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Axon Initial Segment Cytoskeleton: Architecture, Development, and Role in Neuron Polarity - PubMed

Review

Axon Initial Segment Cytoskeleton: Architecture, Development, and Role in Neuron Polarity

Steven L Jones et al. Neural Plast. 2016.

Abstract

The axon initial segment (AIS) is a specialized structure in neurons that resides in between axonal and somatodendritic domains. The localization of the AIS in neurons is ideal for its two major functions: it serves as the site of action potential firing and helps to maintain neuron polarity. It has become increasingly clear that the AIS cytoskeleton is fundamental to AIS functions. In this review, we discuss current understanding of the AIS cytoskeleton with particular interest in its unique architecture and role in maintenance of neuron polarity. The AIS cytoskeleton is divided into two parts, submembrane and cytoplasmic, based on localization, function, and molecular composition. Recent studies using electron and subdiffraction fluorescence microscopy indicate that submembrane cytoskeletal components (ankyrin G, βIV-spectrin, and actin filaments) form a sophisticated network in the AIS that is conceptually similar to the polygonal/triangular network of erythrocytes, with some important differences. Components of the AIS cytoplasmic cytoskeleton (microtubules, actin filaments, and neurofilaments) reside deeper within the AIS shaft and display structural features distinct from other neuronal domains. We discuss how the AIS submembrane and cytoplasmic cytoskeletons contribute to different aspects of AIS polarity function and highlight recent advances in understanding their AIS cytoskeletal assembly and stability.

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Figures

Figure 1
Figure 1

Architecture of the axon initial segment (AIS) and its key protein components. ((a), top) Neuron polarity. Polarized neurons receive synaptic inputs in the somatodendritic domain (green), which transmits the signals through the axon hillock to the axon initial segment (red). The AIS integrates synaptic inputs and initiates an action potential that gets propagated along the distal axon (blue) and amplified at nodes of Ranvier. ((a), bottom) Molecular organization of the AIS. The AIS can be divided into three layers: the plasma membrane, submembrane cytoskeleton, and inner AIS shaft (left), each having AIS-specific features (zoomed view at right). The scaffolding protein ankyrin G (AnkG) recruits many other proteins to the AIS and can interact with components in the different AIS regions. In the plasma membrane, AnkG through its N-terminal membrane-binding domain binds voltage-gated ion channels, which are important for action potential initiation and regulation, and cell adhesion molecules (CAMs). The submembrane cytoskeleton contains AnkG, βIV-spectrin, and actin filaments. These proteins form a periodic network along the entire length of the AIS. Periodic actin is spaced ~190 by at least two βIV-spectrin subunits, which in turn attach to the membrane through interactions with AnkG. In addition to periodic actin, relatively long, randomly oriented, dynamic actin filaments also exist in the submembrane cytoskeleton, and these filaments may have functions distinct from periodic actin. The inner AIS shaft contains microtubules bundles (fascicles), neurofilaments, and potentially also actin filaments (not shown). AnkG can extend its C-terminal tail into the inner AIS shaft where it is predicted to interact with microtubule fascicles. (b) Domain organization of isoforms of ankyrin G (AnkG). AnkG population contains two large neuron-specific isoforms, 270 kDa and 480 kDa, that localize specifically to AIS and nodes of Ranvier. The N-terminal membrane-binding domain of AnkG contains 24 ANK repeats (33-amino acid motif that mediates protein interactions). These 24 ANK repeats fold to form four independent subdomains (each containing 6 ANK repeats) that compose a globular membrane-binding domain. The spectrin-binding domain allows interactions with βIV-spectrin and thereby attachment of the submembrane cytoskeleton to the membrane. The serine-rich domain is glycosylated with N-acetylglucosamine monosaccharides. 480 kDa AnkG contains a 220 kDa insert following the spectrin-binding domain that is predicted to form a random coil. (c) Domain organization of typical α- and β-spectrin isoforms. (Top) α-spectrin comprises an incomplete spectrin repeat at the N-terminus (red), 20 complete spectrin repeats (violet), an SH3 (Src homology 3) domain (dark blue) inserted into spectrin repeat 9, and two EF-hand motifs at the C-terminus. Last two spectrin repeats (dark violet) can interact with the first two spectrin repeats in β-spectrin to form an antiparallel dimer. (Middle) β-spectrin comprises an N-terminal actin-binding domain (BD), 16 full spectrin repeats (violet), an incomplete 17th spectrin repeat (red), a variable region specific for individual β-spectrin isoforms (specific domain, SD), and a C-terminal pleckstrin homology (PH) domain. Spectrin repeats 1 and 2 (dark violet) dimerize with α-spectrin; spectrin repeats 14 and 15 interact with AnkG. (Bottom) A typical spectrin molecule represents an αβ-heterotetramer (two α- and two β-subunits) that form a rod-shaped structure. Two αβ-spectrin dimers, each formed by transverse interaction between α-spectrin repeats 19 and 20 and β-spectrin repeats 1 and 2, associate with each other longitudinally by making complete spectrin repeats through pairwise interaction of the incomplete spectrin repeats at the N-terminus of α-spectrin and the C-terminus of β-spectrin. (d) AIS-specific βIV-spectrin isoforms. βIV∑1 and βIV∑6 spectrins. The full-length βIV∑1 isoform has organization typical to β-spectrins (c). The βIV∑6 isoform lacks the N-terminus and first 10 spectrin repeats.

Figure 2
Figure 2

Platinum replica electron microcopy (PREM) revealing axon initial segment (AIS) structure of 21 DIV hippocampal neuron. (a) A neuron with dendrites and an axon (box B) extending from the soma. (b) Enlargement of box B in (a) showing a segment of the AIS (box C) and a portion of the axon hillock (box D), a region of the neuron that connects the soma and axon. The axon hillock is largely covered by an axon or dendrite (arrow head) from a different neuron. (c) Enlargement of box C in (b). (d) Enlargement of box D in (b) showing a microtubule fascicle (arrow) in the axon hillock that appears to enter the AIS. Most of the microtubules in the axon hillock are nonfasciculated. (e) Enlargement of box E in (c) showing a dense coat of globular and fibrillary structures. This coat corresponds to two upper layers of the AIS cytoskeleton, the immobilized plasma membrane proteins and submembrane cytoskeleton. Many of the globular structures in the coat correspond to voltage-gated ion channels and AnkG bound to its interaction partners. The fibrils represent cell adhesion molecules (CAMs), βIV-spectrin, and some AnkG molecules lacking interacting proteins. A microtubule fascicle can be seen in an opening of the coat where globules and fibrils are lacking (arrow). Note that such occasional openings in the AIS coat create small windows through which the inner AIS shaft can be seen (see Figure 1(a)). Bars: (a) 10 μm; (b) 2 μm: (c) 200 nm; (d) 100 nm; (e) 100 nm.

Figure 3
Figure 3

PREM images showing different stages of assembly of the AIS coat in hippocampal neurons. ((a)-(b)) Proximal axons of early 3 and 4 DIV neurons showing microtubules that are either loosely aligned (a) or tightly bundled (b), respectively. (c) Proximal axon of 7 DIV neuron displaying a loose fibrillar (arrows) coat containing sparse globules (inset) covering the microtubules. (d) Proximal axon of a 10 DIV neuron containing a dense fibrillar-globular coat. (e) A 14 DIV neuron displaying a dense coat containing mostly globules (inset) and few visible fibrils. (f) A 21 DIV neuron revealing a mature coat dominated by globules, although few parallel fibrils are visible (inset).

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