymerization at the barbed end. Relative facilitation of An actin filament consists of double helix of actin protomers actin depolymerization at the pointed end consequently decorated with its binding proteins. The physical and decreases the content of actin filaments. Interestingly, ADF/ biochemical properties of actin filaments are varied among cofilin is known to facilitate the actin depolymerization by different cell types and even among subcellular regions. This increasing the rate of dissociation from the pointed end of is not because actin molecule isoforms are different, but actin filaments as well as sequestering actin monomers because filament-binding proteins, such as tropomyosin and (Carlier et al. 1997). Because its actin-binding activity is drebrin, are so (Shirao et al. 1992; Sekino et al. 2007) and modulated by phosphorylation, the phosphorylation signals actin depolymerizing factor (ADF)/cofilin (Bamburg et al. might cause the locally biased actin-filament depolymerizing 1999). activity of ADF/cofilin. In addition, an actin filament has a polarity. It has a barbed Actin filaments can form various kinds of higher order end and a pointed end, which are named after the electron structures. In cooperation with actin-bundling proteins, such microscopic image of actin filaments decorated with heavy as a-actinin and fascin, they form a straight bundle. a- mero-myosin as a metaphor for the arrowhead (Huxley 1963; actinin-mediated actin bundle is a straight long bundle, Ishikawa et al. 1969). It is known that actin filaments keep typically linking adhesion plaques within a cell. Fascin forms treadmilling reaction (Wegner 1976) when their ends are not a thin actin bundle consisting of five to six filaments, a capped by actin-capping proteins. According to the tread- typical actin structure found in a filopodium of axonal growth milling reaction, actin protomers are continuously added cone (Sasaki et al. 1996). On the other hand, filamin forms a (polymerized) at the barbed end and removed (depolymer- meshwork of actin filaments (Nakamura et al. 2011). ized) at the pointed end (Pollard and Mooseker 1981). Protrusive motility of a cell generally proceeds by a Unique character of actin filaments in dendritic spines treadmilling reaction in cooperation with actin-binding Actin filaments are remarkably highly accumulated in a proteins (Achard et al. 2010), such as ADF/cofilin, actin- dendritic spine (head) compared with the parent dendrite related proteins Arp2 and Arp3 (Arp2/3) complex (Pollard (Fig. 1d and e). Although the accumulation mechanism has and Beltzner 2002), and WASP/Scar family (Symons et al. yet to be elucidated, it is known that actin filaments in 1996), when the filaments show isotropic orientation. dendritic spines have a unique character because of a high Fluorescent recovery after photobleaching revealed the rapid drebrin content (Sekino et al. 2007). turnover of actin in dendritic spines (Star et al. 2002). This Variations in the helical structure of actin filaments can be suggests that the treadmilling reaction of actin filament modulated by the binding of actin-binding proteins (Sharma occurs in dendritic spines. However, because a single- et al. 2012). The pitch of an actin filament plays an important molecule imaging assay indicates that F-actin in dendritic role in modifying the relationship (binding activity) between spines is made mostly of short filaments and is not well actin filaments and actin-regulating proteins. A typical side- aligned (Tatavarty et al. 2009), the treadmilling reaction binding protein of actin filament, tropomyosin, forms a helix might not generate the driving force that changes spine pitch of 36.5 nm, which is similar to the pitch of bared morphology. double helix of actin protomers. In contrast, drebrin forms In vitro studies have demonstrated that the total amount of the 40.0 nm pitch of actin filaments (Sharma et al. 2011). actin filaments is regulated by the amount of monomeric The difference in the structural and dynamic states of actin actin and ATP concentration (Korn et al. 1987), while the filaments is thought to influence their response to other actin- polymerization speed and the final length of actin filaments regulating proteins. While tropomyosin-binding actin fila- are regulated by the number of seeds for polymerization (the ments are biochemically registant against gelsolin, a severing initial number of free ends of the actin filaments). In vivo, protein of actin filaments, drebrin-binding actin (DB-actin) however, the content of actin filaments is much lower than filaments are labile against gelsolin (Ishikawa et al. 1994). what has been deduced from the in vitro study. Although the Similarly, while an actin depolymerizing reagent, cytocha- mechanism is not yet fully elucidated, various kinds of actin- lasin D, induces the depolymerization of actin filaments in binding proteins are thought to play important roles in neuronal cell bodies and dendritic shafts, it paradoxically regulating the content of actin filaments in vivo. When induces the increase in DB-actin filaments in dendritic spines cultured neurons are treated by latrunculin A, which (Takahashi et al. 2009). sequestrates actin monomers, actin filaments disappear. The difference of actin filament character also affects the Thus, a possible regulatory mechanism of biased actin higher structures composed of actin filaments, such as bundle filament distribution within a cell is a local sequestration of formation and network formation. DB-actin filaments form actin monomers by actin sequestering proteins such as thicker winding bundles and sometimes circling bundles, b-thymosin and ADF/cofilin (Pollard and Borisy 2003). while tropomyosin-binding actin filaments form straight

© 2013 International Society for Neurochemistry, J. Neurochem. (2013) 126, 155--164 158 T. Shirao and C. Gonzalez-Billault bundles within a cell, called stress fibers, in cooperation with factor (BDNF) increases the entry of microtubules into a-actinin. Replacing tropomyosin with drebrin through the dendritic spines. Nocodazole inhibits BDNF-induced den- cDNA transfection technique resulted in stress fibers chang- dritic spine formation by impairing microtubule polymeriza- ing into thick winding actin bundles (Shirao et al. 1994). tion. In contrast, a small concentration of taxol, which Thus, it can be concluded that the cell forms many enhances microtubule polymerization, increases BDNF- protrusions, some of which sometimes become much longer induced dendritic spine formation, indicating that microtubule than the cell length (Shirao et al. 1992). (MT) dynamics is regulated by either eliciting the formation A dendritic spine contains two distinct pools of F-actin. of new dendritic spines or inhibiting the disassembly of The dynamic pool of actin filaments is located at the tip of previously formed spines (Gu et al. 2008). The fact that the spine, whereas the stable pool is located in the core of the microtubules are found in dendritic spines in an activity- spine (Honkura et al. 2008). Interestingly, DB-actin fila- dependent manner suggests that this is not a stochastic ments are located in the core of spines (Aoki et al. 2005); process (Hu et al. 2008). Microtubules that penetrate den- they are also capable of forming higher order complexes dritic spines are highly dynamic and are decorated at their (Grintsevich et al. 2010). It is therefore suggested that DB- plus end with the MT plus end tracking protein +TIP protein, actin filaments compose the stable pool of actin filaments in EB3 (Hu et al. 2008; Jaworski et al. 2009). In contrast, the dendritic spine core. microtubules at the dendritic shaft are more stable (Kaech et al. 2001; Jaworski et al. 2009). Interestingly, inactivation Overview of microtubule of EB3 using shRNA reduces dendritic spine formation (Gu Microtubules are polarized heteropolymers composed of a- et al. 2008; Jaworski et al. 2009). and b-tubulin subunits, which are oriented in a head-to-tail arrangement, with b subunit projecting to the fast-growing Coupling of microtubule dynamics to actin filament end. Tubulin subunits polymerize in the presence of GTP dynamics (Vallee 1986). Microtubules display two functionally differ- The presence of microtubules containing EB3 at dendritic ent ends. While the slow growing end, termed the minus end, spines regulates the assembly of actin filaments, as suggested is attached to the microtubule organizing center, the fast- by experiments using jasplakinolide treatments to reverse growing end, termed the plus end, projects to the cell EB3-inactivation (Jaworski et al. 2009). The presence of periphery. The dynamic behavior of microtubules is charac- +TIP proteins decorating dynamic microtubules may have terized by the growing and shrinking of the plus end in a profound implications for the capture process of microtu- process termed dynamic instability (Mitchison and Kirschner bules, coupling MT dynamics with actin polymerization. The 1984). Thus microtubules can polymerize and depolymerize binding activity of EB3 to drebrin may contribute to the according to cell requirements. The half-life of microtubules interaction between microtubule and actin filaments (Geraldo is also contributed by several molecular mechanisms that et al. 2008). Coupling microtubule and actin filament include the binding of microtubule-associated protein dynamics might be essential for temporal and local regula- (MAPs, TIPs), the post-translational modification in tubulin tion of dendritic spines. In this context, other proteins could and the difference of tubulin isotypes (Conde and Caceres mediate such a coupling as well as EB3. 2009; Janke and Bulinski 2011). IQ motif containing GTPase activating protein (IQGAP), a scaffold protein highly expressed in neurons, may Microtubules in dendritic spines coordinate the dynamics of both microtubules and actin The presence of microtubules and their associated proteins in filaments (Jausoro et al. 2012). IQGAP can interact not dendritic spines has been controversial. Electron microscopic only with Rac and Cdc42 (Kuroda et al. 1996) but also studies conducted in the early 1980s suggest that microtu- with cytoplasmic linker protein 170 (Fukata et al. 2002) bules are capable of penetrating dendritic spines (Westrum and EB1 (Watanabe et al. 2009). Recently, it has been et al. 1980; Gray et al. 1982). However, technical caveats shown that IQGAP1 promotes both dendrite development led to the assumption that dendritic spines were devoid of (Swiech et al. 2011) and dendritic spine formation (Gao microtubules (Fiala et al. 2003). In the last 5 years, several et al. 2011). Moreover, IQGAP1 also plays a role in studies have drawn the attention of neurobiologists to a synaptic plasticity and memory formation (Schrick et al. possible role of microtubules in dendritic spines. Using 2007; Gao et al. 2011). As IQGAP can interact with +TIPs fluorescence-tagged proteins coupled with live 3D imaging, proteins in microtubules, it is possible that dynamic Gu et al. (2008) showed the presence of microtubules in microtubules penetrating dendritic spines are necessary to dendritic spines. Microtubules are mainly found in mush- promote IQGAP functions. Interestingly, it has been shown room-type dendritic spines (Fig. 1e). that an IQGAP mutant lacking its C-terminal domain, the The presence of microtubules in dendritic spines is domain involved in CLIP170 binding, does not decrease the correlated with synaptic activity. Brain-derived neurotrophic number of dendritic spines in cultured neurons, but

© 2013 International Society for Neurochemistry, J. Neurochem. (2013) 126, 155--164 Actin and microtubule in dendritic spine 159 decreases the proportion of mushroom-type spines (Jausoro Role of actin filaments in dendritic spine et al. 2013). formation Stable and dynamic microtubules From dendritic filopodia to spines The presence of two populations of stable and dynamic Dendritic filopodia show higher motility than dendritic spines, microtubules at dendrites may reflect some specialization for while dendritic spines show plastic morphological changes microtubules penetrating dendritic spines. MAP2 is a potent depending on synaptic activity. Dendritic filopodia have microtubule stabilizing factor that promotes the formation of network-like organization of actin filaments (Korobova and dense and stable microtubule bundles (Takemura et al. Svitkina 2010), which is unusual for highly elongated 1992). Microtubules at dendritic spines seem dynamic as membrane protrusions, where tight actin filament bundles, live imaging experiments using MAP2-GFP failed to identify such as fascin-bundling actin filaments, are obligatory. The microtubules at dendritic spines (Kaech et al. 2001). Among network-like organization is thought to make dendritic neuronal MAPs, MAP1B does not stimulate microtubule filopodia more elastic, allowing continuous morphological bundling, being a good candidate for maintaining a popula- changes. On the other hand, dendritic spines contain stable tion of more dynamic microtubules (Black et al. 1994; actin filaments in the core region of dendritic spines. The stable Gonzalez-Billault and Avila 2000; Gonzalez-Billault et al. actin filaments probably help keep the spine morphology 2001). Thus, under some conditions, microtubules present at constant until the occurrence of some special synaptic activity, dendritic spines are likely to contain MAP1B (Tortosa et al. such as tetanic stimulation, and the induction of synaptic 2011). plasticity. Although the properties of stable actin filaments in Neurons lacking MAP1B show a robust decrease in dendritic spines are not yet elucidated, drebrin is thought to be mushroom-type spines concomitantly with increased thin- involved in the stabilization of actin filaments (Fig. 2). type spines, suggesting that dendritic spine maturation is impaired. Indeed, neurons lacking MAP1B show decreased Accumulation of DB-actin filaments at the nascent miniature excitatory postsynaptic currents (mEPSCs) (Tor- postsynaptic sites tosa et al. 2011). Interestingly, MAP1B deficiency leads to There are two hypotheses as to how spine synapses are increased stable microtubules at the expense of the more formed. One is that shaft synapses change to spine synapses, dynamic microtubules (Gonzalez-Billault et al. 2001). This while the other maintains that presynaptic contact induces the may be contributed in part by the interaction of MAP1B with change in filopodia into dendritic spines. In either case, soon the enzyme responsible for tubulin tyrosination, tubulin after an axon comes into contact with a dendrite or a tyrosine ligase (Utreras et al. 2008). dendritic filopodium, presynaptic molecules such as bassoon The functional consequences of MTs entry into dendritic and piccolo begin to accumulate. On the other hand, PSD-95 spines remain an open field. In a recent study, Hu et al. have accumulation occurs in 1 h after the contact (Friedman et al. shown that dynamic microtubules promote the accumulation 2000). of postsynaptic density (PSD)-95 in dendritic spines after One of the initial events to occur at the contact sites of BDNF treatments (Hu et al. 2011). NMDA receptor activa- dendritic filopodia or shaft is the accumulation of drebrin tion induces dendritic spine enlargement. Because this both in vivo (Aoki et al. 2005) and in vitro (Takahashi et al. enlargement is more prominent in spines containing micro- 2003). In vitro analysis shows that these filopodia have DB- tubules, it is suggested that NMDA-dependent microtubule actin clusters, but lack scaffold proteins such as PSD-95 polymerization promotes dendritic spine enlargement (Mer- (Takahashi et al. 2003), indicating that these filopodia are riam et al. 2011). Interestingly, chemically induced long different from mature dendritic spines (Fig. 2c and d). Once term depression suppresses the entry of microtubules into DB-actin filament clusters are formed, PSD-95 and gluta- dendritic spines (Kapitein et al. 2011). About 1–2% of mate receptors are accumulated into these clusters (Mizui dendritic spines contain MTs, suggesting that MTs’ entry et al. 2005). On the other hand, when the cluster formation into dendritic spines is a transient and dynamic phenomenon is inhibited by the suppression of drebrin expression, (Gu et al. 2008; Hu et al. 2008; Jaworski et al. 2009; accumulation of PSD-95 at the postsynaptic sites is Tortosa et al. 2011). The presence of dynamic MTs in suppressed (Takahashi et al. 2003). Interestingly, the excit- dendritic spines suggests a role of MT in the conversion of atory synaptic activity is necessary for the cluster formation thin-type spines into mushroom-type spines. A potential of DB-actin filaments at postsynaptic sites (Takahashi et al. microtubule-based transport mechanism occurring at den- 2009). These suggest that the cluster formation of DB-actin dritic spines would be complementary to the widely known filaments is one of the initial events occurring at the nascent myosin-dependent mechanism for material transport into excitatory postsynaptic site and plays a pivotal role in spine spines (Dent et al. 2011). Moreover, microtubules in formation. Although the molecular mechanism is not dendritic spines might be involved in the regulatory mech- fully understood, a-amino-3-hydroxy-5-methyl-4-isoxazole- anism of actin dynamics within dendritic spines. propionic acid (AMPA) receptor-dependent stabilization of

© 2013 International Society for Neurochemistry, J. Neurochem. (2013) 126, 155--164 160 T. Shirao and C. Gonzalez-Billault drebrin has been shown to be involved in the mechanism by contribute to the inhibition of ADF/cofilin activity (Rex means of fluorescence recovery after photobleaching analysis et al. 2009). (Takahashi et al. 2009). Competitive role of drebrin and ADF/cofilin in Roles of Rac1A and RhoA in actin filaments in dendritic spines dendritic spines Drebrin has an ADF/cofilin-homology domain in its As Luo’s group showed that Rac1 and Rho functions are N-terminal region and binds to actin filaments with similar À7 À6 essential to promote dendritic spine formation (Nakayama KD (1.2 9 10 M) to tropomyosin (5 9 10 M) and et al. 2000), the role of small Rho GTPases in the formation ADF/cofilin (2 9 10À7 M) (Ishikawa et al. 1994). While of dendritic spines has been extensively reviewed (Luo 2002; ADF/cofilin binds to both actin monomer and actin filaments, Newey et al. 2005; Tada and Sheng 2006; Yoshihara et al. drebrin does not bind to actin monomer. In addition, drebrin 2009). Rac1 inactivation leads to the progressive elimination binds to actin filaments via a unique actin-binding domain of dendritic spines, while RhoA activation promotes a other than ADF/cofilin-homology domain (Hayashi et al. simplification of dendritic branching (Nakayama et al. 1999). Interestingly, an actin protomer in the filament has 2000). Recently, using two-photon lifetime microscopy, two drebrin-binding sites, and thus each drebrin molecule Murakoshi et al. (2011) measured the activities of small Rho can bind either one protomer or two protomers. DB-actin GTPases at dendritic spines. Both RhoA and Cdc42 activities filaments can form five different filament structures (Grin- rely on transient CamKII-activity to regulate spine dynamic tsevich et al. 2010). properties. MAP1B can also promote actin microfilament Drebrin and ADF/cofilin competitively bind to actin polymerization through a mechanism that involves the filaments, and the pitch of ADF/cofilin-binding actin fila- participation of small GTPases Rac1/Cdc42 and RhoA ments (28.7 nm) is much shorter than that of DB-actin (Montenegro-Venegas et al. 2010). Interestingly, MAP1B filaments (Sharma et al. 2011). Dephosphorylation of ADF/ binds to Tiam1, a guanine exchanging factor that promotes cofilin diminishes its actin-binding activity, but that of Rac1 activity (Henrıquez et al. 2012). drebrin does not affect its actin-binding activity. Thus, it is Morphological changes in dendritic spines are thought to suggested that the regulation of the balance between drebrin be linked to information processing, and the regulation of and ADF/cofilin plays a pivotal role in actin dynamics in actin dynamics, therefore, would be crucial in controlling dendritic spines (Fig. 2e and f). information processing. As a matter of fact, long-term potentiation (LTP) induces actin polymerization, and conse- Dendritic spine abnormalities in neuropsychiatric, quently increases the volume of dendritic spines. A change in neurodevelopmental, and neurodegenerative dendritic spine volume correlates with a shift in the ratio of pathologies actin monomers and actin filaments (Okamoto et al. 2004). In addition, NMDA receptor activation induces the transient Several neuropsychiatric, neurodevelopmental and neurode- translocation of drebrin from dendritic spine head to the generative diseases show alterations in the morphology of parent dendrite, causing the reorganization of actin cytoskel- dendrites. These changes include abnormal dendritic branch- eton in dendritic spines (Sekino et al. 2006). Among several ing, fragmentation of dendrites and altered morphology of actin-regulating proteins that control dendritic spine mor- dendritic spines. This is perfectly exemplified in Fragile X phogenesis, ADF/cofilin is essential to maintain a soluble syndrome and Down syndrome. The pathologies of these pool of actin monomers. ADF/cofilin promotes actin turnover diseases are accompanied by the decrease in mushroom-type and regulates dendritic spine morphology (Hotulainen et al. spines and the concomitant increase in thin-type spines (Irwin 2009). ADF/cofilin functions are regulated by serine 3 et al. 2000; Kaufmann and Moser 2000). Fragile X mental phosphorylation by LIM kinase 1, which inhibits ADF/ retardation protein, the protein responsible for Fragile X cofilin function (Arber et al. 1998). Inactivation of LIMK1 syndrome, binds and regulates the activity of cytoplasmic by gene targeting results in abnormal dendritic spine density FMR1-interacting protein 1 (CYFIP1), a Rac1 effector protein and morphology, which is characterized by the presence of (Schenck et al. 2003). Interestingly, mutations in other genes abundant thin-type spines, impairing normal synaptic trans- related to Rho GTPases functions are linked to non-syndromic mission (Meng et al. 2002). mental retardation such as oligophrenin-1, which is a Rho LIMK and ADF/cofilin are effector proteins, which are GAP protein (Billuart et al. 1998), and a-PAK-interactive downstream of Rac and RhoA signaling pathway (Gonzalez- exchange factor (Pix), a guanine exchange factor for Rac Billault et al. 2012). It has been shown that RhoA activity is (Manser et al. 1998). LIMK, a protein kinase activated by required for LTP in a mechanism involving ADF/cofilin small GTPases RhoA and Rac1, is mutated in Williams phosphorylation (Rex et al. 2009). Therefore, RhoA may syndrome leading to mental retardation (Bellugi et al. 1999). promote actin stabilization by enhancing ADF/cofilin phos- The morphology and density of dendritic spines are altered phorylation. Concomitantly, Rac1 and cdc42 may also also in autism spectrum disorders (ASD). In humans,

© 2013 International Society for Neurochemistry, J. Neurochem. (2013) 126, 155--164 Actin and microtubule in dendritic spine 161 alterations in neurexin and neuroligin genes are implicated in Future implications autism (Sudhof€ 2008). Neuroligin-3 and neuroligin-4 are adhesion proteins present at postsynaptic sites, which are Since their discovery, dendritic spines have captured the mutated in siblings with ASD (Jamain et al. 2003). The attention of neurobiologists. Containing thousands of con- expression of mutated forms of neuroligin-3 and neurologin- centrated proteins, they are the site for excitatory synaptic 4 in cultured neurons increases dendritic spine density (Chih transmission. Over the last few years, newfound information et al. 2004). Similarly, mutations in neurexin 1, the presyn- regarding the local regulation of cytoskeleton, signaling aptic binding partner for neuregulins, also lead to abnormal complex, and receptor endocytosis/recycling has provided dendritic spine density (Sudhof€ 2008). Shank scaffolding vital clues to understanding the normal and pathological proteins have also been linked to ASD (Durand et al. 2007; functions of dendritic spines. However, many questions still Sato et al. 2012). Shank2 and Shank3 mutant variants lead to remain unanswered. What is the molecular mechanism that increased dendritic spine size and density, respectively promotes maturation and/or stabilization of dendritic spines? (Roussignol et al. 2005; Steiner et al. 2008). Can signaling protein complexes locally alter both actin and Finally, neurodegenerative conditions such as Alzheimer’s microtubule dynamics in dendritic spines? Which hubs disease (AD) also display dendritic spine alterations. The would be required to simultaneously control receptor endo- most accepted risk factor for AD is the allele e4 of the cytosis, cytoskeleton dynamics, protein synthesis, and apolipoprotein E (APOE). ApoE e4 shows reduced density of signaling cascades? These and other intriguing questions dendritic spines (Ji et al. 2003). In contrast, the over- will likely be answered in the coming years, helping sustain expression of the allele e2 in the mouse model of AD can the interest of cellular-molecular neurobiologist in this tiny, reverse the dendritic spine pathology (Lanz et al. 2003). but tremendously important domain. Drebrin and cofilin are also involved in AD (for review, see Kojima and Shirao 2007). Drebrin level is markedly reduced in the brains of AD (Harigaya et al. 1996; Hatanpaa Acknowledgements et al. 1999; Counts et al. 2006). Furthermore, various The authors declare no conflict of interest. studies using AD animal models indicate that drebrin is involved in the pathogenesis of AD (Calon et al. 2004; Mahadomrongkul et al. 2005; Lacor et al. 2007). It has been References recently reported that drebrin A has a causal role in Achard V., Martiel J. L., Michelot A., Guerin C., Reymann A. C., compromising activity-dependent glutamate receptor traf- Blanchoin L. and Boujemaa-Paterski R. (2010) A “primer”-based ficking in the AD animal model (Lee and Aoki, 2012). mechanism underlies branched actin filament network formation Similarly, cofilin phosphorylation is altered in cultured cells and motility. Curr. Biol. 20, 423–428. exposed to Ab peptide or in the mouse model of AD Aoki C., Sekino Y., Hanamura K., Fujisawa S., Mahadomrongkul V., Ren Y. and Shirao T. (2005) Drebrin A is a postsynaptic protein (Minamide et al. 2000; Heredia et al. 2006; Mendoza- that localizes in vivo to the submembranous surface of dendritic Naranjo et al. 2012). Interestingly, drebrin is also reduced sites forming excitatory synapses. J. Comp. Neurol. 483, 383–402. in Down syndrome (Shim and Lubec 2002). In addition, the Arber S., Barbayannis F. A., Hanser H., Schneider C., Stanyon C. A., drebrin gene (dbn1) has a cross-talking point of basic helix- Bernard O. and Caroni P. (1998) Regulation of actin dynamics fi 393 loop-helix-PAS transcriptional factor related to Down syn- through phosphorylation of co lin by LIM-kinase. Nature , 805–809. drome pathology, such as NXF (activation) and Sim2 Bamburg J. R., McGough A. and Ono S. (1999) Putting a new twist on (repression), in its promoter regions (Ooe et al. 2004). actin: ADF/cofilins modulate actin dynamics. Trends Cell Biol. 9, Another key molecule to regulate actin dynamics in 364–370. dendritic spines is the protein kinase PAK, which is a Bellugi U., Lichtenberger L., Mills D., Galaburda A. and Korenberg J. downstream effector of Rac1. Both in animal models R. (1999) Bridging cognition, the brain and molecular genetics: evidence from Williams syndrome. Trends Neurosci. 22, 197–207. recapitulating AD and brain samples derived from post- Billuart P., Bienvenu T., Ronce N. et al. (1998) Oligophrenin-1 encodes mortem patients, PAK activation is markedly reduced (Zhao a rhoGAP protein involved in X-linked mental retardation. Nature et al. 2006). Moreover, PAK1 and PAK3 promote the 392, 923–926. formation and growth of dendritic spines by regulating the Black M. M., Slaughter T. and Fischer I. (1994) Microtubule-associated phosphorylation of the myosin II regulatory light chain protein 1b (MAP1b) is concentrated in the distal region of growing axons. J. Neurosci. 14, 857–870. kinase and stimulating myosin II (Zhang et al. 2005). Calon F., Lim G. P., Yang F. et al. (2004) Docosahexaenoic acid Therefore, a likely working hypothesis is that aberrant protects from dendritic pathology in an Alzheimer’s disease mouse regulation of cytoskeleton dynamics in spines contributes to model. Neuron 43, 633–645. several pathological conditions that affect dendritic spine Carlier M. F., Laurent V., Santolini J., Melki R., Didry D., Xia G. X., morphology, thus preventing appropriate synaptic function Hong Y., Chua N. H. and Pantaloni D. (1997) Actin depolymerizing factor (ADF/cofilin) enhances the rate of filament and plasticity.

turnover: implication in actin-based motility. J. Cell Biol. 136, Henrıquez D. R., Bodaleo F. J., Montenegro-Venegas C. and Gonzalez- 1307–1322. Billault C. (2012) The light chain 1 subunit of the microtubule- Chih B., Afridi S. K., Clark L. and Scheiffele P. (2004) Disorder- associated protein 1B (MAP1B) is responsible for Tiam1 binding associated mutations lead to functional inactivation of neuroligins. and Rac1 activation in neuronal cells. PLoS ONE 7, e53123. Hum. Mol. Genet. 13, 1471–1477. Heredia L., Helguera P., de Olmos S. et al. (2006) Phosphorylation of Conde C. and Caceres A. (2009) Microtubule assembly, organization and actin-depolymerizing factor/cofilin by LIM-kinase mediates dynamics in axons and dendrites. Nat. Rev. Neurosci. 10, 319–332. amyloid beta-induced degeneration: a potential mechanism of Counts S. E., Nadeem M., Lad S. P., Wuu J. and Mufson E. J. (2006) neuronal dystrophy in Alzheimer’s disease. J. Neurosci. 26(24), Differential expression of synaptic proteins in the frontal and 6533–6542. temporal cortex of elderly subjects with mild cognitive Honkura N., Matsuzaki M., Noguchi J., Ellis-Davies G. C. and Kasai H. impairment”. J. Neuropathol. Exp. Neurol. 65, 592–601. (2008) The subspine organization of actin fibers regulates the Dent E. W., Merriam E. B. and Hu X. (2011) The dynamic cytoskeleton: structure and plasticity of dendritic spines. Neuron 57, 719–729. backbone of dendritic spine plasticity. Curr. Opin. Neurobiol. 21, Hotulainen P., Llano O., Smirnov S., Tanhuanp€a€a K., Faix J., Rivera C. 175–181. and Lappalainen P. (2009) Defining mechanisms of actin Durand C. M., Betancur C., Boeckers T. M. et al. (2007) Mutations in polymerization and depolymerization during dendritic spine the gene encoding the synaptic scaffolding protein SHANK3 are morphogenesis. J. Cell Biol. 185, 323–339. associated with autism spectrum disorders. Nat. Genet. 39,25–27. Hu X., Viesselmann C., Nam S., Merriam E. and Dent E. W. (2008) Fiala J. C., Kirov S. A., Feinberg M. D., Petrak L. J., George P., Activity-dependent dynamic microtubule invasion of dendritic Goddard C. A. and Harris K. M. (2003) Timing of neuronal and spines. J. Neurosci. 28, 13094–13105. glial ultrastructure disruption during brain slice preparation and Hu X., Ballo L., Pietila L., Viesselmann C., Ballweg J., Lumbard D., recovery in vitro. J. Comp. Neurol. 465,90–103. Stevenson M., Merriam E. and Dent E. W. (2011) BDNF-induced Friedman H. V., Bresler T., Garner C. C. and Ziv N. E. (2000) Assembly increase of PSD-95 in dendritic spines requires dynamic of new individual excitatory synapses: time course and temporal microtubule invasions. J. Neurosci. 31, 15597–15603. order of synaptic molecule recruitment. Neuron 27,57–69. Huxley H. E. (1963) Electron microscopic studies on structure of natural Fukata M., Watanabe T., Noritake J., Nakagawa M., Yamaga M., and synthetic protein filaments from striated muscle. J. Mol. Biol. Kuroda S., Matsuura Y., Iwamatsu A., Perez F. and Kaibuchi K. 7, 281–308. (2002) Rac1 and Cdc42 capture microtubules through IQGAP1 Irwin S. A., Galvez R. and Greenough W. T. (2000) Dendritic spine and CLIP-170. Cell 109, 873–885. structural anomalies in fragile-X mental retardation syndrome. Gao C., Frausto S. F., Guedea A., Tronson N., Jovasevic V., Cereb. Cortex 10, 1038–1044. Leaderbrand K., Corcoran K., Guzman G., Swanson G. T. and Ishikawa H., Bischoff R. and Holtzer H. (1969) Formation of arrowhead Radulovic J. (2011) IQGAP1 regulates NR2A signaling, spine complexes with heavy meromyosin in a variety of cell types. density, and cognitive processes. J. Neurosci. 31, 8533–8542. J. Cell Biol. 43, 312–328. Geraldo S., Khanzada U. K., Parsons M., Chilton J. K. and Gordon- Ishikawa R., Hayashi K., Shirao T., Xue Y., Takagi T., Sasaki Y. and Weeks P. R. (2008) Targeting of the F-actin-binding protein Kohama K. (1994) Drebrin, a development-associated brain drebrin by the microtubule plus-tip protein EB3 is required for protein from rat embryo, causes the dissociation of tropomyosin neuritogenesis. Nat. Cell Biol. 10, 1181–1189. from actin filaments. J. Biol. Chem. 269, 29928–29933. Gonzalez-Billault C. and Avila J. (2000) Molecular genetic approaches Jamain S., Quach H., Betancur C. et al. (2003) Mutations of the X- to microtubule-associated protein function. Histol. Histopathol. 15, linked genes encoding neuroligins NLGN3 and NLGN4 are 1177–1183. associated with autism. Nat. Genet. 34,27–29. Gonzalez-Billault C., Avila J. and Caceres A. (2001) Evidence for the Janke C. and Bulinski J. C. (2011) Post-translational regulation of the role of MAP1B in axon formation. Mol. Biol. Cell 12, 2087–2098. microtubule cytoskeleton: mechanisms and functions. Nat. Rev. Gonzalez-Billault C., Munoz-Llancao~ P., Henriquez D. R., Wojnacki J., Mol. Cell Biol. 12, 773–786. Conde C. and Caceres A. (2012) The role of small GTPases in Jausoro I., Mestres I., Remedi M., Sanchez M. and Caceres A. (2012) neuronal morphogenesis and polarity. Cytoskeleton (Hoboken) 69, IQGAP1: a microtubule-microfilament scaffolding protein with 464–485. multiple roles in nerve cell development and synaptic plasticity. Gray E. G., Westrum L. E., Burgoyne R. D. and Barron J. (1982) Histol. Histopathol. 27, 1385–1394. Synaptic organisation and neuron microtubule distribution. Cell Jausoro I., Mestres I., Quassollo G., Masseroni L., Heredia F. and Tissue Res. 226, 579–588. Caceres A. (2013) Regulation of spine density and morphology by Grintsevich E. E., Galkin V. E., Orlova A., Ytterberg A. J., Mikati M. IQGAP1 protein domains. PLoS ONE 8, e56574. M., Kudryashov D. S., Loo J. A., Egelman E. H. and Reisler E. Jaworski J., Kapitein L. C., Gouveia S. M. et al. (2009) Dynamic (2010) Mapping of drebrin binding site on F-actin. J. Mol. Biol. microtubules regulate dendritic spine morphology and synaptic 398, 542–554. plasticity. Neuron 61,85–100. Gu J., Firestein B. L. and Zheng J. Q. (2008) Microtubules in dendritic Ji Y., Gong Y., Gan W., Beach T., Holtzman D. M. and Wisniewski T. spine development. J. Neurosci. 28, 12120–12124. (2003) Apolipoprotein E isoform-specific regulation of dendritic Harigaya Y., Shoji M., Shirao T. and Hirai S. (1996) Disappearance of spine morphology in apolipoprotein E transgenic mice and actin-binding protein, drebrin, from hippocampal synapses in Alzheimer’s disease patients. Neuroscience 122, 305–315. Alzheimer’s disease. J. Neurosci. Res. 43(1), 87–92. Kaech S., Parmar H., Roelandse M., Bornmann C. and Matus A. (2001) Hatanpaa K., Isaacs K. R., Shirao T., Brady D. R. and Rapoport S. I. Cytoskeletal microdifferentiation: a mechanism for organizing (1999) Loss of proteins regulating synaptic plasticity in normal morphological plasticity in dendrites. Proc. Natl Acad. Sci. USA aging of the human brain and in Alzheimer disease. 98, 7086–7092. J. Neuropathol. Exp. Neurol. 58, 637–643. Kapitein L. C., Yau K. W., Gouveia S. M., van der Zwan W. A., Wulf P. Hayashi K., Ishikawa R., Kawai-Hirai R., Takagi T., Taketomi A. and S., Keijzer N., Demmers J., Jaworski J., Akhmanova A. and Shirao T. (1999) Domain analysis of the actin-binding and actin- Hoogenraad C. C. (2011) NMDA receptor activation suppresses remodeling activities of drebrin. Exp. Cell Res. 253, 673–680. microtubule growth and spine entry. J. Neurosci. 31, 8194–81209.

Kaufmann W. E. and Moser H. W. (2000) Dendritic anomalies in (2010) MAP1B regulates axonal development by modulating Rho- disorders associated with mental retardation. Cereb. Cortex 10, GTPase Rac1 activity. Mol. Biol. Cell 21, 3518–3528. 981–991. Murakoshi H., Wang H. and Yasuda R. (2011) Local, persistent Kojima N. and Shirao T. (2007) Synaptic dysfunction and disruption of activation of Rho GTPases during plasticity of single dendritic postsynaptic drebrin-actin complex: a study of neurological spines. Nature 472, 100–104. disorders accompanied by cognitive deficits. Neurosci. Res. 58, Nakamura F., Stossel T. P. and Hartwig J. H. (2011) The filamins: 1–5. organizers of cell structure and function. Cell Adh. Migr. 5, 160–169. Korn E. D., Carlier M. F. and Pantaloni D. (1987) Actin polymerization Nakayama A. Y., Harms M. B. and Luo L. (2000) Small GTPases Rac and ATP hydrolysis. Science 238, 638–644. and Rho in the maintenance of dendritic spines and branches in Korobova F. and Svitkina T. (2010) Molecular architecture of synaptic hippocampal pyramidal neurons. J. Neurosci. 20, 5329–5338. actin cytoskeleton in hippocampal neurons reveals a mechanism of Newey S. E., Velamoor V., Govek E. E. and Van Aelst L. (2005) Rho dendritic spine morphogenesis. Mol. Biol. Cell 21, 165–176. GTPases, dendritic structure, and mental retardation. J. Neurobiol. Kuroda S., Fukata M., Kobayashi K., Nakafuku M., Nomura N., 64,58–74. Iwamatsu A. and Kaibuchi K. (1996) Identification of IQGAP as a Okamoto K., Nagai T., Miyawaki A. and Hayashi Y. (2004) Rapid and putative target for the small GTPases, Cdc42 and Rac1. J. Biol. persistent modulation of actin dynamics regulates postsynaptic Chem. 271, 23363–23367. reorganization underlying bidirectional plasticity. Nat. Neurosci. 7, Lacor P. N., Buniel M. C., Furlow P. W., Clemente A. S., Velasco P. T., 1104–1112. Wood M., Viola K. L. and Klein W. L. (2007) Abeta oligomer- Ooe N., Saito K., Mikami N., Nakatuka I. and Kaneko H. (2004) induced aberrations in synapse composition, shape, and density Identification of a novel basic helix-loop-helix-PAS factor, NXF, provide a molecular basis for loss of connectivity in Alzheimer’s reveals a Sim2 competitive, positive regulatory role in dendritic- disease. J. Neurosci. 27, 796–807. cytoskeleton modulator drebrin gene expression. Mol. Cell. Biol. Lanz T. A., Carter D. B. and Merchant K. M. (2003) Dendritic spine loss 24, 608–616. in the hippocampus of young PDAPP and Tg2576 mice and its Pollard T. D. and Beltzner C. C. (2002) Structure and function of the prevention by the ApoE2 genotype. Neurobiol. Dis. 13, 246–253. Arp2/3 complex. Curr. Opin. Struct. Biol. 12, 768–774. Lee D. and Aoki C. (2012) Presenilin conditional double knockout mice Pollard T. D. and Borisy G. G. (2003) Cellular motility driven by exhibit decreases in drebrin A at hippocampal CA1 synapses. assembly and disassembly of actin filaments. Cell 112, 453–465. Synapse 66, 870–879. Pollard T. D. and Mooseker M. S. (1981) Direct measurement of actin Luo L. (2002) Actin cytoskeleton regulation in neuronal morphogenesis polymerization rate constants by electron microscopy actin and structural plasticity. Annu. Rev. Cell Dev. Biol. 18, 601–635. filaments nucleated by isolated microvillus cores. J. Cell Biol. Mahadomrongkul V., Huerta P. T., Shirao T. and Aoki C. (2005) 88, 654–659. Stability of the distribution of spines containing drebrin A in the Rex C. S., Chen L. Y., Sharma A., Liu J., Babayan A. H., Gall C. M. and sensory cortex layer I of mice expressing mutated APP and PS1 Lynch G. (2009) Different Rho GTPase-dependent signaling genes. Brain Res. 1064,66–74. pathways initiate sequential steps in the consolidation of long- Manser E., Loo T. H., Koh C. G., Zhao Z. S., Chen X. Q., Tan L., Tan I., term potentiation. J. Cell Biol. 186,85–97. Leung T. and Lim L. (1998) PAK kinases are directly coupled to Rochefort N. L. and Konnerth A. (2012) Dendritic spines: from structure the PIX family of nucleotide exchange factors. Mol. Cell 1, 183– to in vivo function. EMBO Rep. 13, 699–708. 192. Roussignol G., Ango F., Romorini S., Tu J. C., Sala C., Worley P. F., Mendoza-Naranjo A., Contreras-Vallejos E., Henriquez D. R., Otth C., Bockaert J. and Fagni L.(2005)Shank expression is sufficient to Bamburg J. R., Maccioni R. B. and Gonzalez-Billault C. (2012) induce functional dendritic spine synapses in aspiny neurons. Fibrillar amyloid-b1-42 modifies actin organization affecting the J. Neurosci. 25:3560–3570. cofilin phosphorylation state: a role for Rac1/cdc42 effector Sasaki Y., Hayashi K., Shirao T., Ishikawa R. and Kohama K. (1996) proteins and the slingshot phosphatase. J. Alzheimers Dis. 29, Inhibition by drebrin of the actin-bundling activity of brain fascin, 63–77. a protein localized in filopodia of growth cones. J. Neurochem. 66, Meng Y., Zhang Y., Tregoubov V. et al. (2002) Abnormal spine 980–988. morphology and enhanced LTP in LIMK-1 knockout mice. Neuron Sato D., Lionel A. C., Leblond C. S. et al. (2012) SHANK1 deletions in 35, 121–133. males with autism spectrum disorder. Am.J.Hum.Genet.90, 879–887. Merriam E. B., Lumbard D. C., Viesselmann C., Ballweg J., Stevenson Schenck A., Bardoni B., Langmann C., Harden N., Mandel J. L. and M., Pietila L., Hu X. and Dent E. W. (2011) Dynamic microtubules Giangrande A. (2003) CYFIP/Sra-1 controls neuronal connectivity promote synaptic NMDA receptor-dependent spine enlargement. in Drosophila and links the Rac1 GTPase pathway to the fragile X PLoS ONE 6, e27688. protein. Neuron 38, 887–898. Minamide L. S., Striegl A. M., Boyle J. A., Meberg P. J. and Bamburg J. Schrick C., Fischer A., Srivastava D., Tronson N., Penzes P. and R. (2000) Neurodegenerative stimuli induce persistent ADF/ Radulovic J. (2007) N-cadherin regulates cytoskeletally associated cofilin-actin rods that disrupt distal neurite function. Nat. Cell IQGAP1/ERK signaling and memory formation. Neuron 55, 786– Biol. 2, 628–636. 798. Mitchison T. and Kirschner M. (1984) Dynamic instability of Sekino Y., Tanaka S., Hanamura K., Yamazaki H., Sasagawa Y., Xue microtubule growth. Nature 312, 237–242. Y., Hayashi K. and Shirao T. (2006) Activation of N-methyl-d- Mizui T., Sekino Y., Takahashi H., Yamazaki H. and Shirao T. (2005) aspartate receptor induces a shift of drebrin distribution: Overexpression of drebrin A in immature neurons induces the disappearance from dendritic spines and appearance in dendritic accumulation of F-actin and PSD-95 into dendritic filopodia, and shafts. Mol. Cell. Neurosci. 31, 493–504. the formation of large abnormal protrusions. Mol. Cell. Neurosci. Sekino Y., Kojima N. and Shirao T. (2007) Role of actin cytoskeleton in 30, 149–157. dendritic spine morphogenesis. Neurochem. Int. 51,92–104. Montenegro-Venegas C., Tortosa E., Rosso S., Peretti D., Bollati F., Sharma S., Grintsevich E. E., Phillips M. L., Reisler E. and Gimzewski J. Bisbal M., Jausoro I., Avila J., Caceres A. and Gonzalez-Billault C. K. (2011) Atomic force microscopy reveals drebrin induced

remodeling of f-actin with subnanometer resolution. Nano Lett. 11, Takahashi H., Yamazaki H., Hanamura K., Sekino Y. and Shirao T. 825–827. (2009) AMPA receptor inhibition causes abnormal dendritic spines Sharma S., Grintsevich E. E., Hsueh C., Reisler E. and Gimzewski J. K. by destabilizing drebrin. J. Cell Sci. 122, 1211–1229. (2012) Molecular cooperativity of drebrin1-300 binding and Takemura R., Okabe S., Umeyama T., Kanai Y., Cowan N. J. and structural remodeling of F-actin. Biophys. J . 103, 275–283. Hirokawa N. (1992) Increased microtubule stability and alpha Shim K. S. and Lubec G. (2002) Drebrin, a dendritic spine protein, is tubulin acetylation in cells transfected with microtubule-associated manifold decreased in brains of patients with Alzheimer’s disease proteins MAP1B, MAP2 or tau. J. Cell Sci. 103, 953–964. and Down syndrome. Neurosci. Lett. 324, 209–212. Tatavarty V., Kim E. J., Rodionov V. and Yu J. (2009) Investigating Shirao T., Kojima N. and Obata K. (1992) Cloning of drebrin A and sub-spine actin dynamics in rat hippocampal neurons with super- induction of neurite-like processes in drebrin-transfected cells. resolution optical imaging. PLoS ONE 4, e7724. NeuroReport 3, 109–112. Tortosa E., Montenegro-Venegas C., Benoist M., H€artel S., Gonzalez- Shirao T., Hayashi K., Ishikawa R., Isa K., Asada H., Ikeda K. and Billault C., Esteban J. A. and Avila J. (2011) Microtubule-associated Uyemura K. (1994) Formation of thick, curving bundles of actin protein 1B (MAP1B) is required for dendritic spine development and by drebrin A expressed in fibroblasts. Exp. Cell Res. 215, 145– synaptic maturation. J. Biol. Chem. 286, 40638–40648. 153. Utreras E., Jimenez-Mateos E. M., Contreras-Vallejos E., Tortosa E., Star E. N., Kwiatkowski D. J. and Murthy V. N. (2002) Rapid turnover Perez M., Rojas S., Saragoni L., Maccioni R. B., Avila J. and of actin in dendritic spines and its regulation by activity. Nat. Gonzalez-Billault C. (2008) Microtubule-associated protein 1B Neurosci. 5, 239–246. interaction with tubulin tyrosine ligase contributes to the control of Steiner P., Higley M. J., Xu W., Czervionke B. L., Malenka R. C. and microtubule tyrosination. Dev. Neurosci. 30, 200–210. Sabatini B. L. (2008) Destabilization of the postsynaptic density by Vallee R. B. (1986) Reversible assembly purification of microtubules PSD-95 serine 73 phosphorylation inhibits spine growth and without assembly-promoting agents and further purification of synaptic plasticity. Neuron 60, 788–802. tubulin, microtubule-associated proteins, and MAP fragments. Sudhof€ T. C. (2008) Neuroligins and neurexins link synaptic function to Methods Enzymol. 134,89–104. cognitive disease. Nature 455, 903–911. Watanabe T., Noritake J., Kakeno M. et al. (2009) Phosphorylation of Swiech L., Blazejczyk M., Urbanska M., Pietruszka P., Dortland B., CLASP2 by GSK-3beta regulates its interaction with IQGAP1, Malik A., Wulf P., Hoogenraad C. and Jaworski J. (2011) CLIP- EB1 and microtubules. J. Cell Sci. 122, 2969–2979. 170 and IQGAP1 cooperatively regulate dendrite morphology. Wegner A. (1976) Head to tail polymerization of actin. J. Mol. Biol. 108, J. Neurosci. 31, 4555–4568. 139–150. Symons M., Derry J. M., Karlak B., Jiang S., Lemahieu V., Mccormick Westrum L. E., Jones D. H., Gray E. G. and Barron J. (1980) Microtubules, F., Francke U. and Abo A. (1996) Wiskott-Aldrich syndrome dendritic spines and spine appratuses. Cell Tissue Res. 208, 171–181. protein, a novel effector for the GTPase CDC42Hs, is implicated in Yoshihara Y., De Roo M. and Muller D. (2009) Dendritic spine actin polymerization. Cell 84, 723–734. formation and stabilization. Curr. Opin. Neurobiol. 19, 146–153. Tada T. and Sheng M. (2006) Molecular mechanisms of dendritic spine Zhang H., Webb D. J., Asmussen H., Niu S. and Horwitz A. F. (2005) A morphogenesis. Curr. Opin. Neurobiol. 16,95–101. GIT1/PIX/Rac/PAK signaling module regulates spine morphogenesis Takahashi H., Sekino Y., Tanaka S., Mizui T., Kishi S. and Shirao T. and synapse formation through MLC. J. Neurosci. 25, 3379–3388. (2003) Drebrin-dependent actin clustering in dendritic filopodia Zhao L., Ma Q. L., Calon F. et al. (2006) Role of p21-activated kinase governs synaptic targeting of postsynaptic density-95 and dendritic pathway defects in the cognitive deficits of Alzheimer disease. Nat. spine morphogenesis. J. Neurosci. 23, 6586–6595. Neurosci. 9, 234–242.