Actin Regulatory Proteins In Dendritic Spines

Actin filaments are the predominant form of cytoskeleton in dendritic spines, which determines their morphology as well as functional characteristics. Recent FRAP studies have shown that actin in spines of cultured neurons undergoes rapid turnover, with a half-life of less than a minute20. Activity-dependent changes in spine size are associated with changes in the ratio of monomeric G-actin to filamentous F-actin, such that the increase in spine size observed during LTP shifts the balance toward F-actin21. In addition, several studies have indicated that mature dendritic spines are generated from filopodial precursors on dendrites. These long, thin, and highly motile protrusions may function as a "scout" to detect incoming axon terminals. Filopodia are characterized by actin bundles, whereas mature spines are likely to contain a more branched actin network22. Taken together these observations indicate that signals regulating the shape of the actin cytoskeleton are of fundamental importance for any type of structural change occurring in dendrites, either in a developmental phase or during plasticity. Knowledge about actin regulatory processes arises mainly from non-neuronal systems, such as fibroblasts. Here, nucleation of actin filaments, as well as the generation of branches on existing filaments, requires the Arp2/3 complex which is recruited to specific cellular sites by the VCA (verprolin homology, cofilin homology, acidic) domain of WASP (Wiskott-Aldrich-syndrome protein) family members (WASP, N-WASP, WAVE1-3; ref. 23). Activation of WASP proteins in different cellular pathways is achieved by exposure of the VCA region, which otherwise is locked in an intramolecular interaction in the resting state of the protein. In addition, the actin-binding protein cortactin contains an acidic region and may recruit the Arp2/3 complex24. Growth of filaments is stimulated by profilin, which aids the addition of monomeric actin to the growing or (+) end. Actin filaments are stabilized by capping proteins, including CapZ, gelsolin, and Eps8, whereas members of the Ena/VASP family may compete with capping proteins for free ends, allowing further polymerization. Cofilin/actin deploymerizing factor disassembles F-actin at the (-) end; it is thus required for actin treadmilling as it eventually provides the actin monomers that can be reinserted into filaments by profilin. Many of these actin regulatory proteins are present in neuronal dendrites and contribute to the constant turnover of F-actin indicated by the FRAP measurements reported in ref. 20. The equilibrium between the different forms of actin filaments is dictated by various signaling molecules, among which members of the rho GTPase family are most notorious.

5. RHO GTPASES: NATURALLY BORN TRIGGERS OF POSTSYNAPTIC ASSEMBLY

Small GTPases from the rho, ras, rab, and other families are switched "on" by specific guanine nucleotide exchange factors (GEFs) which catalyze the exchange of bound GDP by GTP. GTP binding induces a conformational change which enables the now active protein to bind to and regulate the activity of numerous effector molecules. This occurs usually by changing the conformation of target proteins, thus enabling additional protein/protein interactions. Small GTPases may eventually be switched "off " by their namesake intrinsic GTPase activity. As this activity is rather slow, it may be enhanced by GTPase activating proteins (GAPs), which therefore serve to terminate G-protein signaling. Thus, the G-protein returns to its inactive, GDP-bound state which in general does not bind to any effector molecules. Members of several different GTPase families affect form or function of the postsynaptic specialization, notable examples being rap25 and ras26. For the sake of simplicity, I focus here on the Rho family of small GTPases, which contains among others RhoA, rac, and cdc42. Rho proteins are considered as regulators of dynamic reorganizations of the actin cytoskeleton since individual Rho proteins have been shown to induce rather specific morphological changes in the fibroblast model system27. Given the dependence of both spine development and synaptic plasticity on changes in actin filament assembly, rho family GTPases may be considered as natural born molecular triggers in these processes.

Attempts to establish signaling pathways involving rho proteins which might be initiated by axonal contact have identified cell adhesion molecules from the ephrin family (see Chapter 10). EphB2 is a transmembrane tyrosine kinase which is present at the postsynaptic membrane; it may be activated by membrane-associated ephrinB ligands, or experimentally by clustered ephrinB-Fc fusions.

Several GEFs, in particular kalirin, intersectin, and PPIX are present in dendritic spines or developing dendrites and might transduce the signal to rho GTPases. Activation of rho itself has been implicated in the inhibitory control of dendrite branching, most likely through activation of rho kinase and phosphorylation of myosin light chain28,29. Both rac and cdc42 are likely to play an active role in spine formation and synapse maturation. EphB2 activation has been suggested to locally activate either rac (via kalirin30) or cdc42 (via intersectin31). Kalirin is attached to members of the PSD-95 family via a PDZ-type interaction, and this form of attachment to postsynaptic molecules is required for its effect on spine growth32. Similarly, PPIX can be anchored at postsynaptic sites via a PDZ-type interaction with shank, potentially allowing for localized activation of cdc42 or rac33. The nature of signaling downstream of the GTPases is then determined by the potential effector molecules present, i.e., molecules which can bind the GTP-bound form through cdc42/rac interactive-binding (CRIB) motifs, or other target sequences. Specificity in small GTPase signaling for particular pathways can be achieved when downstream targets are physically associated with the GEFs that initially generate the active GTPase (Figure 17.1).

Receptors

Receptors

colilin/ADF, or MIC

Figure 17.1. Signaling Modules Controlling the Activity of Small GTPases in Dendrites. In case of the GIT1/PAK/PIX module, shank is not required for functional activity but has been described to target this protein complex to postsynaptic sites. Note that EphB2 apparently acts via different exchange factors. Kalirin is translocated to the synapse upon EphB2 activation; however it is not clear if synaptic targeting involves a scaffold such as PSD-95, or if kalirin can interact directly with (Tyr-phosphorylated) active EphB2.

colilin/ADF, or MIC

Figure 17.1. Signaling Modules Controlling the Activity of Small GTPases in Dendrites. In case of the GIT1/PAK/PIX module, shank is not required for functional activity but has been described to target this protein complex to postsynaptic sites. Note that EphB2 apparently acts via different exchange factors. Kalirin is translocated to the synapse upon EphB2 activation; however it is not clear if synaptic targeting involves a scaffold such as PSD-95, or if kalirin can interact directly with (Tyr-phosphorylated) active EphB2.

A remarkable example for this is the so-called PIX/PAK/GIT1 signaling module. p21-activated kinases (PAK1-3) are typically involved in cytoskeletal regulation in other cell systems and have also been implicated in synaptogenesis. PAK is physically associated with one of the aforementioned GEFs, PIX, and both proteins are additionally linked by further interactions with the adaptor protein GIT1. This trimolecular complex is involved in the establishment and regulation of focal adhesions in non-neuronal cells34. PAK, and presumably also GIT1, are targeted to the PSD due to the interaction of the PPIX C-terminal PDZ ligand motif with the PDZ domain of shank proteins33. The role of this signaling module for the establishment of dendritic spines was recently demonstrated in two studies by Zhang et al.35,36 who showed that spine and synapse formation was reduced by interference with either GIT1 or PAK activity. The authors elegantly demonstrated local activation of rac by the use of fluorescence resonance energy transfer (FRET)

probes which combine rac with a G-protein binding motif flanked by different fluorophores36. The FRET signal obtained from this probe, indicative of local exchange activity, was found in dendritic protrusions making contact to presynaptic terminals, while the main shaft of the dendrite exhibited little or no signal.

The downstream activities of locally activated PAK might involve the phosphorylation and activation of either the regulatory light chain of myosin II36, or, as inferred from non-neuronal systems, LIM kinase. LIM kinase would then in turn phosphorylate and thus inhibit cofilin/actin depolymerizing factor. The road from an extracellular stimulus to the activation of LIM kinase and inhibition of cofilin may also be more direct, as a receptor for bone morphogenetic protein, BMPRII, can interact directly with LIM kinase at the cell membrane. By allowing actin polymerization, BMP thus exerts a rather direct influence on dendritic growth37.

Activated cdc42 activates PAK isoforms, and in addition it can interact with N-WASP. Activation of N-WASP leads to recruitment of the Arp2/3 complex, which is required for actin nucleation and the formation of a branched actin network by providing attachment points for new actin filaments alongside preexisting ones. Intersectin (which is activated by EphB2, see above) also interacts with N-WASP and may constitute another signaling module which stimulates Arp2/3 mediated actin branching. Taken together, several of these signaling modules have been identified which are present in neuronal dendrites or spines, and have the ability to transduce an extracellular signal to changes in F-actin (and therefore spine) structure.

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