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Actin Cytoskeleton in Growth Cones, Nerve Terminals, and Dendritic Spines
Actin Cytoskeleton in Growth Cones, Nerve Terminals, and Dendritic Spines 15
Actin Cytoskeleton in Growth Cones, Nerve Terminals, and Dendritic Spines S Halpain, B Calabrese, and L Dehmelt, The Scripps Research Institute, La Jolla, CA, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction The range and complexity of cell morphologies found in neurons are largely defined by the structure and dynamic organization of the underlying neuronal cytoskeleton. Neurons contain three major cytoskeleton types: microtubules, intermediate filaments, and filamentous actin. Here we review the properties and roles of actin in early and late neuronal development. In the brain, neurons develop within an intricate, layered, three-dimensional matrix, which makes it difficult to analyze the detailed organization of the neuronal cytoskeleton. Therefore, many studies on actin organization during neuromorphogenesis have been performed using dissociated neuronal cultures grown on glass coverslips. In such a simplified model system, neurons first display a quasi-symmetrical shape. Actin is concentrated in the cell periphery in one or more flattened, veil-like structures, usually referred to as lamellipodia. During neurite initiation these lamellipodia divide into smaller lamellipodia, which then slowly move outward, trailed by a condensed, microtubule-rich shaft. If the shaft reaches significant length, the whole neuronal protrusion is called a neurite, and the lamellipodium at the neurite tip is called a growth cone (Figure 1, left). In one of the classical neuronal model systems – primary hippocampal neurons – one of the neurites will grow significantly longer and accumulate certain markers, such as antigenicity to the tau-1 antibody. This neurite usually continues to grow rapidly and will become the future axon. During this stage, the remaining smaller neurites are called minor neurites, and these eventually mature and become the future dendrites. As the dendritic arbor develops, many filopodia rapidly protrude and retract from the dendritic shaft, with a lifetime of minutes. Over a protracted period of time, the number of filopodia declines, as the number of synaptic contacts and dendritic spines increase. Dendritic spines are the postsynaptic receptive regions of most excitatory synapses. They consist of a bulbous head connected to the dendritic shaft by a narrow neck (Figure 1, right). Normally each spine is connected across the synapse to a specialized region of an axon called a presynaptic bouton or nerve terminal. It is
thought that the shape of spines is critical for the normal function of such synapses and essential for the normal development of neuronal networks. In addition to being one of the most abundant cellular proteins, actin is also one of the most versatile building blocks in cells. By itself, monomeric actin (G-actin) is a simple globular protein with intrinsic adenosine triphosphatase (ATPase) activity. If ATP is bound, G-actin can polymerize into a linear, twostranded helical filament (F-actin). The intrinsic ATPase activity converts ATP-actin monomers to ADP-actin. The two ends of an actin filament have distinct biochemical activities: the (þ) end has a higher affinity for monomeric actin and therefore elongates faster under standard conditions. The () end has lower monomer-binding activity and elongates slower than the (þ) end does. At equilibrium, this difference of affinities leads to a process termed ‘treadmilling,’ which is characterized by net monomer addition at the (þ) end and net depolymerization at the () end. Filament behavior is regulated by a variety of actinregulating proteins that either bind to the (þ) end, the () end, or along the sides of filaments. The overall organization of actin filaments in cells can be controlled by regulating the local rates of de novo filament formation (nucleation), addition of monomers to existing filaments (polymerization), removal of monomers from filament ends (depolymerization), breaking of intact filaments (severing), sliding of individual filaments, and physical cross-linking of filaments to other cellular structures. Throughout this article, we discuss various examples of how precise tuning of such activities within neurons leads to the specific organization and dynamic properties of key neuronal structures.
Early Development Actin Nucleation and Control of Actin Polymerization Rates in Growth Cones
The ‘dendritic nucleation model’ is a popular model for how actin filaments might maintain and control protrusive cell behavior at the plasma membrane. In this case the term ‘dendritic’ refers not to neurites but to the branched organization of actin filaments that defines this particular mode of actin assembly. The dendritic nucleation model is often thought to be generally applicable; however, most data that led to its proposal were derived from nonneuronal cells, notably fibroblasts. In a nutshell, this model proposes that rapid nucleation and ATP-dependent actin
16 Actin Cytoskeleton in Growth Cones, Nerve Terminals, and Dendritic Spines
Figure 1 Localization of filamentous actin (F-actin) in rat cultured hippocampal neurons. Left: hippocampal neuron (1 day after plating) stained for F-actin (red) and neuronal tubulin (green). Right: hippocampal neurons (3 weeks after plating) stained for F-actin (red) and MAP2 (a microtubule-associated protein specific to dendrites; green). Note that F-actin is enriched in small clusters along the dendrites, which correspond to dendritic spines. Scale bar ¼ 20 mm.
polymerization at the leading edge of motile cells are sufficient to drive membrane protrusion. To be effective, the turnover of actin must be very high, in order to maintain a constant supply of monomers to drive protrusion. Therefore, depolymerization must be ensured shortly after the initial polymerization. The zones of polymerization and depolymerization are separated by a zone of active myosin motor- and actin polymerization-driven flux. In this zone, the level of adhesion is thought to be important to regulate the level of protrusion or slippage – a process first suggested in the ‘clutch hypothesis’ (see below). Some key aspects of this model are likely to apply to growth cones as well. However, there are also some very significant differences. Most notably, the dendritic nucleation model proposes that actin filament nucleation at the leading edge is driven by a actinrelated protein (ARP) assembly termed the ARP2/3 complex, which binds to the sides of existing filaments and primes the nucleation of a new filament in a characteristic 60 angle off the mother filament. In fibroblasts, this mechanism leads to the formation of an intricate network of interdigitated filaments of ‘dendritic’ (branched) appearance. However, such dendritic networks are not observed in neuronal growth cones, suggesting that the way in which actin filaments initially form differs significantly between neuronal and nonneuronal systems. It is believed that in addition to the ARP2/3 complex, another class of actin-binding proteins called formins plays an important role in actin nucleation. In the case of growth cones, formin-mediated actin nucleation might be dominant. In fact, whereas
ARP2/3-induced nucleation is necessary for fibroblast protrusion, in growth cones, this nucleation complex is a negative regulator of neurite protrusion, suggesting a distinct role for ARP2/3 in the growth cone. Interestingly, ARP2/3-mediated cell protrusions are typically characterized by broad, flat lamellipodia, usually found at the leading edge of fibroblasts, whereas formin-based cell protrusions are characterized by actin spike structures called filopodia, which in turn are frequently formed in growth cones (Figures 1 (left) and 2(a)). Apart from this obvious difference in the actin nucleation mechanism, other aspects of actin polymerization regulation appear to be similar in fibroblasts and growth cones. In both systems, for example, rapid polymerization at the leading edge, promoted by actin bound to profilin, is thought to drive membrane protrusion. b-Thymosin is present in both neuronal and nonneuronal systems and is known to sequester actin monomers, thereby limiting polymerization rates. Finally, depolymerization is thought to be the rate-limiting step in actin turnover control (see the section titled ‘Control of actin depolymerization and turnover in early neuromorphogenesis’) and thus is thought to be a key modulator of polymerization rates as well. Substrate Adhesion and Generation of Traction in Axon Outgrowth and Guidance
Once actin is polymerized at the leading edge of the growth cone, it enters a zone of rapid retrograde flow usually referred to as the lamellipodium. According to the ‘clutch hypothesis’ proposed by Mitchison and
Actin Cytoskeleton in Growth Cones, Nerve Terminals, and Dendritic Spines 17
Figure 2 Cytoskeletal organization in the growth cone and the synapse. (a) Within the growth cone, actin is concentrated in the peripheral lamella. Actin-binding proteins, such as profilin, cofilin, and myosin, are thought to play a role in regulating actin dynamics and/ or organizing actin into specific structures. (b) Actin filaments are distributed throughout the presynaptic nerve terminal, preferentially concentrated around synaptic vesicles. However, mature nerve terminals contain much less actin compared to either growth cones or dendritic spines. Actin regulatory molecules (such as cofilin and profilin) control the extent and rate of actin polymerization, and, ultimately, dendritic spine shape or growth cone motility. ADF; actin-depolymerizing factor; N-WASP, neural Wiskott–Aldrich syndrome protein; ARP2/3, actin-related protein complex 2/3. Drawings by James Lim; colorization by Barbara Calabrese and Leif Dehmelt.
Kirschner, this retrograde flow acts as a motor for cell protrusion. By analogy to a car engine clutch, the retrograde flow is constitutively active, even in resting growth cones. This retrograde flow does not produce any forward movement unless it is linked to the substratum by formation of cell adhesions. Thus, the temporal regulation of the extent of adhesion or slippage is thought to control the speed of growth cone progression. The giant growth cones of the sea snail Aplysia have been instrumental in revealing how local manipulations of adhesion affect growth cone behavior and protrusion. Such studies revealed that adhesion to a
bead coated with the signaling molecule ApCAM (Aplysia cell adhesion molecule) is not sufficient by itself to initiate a full response of the growth cone. However, if the coated bead is mechanically restrained to allow the growth cone to pull on it, Src kinase is locally activated at the bead interaction site. This is followed by microtubule targeting toward the bead, modulation of retrograde flow, growth cone advance, and axonal turning behavior. Retrograde flow is driven both by forces generated distally from actin polymerization at the leading edge and proximally by myosin motor activity within the lamellipodium (Figure 2). There is still some
18 Actin Cytoskeleton in Growth Cones, Nerve Terminals, and Dendritic Spines
controversy about the roles of myosin II and myosin I in growth cones. These motors have been suggested either to participate in the organization and maintenance of structural integrity of the lamellipodium or in the generation of the retrograde flow. It is clear, however, that myosin II is mostly localized to the proximal part of the growth cone, where it co-localizes with contractile filamentous structures called actin arcs. These structures accumulate at the lateral sides of the growth cone and are thought to compress microtubules into parallel arrays. Furthermore, actin arcs are also thought to mediate contractile responses during neurite retraction. Roles for Actin–Microtubule Interactions in Early Neurite Development
Several laboratories have reported physical, rigid interactions between microtubules and filamentous actin within cells. This physical coupling might have several nonexclusive roles: (1) As mentioned earlier, actin arcs might shape the distribution of microtubules within growth cones and might even be involved in the formation and/or maintenance of the tight microtubule bundles typically found in neurons. (2) Similar to the clutch hypothesis, physical linkage of actin filaments to a stable scaffold of microtubules might promote protrusion of the leading cell edge. (3) Microtubules might be involved in signaling cross-talk mediated by regulatory molecules found on the two cytoskeletons. (4) Dynein-generated forces on microtubule arrays are suggested to counteract actomyosin-driven neurite retraction. Interestingly, microtubules also appear to have an instructive role in growth cone turning, as local pharmacological manipulations of microtubule dynamics are sufficient to drive growth cone turning behavior. Several potential microtubule-actin cross-linkers or signaling mediators have been identified, including the coronin-like protein pod-1, microtubule-associated protein MAP2c, the dynein–dynactin complex, and the IQGAP1/Rac1 complex. These proteins have been implicated in several stages of early neurite development, including neurite initiation, neurite growth, and axon pathfinding. Control of Actin Depolymerization and Turnover in Early Neuromorphogenesis
The dynamic behavior of actin filaments is not only dependent on the rates of polymerization, nucleation, and motor-dependent filament sliding, but is also dependent on a constant supply of active monomers that can be incorporated at the filament ends. Thus, depolymerization is required for rapid actin turnover, and biochemical studies suggest that this might be the rate-limiting step in the cycle of actin dynamics.
Therefore, actin-depolymerizing factors (ADFs), such as cofilin and ADF, play important roles as regulators of actin dynamics. ADF/cofilin binds filamentous actin and increases its depolymerization rate by altering the helical twist of the filament. The activity of ADF/cofilin is controlled by multiple mechanisms, including phosphorylation of serine 3 by LIM and TES protein kinases, dephosphorylation by the protein phosphatase ‘slingshot’ and ‘chronophin’ and binding of 14-3-3 proteins. Furthermore, another actin-binding protein called tropomyosin protects filaments against ADF/cofilin and thereby adds another level of depolymerization rate regulation. Consistent with the idea that ADF/cofilin regulates a rate-limiting step during actin turnover, overexpression leads to an increase in axonal outgrowth, while inhibition of its activity using dominant-negative mutants or by overexpression of an inactivating kinase slows axon outgrowth.
Late Development Actin Organization and Function during the Development of Synapses
The actin cytoskeleton is the main structural component of both pre- and postsynaptic terminals. Here we focus on excitatory synapses, where the function of the actin cytoskeleton is best understood. The formation of synapses in the vertebrate central nervous system is a complex process that occurs over a protracted period of time. It is generally the case that dendritic filopodia are the structural precursors of dendritic spines during synaptogenesis, although direct emergence of new spines from the dendrite shaft has also been observed. Dendritic filopodia are long, thin protrusions characterized by a highly transient and motile behavior. In these protrusions F-actin is sparse until pre- and postsynaptic transmembrane cell adhesion proteins, such as the cadherin–catenin complex, link adjacent synapse precursors. The F-actin accumulates within early filopodial synaptic contacts, where it recruits synaptic signaling molecules. Simultaneously, the dynamic dendritic filopodia begin to morphologically change into intermediate structures sometimes called ‘protospines.’ These intermediate structures have also been referred to as cluster-type filopodia, or synaptic filopodia, and they can eventually mature into more stable dendritic spines. Only a fraction of the filopodia that emerge from a dendrite persist to become dendritic spines. Mature, postsynaptic dendritic spines are highly enriched in F-actin compared to the dendritic shaft (Figure 1, right). Based on measurements of green
Actin Cytoskeleton in Growth Cones, Nerve Terminals, and Dendritic Spines 19
fluorescent protein (GFP)–actin fluorescence recovery after photobleaching in cultured hippocampal neurons, only approximately 5% of the total actin in spines is relatively stable, while the majority of the actin almost completely turns over in a 2 min period. Unlike F-actin, G-actin is distributed homogeneously throughout axonal and dendritic processes in hippocampal neurons, which indicates that its main role at the synapse is to maintain the synaptic F-actin pool. The characteristic shape of dendritic spine heads is highly dependent on actin dynamics and the fraction of polymerized actin. Disruption of filamentous actin by the toxin latrunculin A transforms dendritic spines into filopodia-like processes. F-actin is found within the presynaptic nerve terminal and is preferentially concentrated around synaptic vesicles (Figure 2(b)). In resting conditions, approximately 30% of actin is in a polymerized state, with a turnover halftime of 20 s. During electrical activity, actin is further polymerized and recruited around vesicles from nearby axonal regions, leading many groups to propose a propulsive role for actin, either in maintaining the vesicle cluster or in guiding vesicle recycling. However, the precise role for actin in nerve terminals remains controversial, and may differ depending on the nature of the synapse. Although actin plays an active role in synaptic vesicle endocytosis in some systems, recent evidence indicates that in mammalian central synapses it passively inhibits vesicle fusion at the active zone, and helps sequester synaptic vesicles in the reserve pool. Synaptic vesicle trafficking from the reserve pool to the readily releasable pool can occur independently of actin. Thus, actin may serve primarily as a scaffold for other regulatory proteins involved in vesicle trafficking within the terminal. The recruitment and retention of presynaptic matrix proteins, such as bassoon and piccolo, also are dependent on actin. The higher content of F-actin in dendritic spines compared to nerve terminals correlates with more dynamic shape changes. Nevertheless, the pre- and postsynaptic cytomatrix components are strongly interconnected, and therefore their movements are physically coupled in established synapses. Engulfment of spinules (membrane extensions emerging from the head of big mushroom spines) by presynaptic axons is a process known as transendocytosis and suggests retrograde signaling or coordinated remodeling of pre- and postsynaptic membranes, which is likely modulated by actin. Actin Dynamics and Spine Morphogenesis
In recent years, imaging techniques both in vivo and in vitro have revealed that a majority of mature dendritic spines are stable over months in the adult brain.
They rarely form and disappear; instead, the dendritic spine head is constantly changing shape (morphing). This rapid spine motility is caused by regulated polymerization and depolymerization of actin, based on studies using drugs and cDNAs that perturb actin polymerization. The Rho family of GTPases is one common denominator controlling spine morphology. Multiple signaling pathways converge on the Rho family of small GTPases (particularly RhoA, Rac1, and Cdc42), which ultimately converge on the actin cytoskeleton to regulate spine morphology and dynamics. Generally speaking, RhoA inhibits, whereas Rac and Cdc42 promote, the growth and/or stability of dendritic spines. An expanding number of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) control these Rho family GTPases. For example, kalirin-7, a GEF for Rac1, mediates effects of the transmembrane signaling molecule EphB on spine maturation by activating Rac1 and its downstream effector p21-activated kinase (PAK). PAK1 and PAK3 are Rac effectors that promote formation and/or growth of spines. PAK also stimulates LIM kinase (LIMK), which phosphorylates and inactivates ADF/cofilin, an actindepolymerizing protein that mediates reorganization of the actin cytoskeleton. Recently, cofilin has been detected by immunoelectron microscopy to concentrate in the periphery of the spine head and within the postsynaptic density. Mice expressing dominantnegative PAK in the forebrain showed reduced spine density in cortical neurons, impaired long-term synaptic plasticity, and reduced memory consolidation. PAK activity is markedly reduced in Alzheimer’s disease due to a reduction in phospho-PAK, which in turn results in an increased cofilin activity and downstream loss of the spine actin-regulatory protein drebrin. Insulin receptor substrate p53 (IRSp53), an adaptor protein that connects Rac1 to the Wiskott– Aldrich syndrome protein WAVE2, is implicated in filopodia and lamellipodia formation in nonneural cells. IRSp53 is enriched in the postsynaptic density (PSD) by binding to Shank and PSD-95 family scaffolds. Overexpression of IRSp53 increases spine density, whereas RNA interference (RNAi) or dominant-negative inhibition of IRSp53 causes reduction of spine density and size. The Abl-interactor (Abi) adaptor proteins, so-named because they bind to the Abl tyrosine kinases, also play a part in Rac GTPase signaling and actin regulation. Abi2 is highly expressed in brain; and Abi2-deficient mice show reduced spine density and a decrease in the relative proportion of mushroom spines, which is thought to be associated with deficits in memory.
20 Actin Cytoskeleton in Growth Cones, Nerve Terminals, and Dendritic Spines
It is worth noting that components of Rho GTPase signaling pathways are highly represented among the genes so far identified in hereditary forms of human nonsyndromic mental retardation. These mutant genes include X-linked genes, such as PAK3 (see earlier) and oligophrenin, which is a Rho GAP. RNAi suppression of oligophrenin decreases spine length, mimicking the effect of active RhoA. Numerous scaffold proteins and actin-binding proteins are concentrated in spines and are known to control spine morphogenesis (Figure 2(b)). Many of these are regulated by the small GTPases described earlier. Overexpression of a-actinin 2, a protein linking N-methyl-D-aspartate (NMDA) receptors to the actin cytoskeleton, increases the length and density of dendritic protrusions in cultured hippocampal neurons. Clustering of the actin-binding protein drebrin occurs early in development, supporting its involvement in spinogenesis and synaptogenesis. Cortactin is an activator of the ARP2/3 actin nucleation machinery that interacts with the postsynaptic density scaffold Shank. RNAi knockdown of cortactin results in depletion of dendritic spines, whereas overexpression of cortactin causes enlargement of spines. Myosin II is also enriched in postsynaptic density preparations and acts as a molecular motor that produces tension on actin filaments. Inhibition of myosin IIB destabilizes mushroom spines, suggesting that the structure and function of spines are regulated by an actin-based motor in addition to actin polymerization. Effects of Synaptic Activity on Actin Polymerization in Dendritic Spines
Synaptic plasticity is associated with changes in actin polymerization and spine morphology. Long-term potentiation (LTP) is associated with formation of new spines, enlargement of existing spines, and a shift of the actin turnover equilibrium toward polymerization of F-actin. By contrast, long-term depression (LTD) is associated with shrinkage and/or retraction of spines due to an increase in actin depolymerization. In addition, synaptic activity is thought to play an important role in determining whether a synapse will be stabilized or eliminated. During induction of LTP, a-amino-3-hydroxy-5methyl-4-isoxazole propionic acid (AMPA) receptors are rapidly recruited to dendritic spines through Ca2þ-permeable NMDA receptor activation. In response to NMDA receptor activation, profilin, a promoter of F-actin assembly, is targeted to spine heads, while cortactin redistributes from spines to the dendritic shaft. Distinct but overlapping signaling pathways are involved in LTD and spine shrinkage. During induction of LTD, NMDA receptor-mediated
calcium influx triggers phosphatase 2B (calcineurin) activity. Calcineurin in turn activates a downstream phosphatase ‘slingshot’ and promotes actin severing and depolymerization through ADF/cofilin. Synaptic activity can also affect the relationship between F-actin and the plasma membrane of dendritic spines. Myristoylated alanine-rich protein kinase C substrate (MARCKS), a major target of protein kinase C (PKC), might be involved in local coupling of activity-dependent calcium entry with regulation of actin assembly–disassembly cycles. During synaptic activity, when calcium levels increase and stimulate PKC, MARCKS detaches from the plasma membrane, thereby freeing phosphatidylinositol 4,5-bisphosphate to interact with actin-binding proteins such as profilin and ARP2/3, which in turn can locally alter actin dynamics and membrane trafficking.
Comparing Growth Cones, Dendritic Spines, and Nerve Terminals One might be tempted to generalize the role of actin in growth cones, synaptic terminals, and spines, and introductions to this topic often try to point out similarities. However, the current state of our knowledge does not allow an in-depth comparison. Nonetheless, certain parellels exist (Figures 2(a) and 2(b)). In growth cones, a relatively stable, less dynamic F-actin population is found toward the growth cone center, which is thought to be involved in contractile behavior. Highly dynamic F-actin is concentrated in the periphery of the growth cone, where it is thought to mediate membrane protrusion, resulting in motile filopodia and lamellipodia. Similarly, a stable pool of F-actin is present in the dendritic spine core, while a dynamic pool lies peripherally in its shell, which exhibits highly dynamic, sometime protrusive behavior. In growth cones, the organization and dynamics of the underlying actin structures have been analyzed using both high-contrast electron microscopy and dynamic speckle microscopy. These analyses converge in a detailed working model of growth cone behavior. In spines, defining the detailed actin organization and dynamics has been more difficult. Ultrastructural analysis of filament polarity suggests that most actin filaments are oriented with their barbed end pointing toward the head of the spine protrusion, similar to the preferred orientation of actin filaments toward the growth cone leading edge. One notable difference is that spines generally lack the dense parallel organization of F-actin that characterizes growth cone filopodia. However, at times, rapidly growing, elongated filopodia-like
Actin Cytoskeleton in Growth Cones, Nerve Terminals, and Dendritic Spines 21
structures are observed on spine heads. The purpose of such filopodial-like protrusions emerging from spine heads remains unclear. In growth cones, and along developing dendrites, such filopodial protrusions are thought to extend the structure’s sensory radius to probe the environment for guidance cues or neurotransmitters. Speckle microscopic analysis of actin dynamics has not been performed in spines; however, indirect data on spine actin dynamics have been derived from photobleaching recovery experiments (see the section titled ‘Actin organization and function during the development of synapses’). These experiments reveal a highly dynamic F-actin population, which may be roughly comparable to the rapidly turning over F-actin of growth cone lamellipodia. While there might be some similarities between actin behavior in growth cones and spines, the role and organization of actin in nerve terminals appear to be distinct. F-Actin is distributed throughout the presynaptic nerve terminals and is preferentially concentrated around synaptic vesicles. However, mature nerve terminals do not exhibit significant protrusive behavior and contain much less actin compared to either growth cones or spines. In nerve terminals, a specific role for actin remains unclear. See also: Axonal Regeneration: Role of Growth and Guidance Cues; Axonal and Dendritic Identity and Structure: Control of; Cytoskeletal Interactions in the Neuron; Cytoskeleton in Plasticity; Dendritic Spine History; Glutamate Regulation of Dendritic Spine Form and Function; Growth Cones; LIM Kinase and Actin Regulation of Spines; Microtubules: Organization and Function in Neurons; Spine Plasticity.
Further Reading Calabrese B, Wilson MS, and Halpain S (2006) Development and regulation of dendritic spine synapses. Physiology (Bethesda) 21: 38–47. Carlisle HJ and Kennedy MB (2005) Spine architecture and synaptic plasticity. Trends in Neurosciences 28: 182–187. Dehmelt L and Halpain S (2004) Actin and microtubules in neurite initiation: Are MAPs the missing link? Journal of Neurobiology 58: 18–33. Dent EW and Gertler FB (2003) Cytoskeletal dynamics and transport in growth cone motility and axon guidance. Neuron 40: 209–227. Ethell IM and Pasquale EB (2005) Molecular mechanisms of dendritic spine development and remodeling. Progress in Neurobiology 75: 161–205. Lippman J and Dunaevsky A (2005) Dendritic spine morphogenesis and plasticity. Journal of Neurobiology 64: 47–57. Luo L (2002) Actin cytoskeleton regulation in neuronal morphogenesis and structural plasticity. Annual Review of Cell and Developmental Biology 18: 601–635. Oertner TG and Matus A (2005) Calcium regulation of actin dynamics in dendritic spines. Cell Calcium 37: 477–482. Pollard TD and Borisy GG (2003) Cellular motility driven by assembly and disassembly of actin filaments. Cell 112: 453–465. Sarmiere PD and Bamburg JR (2004) Regulation of the neuronal actin cytoskeleton by ADF/cofilin. Journal of Neurobiology 58: 103–117. Strasser GA, Rahim NA, VanderWaal KE, et al. (2004) Arp2/3 is a negative regulator of growth cone translocation. Neuron 43: 81–94. Suter DM, Schaefer AW, and Forscher P (2004) Microtubule dynamics are necessary for SRC family kinase-dependent growth cone steering. Current Biology 14: 1194–1199. Zhang W and Benson DL (2002) Developmentally regulated changes in cellular compartmentation and synaptic distribution of actin in hippocampal neurons. Journal of Neuroscience Research 69: 427–436. Zhang XF, Schaefer AW, Burnette DT, et al. (2003) Rho-dependent contractile responses in the neuronal growth cone are independent of classical peripheral retrograde actin flow. Neuron 40: 931–944.
Actin Cytoskeleton in Growth Cones, Nerve Terminals, and Dendritic Spines S Halpain, B Calabrese, and L Dehmelt, The Scripps Research Institute, La Jolla, CA, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction The range and complexity of cell morphologies found in neurons are largely defined by the structure and dynamic organization of the underlying neuronal cytoskeleton. Neurons contain three major cytoskeleton types: microtubules, intermediate filaments, and filamentous actin. Here we review the properties and roles of actin in early and late neuronal development. In the brain, neurons develop within an intricate, layered, three-dimensional matrix, which makes it difficult to analyze the detailed organization of the neuronal cytoskeleton. Therefore, many studies on actin organization during neuromorphogenesis have been performed using dissociated neuronal cultures grown on glass coverslips. In such a simplified model system, neurons first display a quasi-symmetrical shape. Actin is concentrated in the cell periphery in one or more flattened, veil-like structures, usually referred to as lamellipodia. During neurite initiation these lamellipodia divide into smaller lamellipodia, which then slowly move outward, trailed by a condensed, microtubule-rich shaft. If the shaft reaches significant length, the whole neuronal protrusion is called a neurite, and the lamellipodium at the neurite tip is called a growth cone (Figure 1, left). In one of the classical neuronal model systems – primary hippocampal neurons – one of the neurites will grow significantly longer and accumulate certain markers, such as antigenicity to the tau-1 antibody. This neurite usually continues to grow rapidly and will become the future axon. During this stage, the remaining smaller neurites are called minor neurites, and these eventually mature and become the future dendrites. As the dendritic arbor develops, many filopodia rapidly protrude and retract from the dendritic shaft, with a lifetime of minutes. Over a protracted period of time, the number of filopodia declines, as the number of synaptic contacts and dendritic spines increase. Dendritic spines are the postsynaptic receptive regions of most excitatory synapses. They consist of a bulbous head connected to the dendritic shaft by a narrow neck (Figure 1, right). Normally each spine is connected across the synapse to a specialized region of an axon called a presynaptic bouton or nerve terminal. It is
thought that the shape of spines is critical for the normal function of such synapses and essential for the normal development of neuronal networks. In addition to being one of the most abundant cellular proteins, actin is also one of the most versatile building blocks in cells. By itself, monomeric actin (G-actin) is a simple globular protein with intrinsic adenosine triphosphatase (ATPase) activity. If ATP is bound, G-actin can polymerize into a linear, twostranded helical filament (F-actin). The intrinsic ATPase activity converts ATP-actin monomers to ADP-actin. The two ends of an actin filament have distinct biochemical activities: the (þ) end has a higher affinity for monomeric actin and therefore elongates faster under standard conditions. The () end has lower monomer-binding activity and elongates slower than the (þ) end does. At equilibrium, this difference of affinities leads to a process termed ‘treadmilling,’ which is characterized by net monomer addition at the (þ) end and net depolymerization at the () end. Filament behavior is regulated by a variety of actinregulating proteins that either bind to the (þ) end, the () end, or along the sides of filaments. The overall organization of actin filaments in cells can be controlled by regulating the local rates of de novo filament formation (nucleation), addition of monomers to existing filaments (polymerization), removal of monomers from filament ends (depolymerization), breaking of intact filaments (severing), sliding of individual filaments, and physical cross-linking of filaments to other cellular structures. Throughout this article, we discuss various examples of how precise tuning of such activities within neurons leads to the specific organization and dynamic properties of key neuronal structures.
Early Development Actin Nucleation and Control of Actin Polymerization Rates in Growth Cones
The ‘dendritic nucleation model’ is a popular model for how actin filaments might maintain and control protrusive cell behavior at the plasma membrane. In this case the term ‘dendritic’ refers not to neurites but to the branched organization of actin filaments that defines this particular mode of actin assembly. The dendritic nucleation model is often thought to be generally applicable; however, most data that led to its proposal were derived from nonneuronal cells, notably fibroblasts. In a nutshell, this model proposes that rapid nucleation and ATP-dependent actin
16 Actin Cytoskeleton in Growth Cones, Nerve Terminals, and Dendritic Spines
Figure 1 Localization of filamentous actin (F-actin) in rat cultured hippocampal neurons. Left: hippocampal neuron (1 day after plating) stained for F-actin (red) and neuronal tubulin (green). Right: hippocampal neurons (3 weeks after plating) stained for F-actin (red) and MAP2 (a microtubule-associated protein specific to dendrites; green). Note that F-actin is enriched in small clusters along the dendrites, which correspond to dendritic spines. Scale bar ¼ 20 mm.
polymerization at the leading edge of motile cells are sufficient to drive membrane protrusion. To be effective, the turnover of actin must be very high, in order to maintain a constant supply of monomers to drive protrusion. Therefore, depolymerization must be ensured shortly after the initial polymerization. The zones of polymerization and depolymerization are separated by a zone of active myosin motor- and actin polymerization-driven flux. In this zone, the level of adhesion is thought to be important to regulate the level of protrusion or slippage – a process first suggested in the ‘clutch hypothesis’ (see below). Some key aspects of this model are likely to apply to growth cones as well. However, there are also some very significant differences. Most notably, the dendritic nucleation model proposes that actin filament nucleation at the leading edge is driven by a actinrelated protein (ARP) assembly termed the ARP2/3 complex, which binds to the sides of existing filaments and primes the nucleation of a new filament in a characteristic 60 angle off the mother filament. In fibroblasts, this mechanism leads to the formation of an intricate network of interdigitated filaments of ‘dendritic’ (branched) appearance. However, such dendritic networks are not observed in neuronal growth cones, suggesting that the way in which actin filaments initially form differs significantly between neuronal and nonneuronal systems. It is believed that in addition to the ARP2/3 complex, another class of actin-binding proteins called formins plays an important role in actin nucleation. In the case of growth cones, formin-mediated actin nucleation might be dominant. In fact, whereas
ARP2/3-induced nucleation is necessary for fibroblast protrusion, in growth cones, this nucleation complex is a negative regulator of neurite protrusion, suggesting a distinct role for ARP2/3 in the growth cone. Interestingly, ARP2/3-mediated cell protrusions are typically characterized by broad, flat lamellipodia, usually found at the leading edge of fibroblasts, whereas formin-based cell protrusions are characterized by actin spike structures called filopodia, which in turn are frequently formed in growth cones (Figures 1 (left) and 2(a)). Apart from this obvious difference in the actin nucleation mechanism, other aspects of actin polymerization regulation appear to be similar in fibroblasts and growth cones. In both systems, for example, rapid polymerization at the leading edge, promoted by actin bound to profilin, is thought to drive membrane protrusion. b-Thymosin is present in both neuronal and nonneuronal systems and is known to sequester actin monomers, thereby limiting polymerization rates. Finally, depolymerization is thought to be the rate-limiting step in actin turnover control (see the section titled ‘Control of actin depolymerization and turnover in early neuromorphogenesis’) and thus is thought to be a key modulator of polymerization rates as well. Substrate Adhesion and Generation of Traction in Axon Outgrowth and Guidance
Once actin is polymerized at the leading edge of the growth cone, it enters a zone of rapid retrograde flow usually referred to as the lamellipodium. According to the ‘clutch hypothesis’ proposed by Mitchison and
Actin Cytoskeleton in Growth Cones, Nerve Terminals, and Dendritic Spines 17
Figure 2 Cytoskeletal organization in the growth cone and the synapse. (a) Within the growth cone, actin is concentrated in the peripheral lamella. Actin-binding proteins, such as profilin, cofilin, and myosin, are thought to play a role in regulating actin dynamics and/ or organizing actin into specific structures. (b) Actin filaments are distributed throughout the presynaptic nerve terminal, preferentially concentrated around synaptic vesicles. However, mature nerve terminals contain much less actin compared to either growth cones or dendritic spines. Actin regulatory molecules (such as cofilin and profilin) control the extent and rate of actin polymerization, and, ultimately, dendritic spine shape or growth cone motility. ADF; actin-depolymerizing factor; N-WASP, neural Wiskott–Aldrich syndrome protein; ARP2/3, actin-related protein complex 2/3. Drawings by James Lim; colorization by Barbara Calabrese and Leif Dehmelt.
Kirschner, this retrograde flow acts as a motor for cell protrusion. By analogy to a car engine clutch, the retrograde flow is constitutively active, even in resting growth cones. This retrograde flow does not produce any forward movement unless it is linked to the substratum by formation of cell adhesions. Thus, the temporal regulation of the extent of adhesion or slippage is thought to control the speed of growth cone progression. The giant growth cones of the sea snail Aplysia have been instrumental in revealing how local manipulations of adhesion affect growth cone behavior and protrusion. Such studies revealed that adhesion to a
bead coated with the signaling molecule ApCAM (Aplysia cell adhesion molecule) is not sufficient by itself to initiate a full response of the growth cone. However, if the coated bead is mechanically restrained to allow the growth cone to pull on it, Src kinase is locally activated at the bead interaction site. This is followed by microtubule targeting toward the bead, modulation of retrograde flow, growth cone advance, and axonal turning behavior. Retrograde flow is driven both by forces generated distally from actin polymerization at the leading edge and proximally by myosin motor activity within the lamellipodium (Figure 2). There is still some
18 Actin Cytoskeleton in Growth Cones, Nerve Terminals, and Dendritic Spines
controversy about the roles of myosin II and myosin I in growth cones. These motors have been suggested either to participate in the organization and maintenance of structural integrity of the lamellipodium or in the generation of the retrograde flow. It is clear, however, that myosin II is mostly localized to the proximal part of the growth cone, where it co-localizes with contractile filamentous structures called actin arcs. These structures accumulate at the lateral sides of the growth cone and are thought to compress microtubules into parallel arrays. Furthermore, actin arcs are also thought to mediate contractile responses during neurite retraction. Roles for Actin–Microtubule Interactions in Early Neurite Development
Several laboratories have reported physical, rigid interactions between microtubules and filamentous actin within cells. This physical coupling might have several nonexclusive roles: (1) As mentioned earlier, actin arcs might shape the distribution of microtubules within growth cones and might even be involved in the formation and/or maintenance of the tight microtubule bundles typically found in neurons. (2) Similar to the clutch hypothesis, physical linkage of actin filaments to a stable scaffold of microtubules might promote protrusion of the leading cell edge. (3) Microtubules might be involved in signaling cross-talk mediated by regulatory molecules found on the two cytoskeletons. (4) Dynein-generated forces on microtubule arrays are suggested to counteract actomyosin-driven neurite retraction. Interestingly, microtubules also appear to have an instructive role in growth cone turning, as local pharmacological manipulations of microtubule dynamics are sufficient to drive growth cone turning behavior. Several potential microtubule-actin cross-linkers or signaling mediators have been identified, including the coronin-like protein pod-1, microtubule-associated protein MAP2c, the dynein–dynactin complex, and the IQGAP1/Rac1 complex. These proteins have been implicated in several stages of early neurite development, including neurite initiation, neurite growth, and axon pathfinding. Control of Actin Depolymerization and Turnover in Early Neuromorphogenesis
The dynamic behavior of actin filaments is not only dependent on the rates of polymerization, nucleation, and motor-dependent filament sliding, but is also dependent on a constant supply of active monomers that can be incorporated at the filament ends. Thus, depolymerization is required for rapid actin turnover, and biochemical studies suggest that this might be the rate-limiting step in the cycle of actin dynamics.
Therefore, actin-depolymerizing factors (ADFs), such as cofilin and ADF, play important roles as regulators of actin dynamics. ADF/cofilin binds filamentous actin and increases its depolymerization rate by altering the helical twist of the filament. The activity of ADF/cofilin is controlled by multiple mechanisms, including phosphorylation of serine 3 by LIM and TES protein kinases, dephosphorylation by the protein phosphatase ‘slingshot’ and ‘chronophin’ and binding of 14-3-3 proteins. Furthermore, another actin-binding protein called tropomyosin protects filaments against ADF/cofilin and thereby adds another level of depolymerization rate regulation. Consistent with the idea that ADF/cofilin regulates a rate-limiting step during actin turnover, overexpression leads to an increase in axonal outgrowth, while inhibition of its activity using dominant-negative mutants or by overexpression of an inactivating kinase slows axon outgrowth.
Late Development Actin Organization and Function during the Development of Synapses
The actin cytoskeleton is the main structural component of both pre- and postsynaptic terminals. Here we focus on excitatory synapses, where the function of the actin cytoskeleton is best understood. The formation of synapses in the vertebrate central nervous system is a complex process that occurs over a protracted period of time. It is generally the case that dendritic filopodia are the structural precursors of dendritic spines during synaptogenesis, although direct emergence of new spines from the dendrite shaft has also been observed. Dendritic filopodia are long, thin protrusions characterized by a highly transient and motile behavior. In these protrusions F-actin is sparse until pre- and postsynaptic transmembrane cell adhesion proteins, such as the cadherin–catenin complex, link adjacent synapse precursors. The F-actin accumulates within early filopodial synaptic contacts, where it recruits synaptic signaling molecules. Simultaneously, the dynamic dendritic filopodia begin to morphologically change into intermediate structures sometimes called ‘protospines.’ These intermediate structures have also been referred to as cluster-type filopodia, or synaptic filopodia, and they can eventually mature into more stable dendritic spines. Only a fraction of the filopodia that emerge from a dendrite persist to become dendritic spines. Mature, postsynaptic dendritic spines are highly enriched in F-actin compared to the dendritic shaft (Figure 1, right). Based on measurements of green
Actin Cytoskeleton in Growth Cones, Nerve Terminals, and Dendritic Spines 19
fluorescent protein (GFP)–actin fluorescence recovery after photobleaching in cultured hippocampal neurons, only approximately 5% of the total actin in spines is relatively stable, while the majority of the actin almost completely turns over in a 2 min period. Unlike F-actin, G-actin is distributed homogeneously throughout axonal and dendritic processes in hippocampal neurons, which indicates that its main role at the synapse is to maintain the synaptic F-actin pool. The characteristic shape of dendritic spine heads is highly dependent on actin dynamics and the fraction of polymerized actin. Disruption of filamentous actin by the toxin latrunculin A transforms dendritic spines into filopodia-like processes. F-actin is found within the presynaptic nerve terminal and is preferentially concentrated around synaptic vesicles (Figure 2(b)). In resting conditions, approximately 30% of actin is in a polymerized state, with a turnover halftime of 20 s. During electrical activity, actin is further polymerized and recruited around vesicles from nearby axonal regions, leading many groups to propose a propulsive role for actin, either in maintaining the vesicle cluster or in guiding vesicle recycling. However, the precise role for actin in nerve terminals remains controversial, and may differ depending on the nature of the synapse. Although actin plays an active role in synaptic vesicle endocytosis in some systems, recent evidence indicates that in mammalian central synapses it passively inhibits vesicle fusion at the active zone, and helps sequester synaptic vesicles in the reserve pool. Synaptic vesicle trafficking from the reserve pool to the readily releasable pool can occur independently of actin. Thus, actin may serve primarily as a scaffold for other regulatory proteins involved in vesicle trafficking within the terminal. The recruitment and retention of presynaptic matrix proteins, such as bassoon and piccolo, also are dependent on actin. The higher content of F-actin in dendritic spines compared to nerve terminals correlates with more dynamic shape changes. Nevertheless, the pre- and postsynaptic cytomatrix components are strongly interconnected, and therefore their movements are physically coupled in established synapses. Engulfment of spinules (membrane extensions emerging from the head of big mushroom spines) by presynaptic axons is a process known as transendocytosis and suggests retrograde signaling or coordinated remodeling of pre- and postsynaptic membranes, which is likely modulated by actin. Actin Dynamics and Spine Morphogenesis
In recent years, imaging techniques both in vivo and in vitro have revealed that a majority of mature dendritic spines are stable over months in the adult brain.
They rarely form and disappear; instead, the dendritic spine head is constantly changing shape (morphing). This rapid spine motility is caused by regulated polymerization and depolymerization of actin, based on studies using drugs and cDNAs that perturb actin polymerization. The Rho family of GTPases is one common denominator controlling spine morphology. Multiple signaling pathways converge on the Rho family of small GTPases (particularly RhoA, Rac1, and Cdc42), which ultimately converge on the actin cytoskeleton to regulate spine morphology and dynamics. Generally speaking, RhoA inhibits, whereas Rac and Cdc42 promote, the growth and/or stability of dendritic spines. An expanding number of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) control these Rho family GTPases. For example, kalirin-7, a GEF for Rac1, mediates effects of the transmembrane signaling molecule EphB on spine maturation by activating Rac1 and its downstream effector p21-activated kinase (PAK). PAK1 and PAK3 are Rac effectors that promote formation and/or growth of spines. PAK also stimulates LIM kinase (LIMK), which phosphorylates and inactivates ADF/cofilin, an actindepolymerizing protein that mediates reorganization of the actin cytoskeleton. Recently, cofilin has been detected by immunoelectron microscopy to concentrate in the periphery of the spine head and within the postsynaptic density. Mice expressing dominantnegative PAK in the forebrain showed reduced spine density in cortical neurons, impaired long-term synaptic plasticity, and reduced memory consolidation. PAK activity is markedly reduced in Alzheimer’s disease due to a reduction in phospho-PAK, which in turn results in an increased cofilin activity and downstream loss of the spine actin-regulatory protein drebrin. Insulin receptor substrate p53 (IRSp53), an adaptor protein that connects Rac1 to the Wiskott– Aldrich syndrome protein WAVE2, is implicated in filopodia and lamellipodia formation in nonneural cells. IRSp53 is enriched in the postsynaptic density (PSD) by binding to Shank and PSD-95 family scaffolds. Overexpression of IRSp53 increases spine density, whereas RNA interference (RNAi) or dominant-negative inhibition of IRSp53 causes reduction of spine density and size. The Abl-interactor (Abi) adaptor proteins, so-named because they bind to the Abl tyrosine kinases, also play a part in Rac GTPase signaling and actin regulation. Abi2 is highly expressed in brain; and Abi2-deficient mice show reduced spine density and a decrease in the relative proportion of mushroom spines, which is thought to be associated with deficits in memory.
20 Actin Cytoskeleton in Growth Cones, Nerve Terminals, and Dendritic Spines
It is worth noting that components of Rho GTPase signaling pathways are highly represented among the genes so far identified in hereditary forms of human nonsyndromic mental retardation. These mutant genes include X-linked genes, such as PAK3 (see earlier) and oligophrenin, which is a Rho GAP. RNAi suppression of oligophrenin decreases spine length, mimicking the effect of active RhoA. Numerous scaffold proteins and actin-binding proteins are concentrated in spines and are known to control spine morphogenesis (Figure 2(b)). Many of these are regulated by the small GTPases described earlier. Overexpression of a-actinin 2, a protein linking N-methyl-D-aspartate (NMDA) receptors to the actin cytoskeleton, increases the length and density of dendritic protrusions in cultured hippocampal neurons. Clustering of the actin-binding protein drebrin occurs early in development, supporting its involvement in spinogenesis and synaptogenesis. Cortactin is an activator of the ARP2/3 actin nucleation machinery that interacts with the postsynaptic density scaffold Shank. RNAi knockdown of cortactin results in depletion of dendritic spines, whereas overexpression of cortactin causes enlargement of spines. Myosin II is also enriched in postsynaptic density preparations and acts as a molecular motor that produces tension on actin filaments. Inhibition of myosin IIB destabilizes mushroom spines, suggesting that the structure and function of spines are regulated by an actin-based motor in addition to actin polymerization. Effects of Synaptic Activity on Actin Polymerization in Dendritic Spines
Synaptic plasticity is associated with changes in actin polymerization and spine morphology. Long-term potentiation (LTP) is associated with formation of new spines, enlargement of existing spines, and a shift of the actin turnover equilibrium toward polymerization of F-actin. By contrast, long-term depression (LTD) is associated with shrinkage and/or retraction of spines due to an increase in actin depolymerization. In addition, synaptic activity is thought to play an important role in determining whether a synapse will be stabilized or eliminated. During induction of LTP, a-amino-3-hydroxy-5methyl-4-isoxazole propionic acid (AMPA) receptors are rapidly recruited to dendritic spines through Ca2þ-permeable NMDA receptor activation. In response to NMDA receptor activation, profilin, a promoter of F-actin assembly, is targeted to spine heads, while cortactin redistributes from spines to the dendritic shaft. Distinct but overlapping signaling pathways are involved in LTD and spine shrinkage. During induction of LTD, NMDA receptor-mediated
calcium influx triggers phosphatase 2B (calcineurin) activity. Calcineurin in turn activates a downstream phosphatase ‘slingshot’ and promotes actin severing and depolymerization through ADF/cofilin. Synaptic activity can also affect the relationship between F-actin and the plasma membrane of dendritic spines. Myristoylated alanine-rich protein kinase C substrate (MARCKS), a major target of protein kinase C (PKC), might be involved in local coupling of activity-dependent calcium entry with regulation of actin assembly–disassembly cycles. During synaptic activity, when calcium levels increase and stimulate PKC, MARCKS detaches from the plasma membrane, thereby freeing phosphatidylinositol 4,5-bisphosphate to interact with actin-binding proteins such as profilin and ARP2/3, which in turn can locally alter actin dynamics and membrane trafficking.
Comparing Growth Cones, Dendritic Spines, and Nerve Terminals One might be tempted to generalize the role of actin in growth cones, synaptic terminals, and spines, and introductions to this topic often try to point out similarities. However, the current state of our knowledge does not allow an in-depth comparison. Nonetheless, certain parellels exist (Figures 2(a) and 2(b)). In growth cones, a relatively stable, less dynamic F-actin population is found toward the growth cone center, which is thought to be involved in contractile behavior. Highly dynamic F-actin is concentrated in the periphery of the growth cone, where it is thought to mediate membrane protrusion, resulting in motile filopodia and lamellipodia. Similarly, a stable pool of F-actin is present in the dendritic spine core, while a dynamic pool lies peripherally in its shell, which exhibits highly dynamic, sometime protrusive behavior. In growth cones, the organization and dynamics of the underlying actin structures have been analyzed using both high-contrast electron microscopy and dynamic speckle microscopy. These analyses converge in a detailed working model of growth cone behavior. In spines, defining the detailed actin organization and dynamics has been more difficult. Ultrastructural analysis of filament polarity suggests that most actin filaments are oriented with their barbed end pointing toward the head of the spine protrusion, similar to the preferred orientation of actin filaments toward the growth cone leading edge. One notable difference is that spines generally lack the dense parallel organization of F-actin that characterizes growth cone filopodia. However, at times, rapidly growing, elongated filopodia-like
Actin Cytoskeleton in Growth Cones, Nerve Terminals, and Dendritic Spines 21
structures are observed on spine heads. The purpose of such filopodial-like protrusions emerging from spine heads remains unclear. In growth cones, and along developing dendrites, such filopodial protrusions are thought to extend the structure’s sensory radius to probe the environment for guidance cues or neurotransmitters. Speckle microscopic analysis of actin dynamics has not been performed in spines; however, indirect data on spine actin dynamics have been derived from photobleaching recovery experiments (see the section titled ‘Actin organization and function during the development of synapses’). These experiments reveal a highly dynamic F-actin population, which may be roughly comparable to the rapidly turning over F-actin of growth cone lamellipodia. While there might be some similarities between actin behavior in growth cones and spines, the role and organization of actin in nerve terminals appear to be distinct. F-Actin is distributed throughout the presynaptic nerve terminals and is preferentially concentrated around synaptic vesicles. However, mature nerve terminals do not exhibit significant protrusive behavior and contain much less actin compared to either growth cones or spines. In nerve terminals, a specific role for actin remains unclear. See also: Axonal Regeneration: Role of Growth and Guidance Cues; Axonal and Dendritic Identity and Structure: Control of; Cytoskeletal Interactions in the Neuron; Cytoskeleton in Plasticity; Dendritic Spine History; Glutamate Regulation of Dendritic Spine Form and Function; Growth Cones; LIM Kinase and Actin Regulation of Spines; Microtubules: Organization and Function in Neurons; Spine Plasticity.
Further Reading Calabrese B, Wilson MS, and Halpain S (2006) Development and regulation of dendritic spine synapses. Physiology (Bethesda) 21: 38–47. Carlisle HJ and Kennedy MB (2005) Spine architecture and synaptic plasticity. Trends in Neurosciences 28: 182–187. Dehmelt L and Halpain S (2004) Actin and microtubules in neurite initiation: Are MAPs the missing link? Journal of Neurobiology 58: 18–33. Dent EW and Gertler FB (2003) Cytoskeletal dynamics and transport in growth cone motility and axon guidance. Neuron 40: 209–227. Ethell IM and Pasquale EB (2005) Molecular mechanisms of dendritic spine development and remodeling. Progress in Neurobiology 75: 161–205. Lippman J and Dunaevsky A (2005) Dendritic spine morphogenesis and plasticity. Journal of Neurobiology 64: 47–57. Luo L (2002) Actin cytoskeleton regulation in neuronal morphogenesis and structural plasticity. Annual Review of Cell and Developmental Biology 18: 601–635. Oertner TG and Matus A (2005) Calcium regulation of actin dynamics in dendritic spines. Cell Calcium 37: 477–482. Pollard TD and Borisy GG (2003) Cellular motility driven by assembly and disassembly of actin filaments. Cell 112: 453–465. Sarmiere PD and Bamburg JR (2004) Regulation of the neuronal actin cytoskeleton by ADF/cofilin. Journal of Neurobiology 58: 103–117. Strasser GA, Rahim NA, VanderWaal KE, et al. (2004) Arp2/3 is a negative regulator of growth cone translocation. Neuron 43: 81–94. Suter DM, Schaefer AW, and Forscher P (2004) Microtubule dynamics are necessary for SRC family kinase-dependent growth cone steering. Current Biology 14: 1194–1199. Zhang W and Benson DL (2002) Developmentally regulated changes in cellular compartmentation and synaptic distribution of actin in hippocampal neurons. Journal of Neuroscience Research 69: 427–436. Zhang XF, Schaefer AW, Burnette DT, et al. (2003) Rho-dependent contractile responses in the neuronal growth cone are independent of classical peripheral retrograde actin flow. Neuron 40: 931–944.