BookID 400_2009 ChapID 15_Proof# 1 - 26/5/2009 Actin in Axons: Stable Scaffolds and Dynamic Filaments Paul C. Letourneau Abstract Actin filaments are thin polymers of the 42 kD protein actin. In mature axons a network of subaxolemmal actin filaments provide stability for membrane integrity and a substrate for short distance transport of cargos. In developing neurons dynamic regulation of actin polymerization and organization mediates axonal morphogenesis and axonal pathfinding to synaptic targets. Other changes in axonal shape, collateral branching, branch retraction, and axonal regeneration, also depend on actin filament dynamics. Actin filament organization is regulated by a diversity of actin-binding proteins (ABP). ABP are the focus of complex extrinsic and intrinsic signaling pathways, and many neurological pathologies and dysfunctions arise from defective regulation of ABP function. 1 Introduction Polymerized filaments of the protein actin are a major cytoskeletal component of axons, along with microtubules and neurofilaments. The most significant function of actin filaments in mature axons is to create sub-plasmalemmal scaffolding that stabilizes the plasma membrane and provides docking for protein complexes of several membrane specializations. In addition, this cortical actin scaffold interacts with myosin motor proteins to transport organelles to short distances. These functions engage actin filaments as a stable structural component in axonal homeostasis. However, during axonal development, the dynamic organization of actin filaments and their interactions with myosins play critical roles in axonal elongation and branching, and in mediating the extrinsic influences that guide axons to their synaptic targets. This chapter will first briefly discuss the characteristics of actin and its regulation by actin-binding proteins (ABPs). Then, actin organization and functions P.C. Letourneau Department of Neuroscience, 6–145 Jackson Hall, University of Minnesota, Minneapolis, MN, 55455 e-mail: [email protected] Results Probl Cell Differ, DOI 10.1007/400_2009_15 65 © Springer-Verlag Berlin Heidelberg 2009 BookID 400_2009 ChapID 15_Proof# 1 - 26/5/2009 BookID 400_2009 ChapID 15_Proof# 1 - 26/5/2009 66 P.C. Letourneau in mature axons will be discussed, and finally, the dynamic roles of actin filaments in growing and regenerating axons will be discussed. 2 The Properties of Actin 2.1 Actin Protein Actin is a globular 42 kD protein and one of the most highly conserved eukaryotic protein. Mammals have three actin isoforms, a, b and g, of which a and mostly b are expressed in neurons. Under proper buffer conditions and in sufficient concen- tration, actin monomers (G-actin) spontaneously assemble into filaments (F-actin) with a diameter of 7 nm and lengths of a few µm. The bonds between actin monomers are specific but not of high strength, allowing actin solutions to be freely shifted from polymerized to unpolymerized states. Due to the intrinsic orientation of each monomer, an actin filament is polarized, in which one end, the barbed end, adds subunits at a lower monomer concentration than that occurs at the opposite end, the pointed end; while the latter end loses monomers at a lower concentration. If ATP is furnished to an actin solution, actin filaments form after nucleating, and reach a steady state, in which filaments undergo a dynamic “treadmilling,” continually adding ATP-actin at the barbed ends, and losing ADP-actin at the pointed ends. Actin is abundantly expressed in all tissues, which is critical for cell division, adhesion, and motility. In neurons actin comprises about 4–5% of total protein (Clark et al. 1983), though during brain development actin expression rises to 7–8% of cell protein (Santerre and Rich 1976). The critical concentration of actin in vitro is the G-actin concentration, in which monomers are in equilibrium with F-actin. For skeletal muscle actin-ATP, this is about 0.1 µM, and in the muscle essentially all actin is polymerized. The intracellular G-actin concentration of chick brain has been estimated as 30–37 µM, but only 50–60% of actin is estimated to be F-actin (Clark et al. 1983; Devineni et al. 1999), rather than >99%, as predicted by the actin content. This lower-than-expected degree of polymerization is due to neuronal G-actin binding proteins that sequester G-actin from being available for polymeri- zation. G-actin sequestering proteins are just a few of the many ABPs that regulate the organization and functions of neuronal actin. 2.2 Actin Binding Proteins Actin does not function in neurons in a “naked” state, as portrayed in models or cartoons. Rather, many intracellular proteins specifically bind F-actin or G-actin (see Table 1 in Dent and Gertler 2003; Pak et al. 2008). These ABPs regulate all aspects of actin organization and dynamics (Dos Remedios et al. 2003). In regulating actin BookID 400_2009 ChapID 15_Proof# 1 - 26/5/2009 Actin in Axons: Stable Scaffolds and Dynamic Filaments 67 functions, ABPs are the targets of extrinsic and intrinsic signaling pathways. ABPs are categorized by function: (1) sequester, or bind G-actin subunits; (2) nucleate actin polymerization; (3) cap F-actin barbed ends to inhibit polymerization; (4) cap pointed ends to inhibit depolymerization; (5) bind barbed ends to inhibit capping; (6) bind pointed ends to promote depolymerization; (7) bundle, crosslink or stabilize F-actin; (8) sever actin filaments; (9) move cargo along actin filaments, or move actin fila- ments; and (10) anchor F-actin to other cellular components. Important examples of ABPs in each class described above include the following: (1) b-thymosin, which holds actin in a nonpolymerizable form, and profilin, which catalyzes exchange of actin-ADP to actin-ATP to promote polymerization; (2) Arp2/3 complex, which binds F-actin, and nucleates a new filament oriented at 70° to an existing actin filament, and formins, which promote polymerization of long actin filaments in filopodia; (3) capZ, which caps actin barbed ends in the Z-band of muscle cells; (4) tropomodulin, which binds pointed ends; (5) ena (Drosophila) (mena; murine homolog), which prevents barbed end capping to promote polymeri- zation; (6) actin depolymerizing factor (ADF)/cofilin, which binds pointed ends and promotes depolymerization; (7) filamin, which crosslinks actin filaments into networks, in which fascin crosslinks actin filaments into close bundles, and tropo- myosin, which binds along actin filaments to block other ABPs to stabilize them, and thereby regulates contraction/depolymerization, etc.; (8) ADF/cofilin, which binds and severs F-actin, and gelsolin, which depends on Ca2+ for actin filament severing; (9) multiple myosin motors that bind actin and move cargo toward the barbed or pointed ends; (10) spectrin, which mediates F-actin binding to the intra- cellular side of the plasma membrane, vinculin, which binds F-actin to integrin- mediated adhesion sites, and ERM proteins (ezrin, radixin, moesin), which bind actin to several membrane proteins. Some ABPs are expressed ubiquitously, such as b-thymosin, ADF/cofilin, and spectrin, while other ABPs are tissue-specific, such as muscle proteins Xin or myopodin, or the neuron-specific, drebrin A, which is only in the postsynaptic side of excitatory synapses. 3 Actin in Axons 3.1 The Sources of Axonal Actin Actin is synthesized at high levels on polyribosomes in neuronal perikarya. However, actin is also synthesized in axons, and in vitro studies with developing neurons estimated that 1–5% of total neuronal actin is made in axons (Eng et al. 1999; Lee and Hollenbeck 2003). Though this is a small fraction of total neuronal actin, the temporal and spatial regulation of axonal actin mRNA translation is critical to functions of developing axons, mature terminals, and regenerating axons (Lin and Holt 2007). Within axons b-actin mRNA, complexed with a regu- latory protein, zipcode-binding protein1, is localized in periaxoplasmic ribosomal BookID 400_2009 ChapID 15_Proof# 1 - 26/5/2009 BookID 400_2009 ChapID 15_Proof# 1 - 26/5/2009 68 P.C. Letourneau plaques (PARPs), which are sites of protein synthesis in myelinated axons (Koenig 2009). Actin mRNA is also concentrated in developing axonal growth cones, where local b-actin synthesis in response to axonal guidance cues may be critical in wiring neural circuits (see Yoon et al. 2009). Local actin synthesis in presynaptic terminals may contribute to synaptic rearrangements underlying neu- ral plasticity, and axonal injury triggers local actin synthesis that promotes axonal regeneration. 3.2 Axonal Transport of Actin and Actin mRNA Both actin protein and actin mRNA are transported in axons. Actin mRNA is sorted for axonal transport by the recognition of 3¢ untranslated regions (UTR), comprising b-actin mRNA sequences (i.e., zipcode) that are bound by a zipcode-binding protein (ZBP1), and assembled with other mRNA into ribonucleoprotein particles (RNPs). These complexes are bound by kinesin family motor proteins and transported along microtubules at fast rates up to >150 mm per day (Wang et al. 2007). Actin protein monomers or oligomers, synthesized in the perikaryon, are transported in the axons in macromolecular complexes associated with the slow component-b (Scb) transport group, which includes »200 proteins that are cotransported along microtubules at rates of 2–8 mm/day (Galbraith and Gallant 2000; Roy et al. 2008). The difference is striking between the transport rates of b-actin mRNA in RNP and transport of actin monomer in Scb. In addition to the transport and translation of actin mRNA in axons, mRNAs for several ABP are also translated in developing and maturing axons (Lin and Holt 2007). There may be advantages for regulating actin structures by locally translating mRNA for actin and ABP after rapid mRNA delivery rather than by relying on slower transport of Scb proteins from the peri- karyon. A greater immediacy and specificity of coordinating protein activities may result, which may be important in regulation of specialized axonal domains (see chapter by Yoon et al. 2009). 3.3 Actin Filament Organization in Axons: Ultrastructure of Axonal F-Actin It has been a challenge to clarify the organization of axonal actin filaments.