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ScienceDirect Neurofilament assembly and function during neuronal development Adi Laser-Azogui1, Micha Kornreich1, Eti Malka-Gibor2 and Roy Beck1 Studies on the assembly of neuronal intermediate filaments (IFs) date back to the early work of Alzheimer. Developing neurons express a series of IF proteins, sequentially, at distinct stages of mammalian cell differentiation. This correlates with altered morphologies during the neuronal development, including axon outgrowth, guidance and conductivity. Importantly, neuronal IFs that fail to properly assemble into a filamentous network are a hallmark of neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer’s, and Parkinson’s disease. Traditional structural methodologies fail to fully describe neuronal IF assembly, interactions and resulting function due to IFs structural plasticity, particularly in their C-terminal domains. We review here current progress in the field of neuronal-specific IFs, a dominant component affecting the cytoskeletal structure and function of neurons. Addresses 1 The Raymond and Beverly Sackler School of Physics and Astronomy, Tel-Aviv University, Tel-Aviv 69978, Israel 2 Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel Corresponding author: Beck, Roy ([email protected])
Current Opinion in Cell Biology 2015, 32:92–101
III IF protein of mesenchymal cells, is transiently coexpressed with nestin in neural precursor cells. Its expression declines during differentiation [2]. Neuronal-specific IFs include five principal subunit proteins: the type IV light, medium, and heavy molecular mass neurofilament (NF) triplet proteins (NF-L, NF-M, and NF-H, respectively), a-internexin, and the type III peripherin. Mature filaments are composed of several combinations of these five subunits. a-Internexin is expressed in most differentiated neurons; its expression precedes that of the NF triplet and declines somewhat postnatally, while expression of the NF triplet sharply rises [3]. In mature neurons, the main role recognized for NFs is to increase the axonal caliber of myelinated axons and consequently their conduction velocity [4]. NF proteins represent the main cytoskeletal elements in mature neurons, however, at the initiation of axonal radial growth, NF-H expression is usually delayed relative to NF-L and NF-M [4]. It is speculated that delayed NF-H expression allows the cytoskeleton network to establish in a distal to proximal gradient [5]; although cases of varied sequential expression have been reported (Figure 1).
This review comes from a themed issue on Cell architecture Edited by Sandrine Etienne-Manneville and Elly M Hol
http://dx.doi.org/10.1016/j.ceb.2015.01.003 0955-0674/# 2015 Elsevier Ltd. All rights reserved.
Introduction Differentiated neurons live longer than any other cell type, but unlike other cell types, they do not proliferate. Throughout the development of neurons, their cytoskeleton undergoes modifications to sustain its mechanoelastic properties necessary for proper function. Striking evidence for such remodeling is given by the sequential expression of IF throughout neuronal development and maturation (Figure 1). Nestin, a type IV IF protein, is expressed in neural stem cells at the earliest stage of embryonic development. Nestin is down-regulated after differentiation and replaced by cell type-specific IFs [1]. Vimentin, a type Current Opinion in Cell Biology 2015, 32:92–101
Peripherin, a type III IF, is co-expressed with NF triplet in the adult peripheral nerve system, in a relatively fixed stoichiometry and exhibits a distribution pattern identical to those of triplet NF proteins in sciatic axons [6]. Peripherin is also found in neurons of the central nerve system (CNS) that have projections toward peripheral structures, such as spinal motor neurons [6]. During development, peripherin is differentially regulated in sensory neurons, and plays a role in motor axons’ growth [7]. Synemin, a type IV IF, is not a neuronal-specific protein. Nevertheless, it forms obligate heteropolymers with vimentin or the NF triplet in neurons. The three synemin isoforms are differentially expressed in neuronal subpopulations, and also show developmental stage specificity. Since they are obligate heteropolymers they always co-express with either vimentin (at early developmental stage) or any neuronal IF, that is NF-L, a-internexin or peripherin [8,9]. The sequential IF expression during development raises fundamental challenges in understanding the differential role of each of the IF proteins. Here, we review the recent progress made to understand the assembly, transport, and function of neuronal-specific IFs from a developmental perspective. We begin with a short introduction to neuronal IF self-assembly into filaments, and continue with www.sciencedirect.com
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Figure 1
Central nervous system neurons (c) α -int, NFL, NFM Precursor nerve cells (a) Nestin, Vimentin
(b) α -int
(d)
α -int, NFM
(f) α -int, NFL, NFM, NFH
(g) Tail Phosphorylation: NFM and NFH
(e) α -int, NFL, NFH (h) Distinct CNS neurons
(i)
Peripheral neurons
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Post-natal
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Sequential expression of IF during development of the nervous system. Neuronal IF proteins are sequentially expressed during development to accommodate for the changing mechanoelastic needs of the developing neurons. Several studies have shown that different IF subunits and expression levels are further specified according to spatial location within the nervous system, exact developmental stage, and organism species. The above scheme demonstrates the main combinations with respect to development, but does not reflect all the different pathways. For example, discrepancy between expression levels of NF-M in chicken embryo has been recorded [65,66]. This discrepancy might depend on specific location within the CNS and/or exact developmental stages. References for each stage are as follow: a, [2,67]; b, [3]; c, [68]; d, [69]; e, [66]; f, [13]; g, [4]; h, [8,70,71] (a-internexin, synemin and peripherin respectively); i, [6].
the role of post-translational modifications (PTM). Recent progress in the characterization of NF axonal transport and the resultant axonal growth will then be discussed. Last, we will review current measurements and theories addressing the organization of NFs using methods adopted from polymer physics. We will conclude with some remaining open questions and perspectives on the future of the field.
From subunit proteins to filament assembly Self-assembly of neuronal IF proteins into mature filaments follows the principle paradigm of all cytoplasmic IFs [10]. Currently, atomic structural studies are limited to vimentin [11,12], as presumably the long disordered Cterminal tails of neuronal IFs hamper structural studies. IF proteins share a common tripartite molecular structure; a conserved central a-helical rod domain, flanked by intrinsically disordered N-terminal head, and C-terminal tail domains. The rod domain drives coiled-coil interaction and co-assembly with other subunits to form dimers, protofilaments, and filaments (Box 1). The major differences between the neuronal IF subunits are the length and sequence of their C-terminal tails. These differences are of www.sciencedirect.com
great importance since those intrinsically disordered tails form flexible extensions that link the filaments to each other and to other elements in the cytoplasm (Figure 2). NF-M and NF-H can dimerize to form filaments only in association with either NF-L or a-internexin. NF-L and a-internexin can either self-assemble or co-assemble with any neuronal IF subunit in vitro [13]. In addition, only human NF-L is able to self-assemble in vivo [14], yet in other mammals NF-L does not self-assemble. In vitro, NF-L homopolymers of bovine, porcine, and rodents have been observed. In contrast to the NF triplet, both a-internexin [13] and peripherin [6] are able to homopolymerize, which coincides with the observation that in some neurons they are the sole NF protein (Figure 1). NF assembly depends heavily on their head domains, and especially their PTM. Of special importance are specific serine residues in NF-L; their phosphorylation state and the rapid turnover of these phosphates regulate NF polymer assembly and affect their axonal transport [15]. Similar to NF-L, phosphorylation of the head domain of a-internexin causes filament disassembly [16]. Current Opinion in Cell Biology 2015, 32:92–101
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Box 1
Type III IF
(a)
Central α-helical core
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Tail
Key: Peripherin N
C Coil 1a
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Coil
Coil 2a/b
KSP repeats
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NF triplet
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C C
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(d)
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MTs
2a 2b Unit length filament
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Dimer
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Self-assembly of filaments: (a) In homology to the other IF proteins, neuronal IFs contain three domains: an N-terminal head domain, a central ahelical core which is further divided into coils 1a, 1b and 2a/b, and a C-terminal tail domain. (b) The first step of neuronal IF formation is the dimerization of NF-L or a-internexin with any other neuronal IF proteins, through association of their conserved rod domains to form parallel, sideto-side, coiled-coil dimers. Two coiled-coil dimers line up in a half-staggered manner, forming an anti-parallel tetramer. Then, a rapid lateral aggregation of about eight tetramers occurs to form a unit-length-filament (ULF) of approximately 55 nm in length [73]. An elongation step occurs through the axial aggregation of ULFs to form immature filaments of about 16 nm in diameter and many microns in length. (c) Radial compaction
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Ser23 of NF-M exhibits early phosphorylation and rapid turnover similar to what is observed for NF-L [17], although no substantial evidence has been presented regarding the effect of head domain phosphorylation of NF-M or NF-H on filament assembly. NF-M and NF-H head domains are also glycosylated on sites close to the phosphorylation sites [18,19]. This PTM proximity suggests that glycosylation also affects filament assembly, perhaps by regulating phosphorylation and modulating the early protein–protein interaction of the assembly cascade.
Figure 2
(a)
(b)
The tail domain of a-internexin and NF-L is short, while that of NF-M and NF-H is much longer and contains many PTM sites. In mature axons, NF-M and NF-H are heavily phosphorylated on their tail domains, primarily on serine residues of multiple lys–ser–pro (KSP) repeats. Tail phosphorylation has no effect on filament assembly, but rather contributes to lateral extension and formation of the cytoskeletal lattice [4]. Assembly of axonal cytoarchitecture is a complex process, involving dynamic short and long range interactions between phosphorylated tails of NF-H and NF-M with microtubules, actin, and other NF-associated proteins, such as plectins [20]. Crossbridging between NFs through the tail domains of NF-H and NF-M is influenced by the phosphorylation level of these tails [4] and mediated by divalent metal cations [21]. Phosphorylated NFs are localized mainly in the axon, whereas the dephosphorylated forms are found in the cell body and dendrites [22]. Pant et al. [23] demonstrated that head domain phosphorylation of NF-M inhibits its tail domain phosphorylation, suggesting that KSP phosphorylation of NF-M in axons depends on prior dephosphorylation of head domain sites. These findings support the idea that phosphorylation of head domains regulates both NF assembly and phosphorylation of specific tail domains, thus preventing premature NF assembly and tail phosphorylation in cell bodies. The different phosphorylation patterns in cell bodies versus axons also indicate the role of phosphorylation in NF transport and perhaps in interaction with other cytoskeletal proteins. Glycosylation sites are present near KSP phosphorylation sites in NF-M and NF-H tails [18,19]. O-linked glycosylation is believed to act in competition with phosphorylation in a dynamic cycle. Dysregulation of this relationship,
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Axonal and dendritic NFs. In axons (a), NFs fill the axoplasm. They are more evenly spaced and their C-terminal tail cross-bridges are longer and more numerous. In dendrites (b), NFs are tightly packed. Although there are cross-bridges between NFs, they appear to be shorter and less frequent than those in axons. Bar, 0.1 mm. Figure is reprinted with permission from Rockefeller University Press, Hirokawa et al.: J Cell Biol 1984, 98:1523–1536 [72].
observed in the brains of a rat model for Alzheimer’s disease, led to NF-M hyper-phosphorylation and accumulation [24]. Similarly, a study on the role of NF-M posttranslational modifications in ALS [25] demonstrated reciprocal changes in phosphorylation and O-linked glycosylation of NF-M tail, which was characterized by aggregated NF proteins. The NF-M glycosylation site in question was localized to the KSP repeat region. NF-H is also extensively modified by O-linked glycosylation in the KSP repeats in the tail domain [19]. The proximity of O-linked glycosylation and phosphorylation sites in both head and tail domains indicate that these modifications may influence one another and play a role in filament assembly and network formation.
Axonal transport of NFs NFs are synthesized in the cell body and travel long distances along axons to reach their sites of function. The mechanisms that underlie their axonal transport are the
( Box 1 Legend Continued ) then takes place resulting in close packing of the molecular filaments to constitute the final 10 nm ‘bottlebrush’ filament [4,10]. Filaments are lengthened by dynamically joining ends of shorter filaments [74]. The core of the filament is composed of the central rod domain, while the flexible C-terminal tails decorate it in a bottlebrush architecture [75]. (d) Electron micrograph cross-sections from different types of axons showing heterogeneous NF cytoarchitecture of myelinated sensory axons (top), unmyelinated axons (middle) and myelinated motor axons (bottom). Reprinted from Szaro BG, Strong MJ: Post-transcriptional control of neurofilaments: new roles in development, regeneration and neurodegenerative disease. Trends Neurosci 2010, 33 (1):27–37, Copyright (2010), with permission from Elsevier [76]. www.sciencedirect.com
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subject of intense study. The state of assembly of NFs during axonal transport has been under debate, with studies that demonstrate movement of non-filamentous assemblies of NF subunits [26], short filaments [27], or both [28]. Yuan et al. showed that at proximal regions of the axon, the transport of subunit assemblies predominates, whereas the transport of short filaments predominates at distal regions of the same axon. It is speculated that NF proteins exist in multiple assembly forms during axonal transport and that the transported NF subunits assemble into filaments during the transport [28]. Yabe et al. suggested that the controversies arise from the use of neurons at different developmental stages, and that the predominant form in which NF subunits undergo axonal transport varies in accordance with the rate of axonal elongation and accumulation of NFs within developing axons [29]. Another long-standing controversy regards NF transport dynamics. The ‘stop-and-go’ transport model suggests that axons contain a single population of NFs that all move in a stochastic, bidirectional and intermittent manner [27,30]. Here, the instantaneous rate of movement is fast, but the overall rate is slow because the movements are interrupted by prolonged pauses [31]. The ‘stop-andgo’ hypothesis is challenged by the stationary hypothesis, suggesting that 10% of all NFs in a given axon are transported in assemblies [28]. These are deposited into a stationary and cross-linked NF cytoskeleton that constitute about 90% of NFs. The stationary NFs remain fixed in place for many months without movement, integrated with other cytoskeletal components [28]. These different perspectives have significant implications for the understanding of NF dynamics and reorganization in axons. The movement of NFs in axons occurs along microtubule tracks, by the retrograde motor dynein and the anterograde motor kinesin-1 [4]. Little is known about the nature of NF interaction with motors. Based on recent observations that NFs move in a fully extended configuration, it is suggested that they may be pulled from their leading end during both anterograde and retrograde motion [32].
Axonal radial growth Following the formation of stable synapses, NFs accumulate and the axonal diameter increases. The axonal diameter influences the rate of axonal impulse propagation. Radial growth is also dependent upon the formation of compact myelin [33]. NF-M and NF-L are required for radial growth, as loss of each results in small caliber axons [34–36]. As NF-H expression increases, radial growth of only the largest axons becomes somewhat dependent on NF-H [37]. The expression of tail-deleted NF-H affects the rate, but not the final magnitude, of radial axonal growth [36]. Current Opinion in Cell Biology 2015, 32:92–101
The mechanism for this radial axonal growth has been thought to involve myelin-dependent phosphorylation of KSP sites in NF-M tail domains; the formation of compact myelin and NF accumulation were both shown to be necessary for radial axonal growth [36]. However, recent studies have shown that KSP phosphorylation within the NF-M tail is not a determinant of radial axonal growth. Axonal diameters of mice expressing full-length, but KSP phospho-incompetent NF-M, are indistinguishable from those of control mice [38,39]. Together, these findings demonstrate the dominant role of NF-M tail domain, but not its phosphorylation, in determining axonal caliber. Nevertheless, NF protein expression by itself has not been found to play a key role for nerve development and function [34–38,39]. A different explanation of NF structural function, phosphorylation and the interplay between those and myelination to determining axonal caliber is required. A possible mechanism suggested by Barry et al. [39] is that the NF-M tail domain serves as a scaffold for cytoskeletal linking proteins, such as plectin. In agreement, deletion of the neuron specific isoform of plectin [40] resulted in a phenotype similar to that observed in NF-Mdeleted mice [41] and mice expressing C-terminally truncated NF-M [36]. Thus, increasing the distance that NF-M C-terminus extends from the filament core may have allowed a longer-range interaction between NF-M tail domain and plectin, resulting in larger axonal diameters. Interestingly, positive correlations were found between the number of KSP repeats of NF-M and the overall animal length, in a subset of mammalian species [42].
Neurofilaments as interacting bottlebrushes The dynamics, structure, and mechanical properties of NF networks are largely determined by their bottlebrush architecture (Box 1c), a well-studied topic in polymer physics. However, due to the complex nature of the NF tails, which include hydrophobic, hydrophilic, and many negative and positive charged residues, a full physical understanding is still missing. This problem was tackled by several groups using polymer physics techniques, including theoretical, computational and controlled in vitro experiments (Figure 3). The theoretical models and simulations currently account for the best available molecular understanding of NF network organization. The theoretical works predict the conformations of the tails during neuronal development, when subunit compositions are different from the stable composition of mature neuron. In addition, those studies examine the role of KSP phosphorylation on the tail expansion. Selfconsistent mean field calculations of NF networks, pioneered by Zhulina and Leermakers [43], studied several NF compositions and phosphorylation levels. These studies predict a dominant role for NF-M in determining www.sciencedirect.com
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Figure 3
(a)
(c)
T=0h
24 h
Cs=2.5 mM, 0.9 wt%
Cs=4.2 mM, 1.5 wt%
BG
BG (d)
(e)
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Cs (mM) 40
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In vitro and simulation studies on NF interactions and self-assembly. (a) MC simulation snapshot of two opposing NFs in physiological condition and 40 nm separation. Higher mutual interpenetration of the C-terminal tails is observed when compared to larger NF separations. Colors indicate different subunit proteins [51]. Color-coding for the different subunit protein: NF-L (green), NF-M (blue), NF-H (red) and backbone (gray). (b) NFs spacing under compaction generated by osmotic pressure and measured by small angle X-ray scattering. The results show a transition from an expanded state at low osmotic pressure to a condensed state at high osmotic pressure where NFs are strongly entangled. Full (empty) symbols stand for nematic (isotropic) hydrogels in various monovalent salt concentrations (Cs). The X symbols represent a second, salt-independent, correlation peak that follows the NF-L homopolymer trend [52]. (c) NFs show water retention ability over many hours. At low salt concentration, within the blue phase, and at high salt concentration of the nematic phase, the hydrogels retain their shape and volume in comparison to faster water release in the isotropic phase [54]. (d) NFs are the softest filament within the cytoskeleton network. NF’s persistence length, measured by AFM, is shown to be tunable by the subunit protein composition which alter intra-filament interactions [60]. Scale bar = 50 nm. (e) Cross polarized microscopy images reveal the macroscopic ordering within the NF hydrogels. For example, after 30 days incubation, NF-L homopolymers hydrogel (top) show more swelling and weaker nematic alignment than NF-L:NF-M hydrogels (bottom) [53]. (f) Rheological measurements of cross-linked NF networks showing variation of the linear viscoelastic moduli at different protein (left) and Mg2+ (right) ion concentrations [57].Plots are reprinted with permission from Jayanthi et al.: J Biol Phys 2013, 39:343–346; Beck et al.: Nat Mater 2010, 9:40–46; Deek et al.: Nat Commun 2013, 4:2224; Beck et al.: Langmuir 2010, 26:18595–18599; Jones and Safinya: Biophys J 2008, 95:823–835; Yao et al.: Biophys J 2010, 98:2147– 2153.
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the inter-filament spacing and non-specific attraction between adjacent filaments. Moreover, recent calculations offer the only theoretical study, to date, which takes a-internexin into account [44]. This study shows that the inclusion of a-internexin subunits regulates the structural properties including tail extensions from the backbone, compressibility and frequency of cross-bridges. The effects of NF phosphorylation are also examined by Monte Carlo (MC) simulations, showing milder effects of NF-H phosphorylation on tail extension compared to the mean field model predictions [45]. Further MC simulations examined the NF triplet under varying ionic strengths, hydrophobicity, grafting locations [46,47], and inclusion of NF-M variant with a different number of KSP repeats [39]. These studies suggest that NF-M tails, regardless of their phosphorylation state, are more extended than NF-H tails, and that the attraction between tails can be regulated by Ca2+ ions and not by hydrophobic interactions. A recent study includes the added effect of backbone charge and explicit salt ions on NF tails conformation. This study demonstrates a tendency to metastability with the tails folding back and forming loops [48], in agreement with previous molecular dynamics simulations [49,50]. Simulations of the interactions of two opposing filaments show that at physiological salt concentration, tails from opposing filaments exhibit coiled conformations with increasing overlap upon compression (Figure 3a) [51]. The structure of reconstituted bovine NF networks, including NF-L homopolymers and co-assemblies with NF-M and/or NF-H, were experimentally studied using small angle X-ray scattering and microscopy (Figure 3b– e) [52,53,54]. These in vitro studies quantify the interfilament distance and macroscopic nematic alignment under controlled conditions, and directly coupled to the above-mentioned theoretical models. Nonetheless, some experimental results still lack a theoretical explanation, such as an unexpected collapsed conformation of NF-L:NF-M networks, liquid crystalline phase transitions and a collapsed condensed state at higher osmotic pressure [55]. Such discrepancies may arise since most simulations do not, for now, fully consider the complex inter-filament interactions and backbone flexibility (Figure 3d). The importance of multivalent ions in cross-linking opposing filaments has been known for a long time. It was demonstrated that divalent ions behave as effective crosslinkers for vimentin and NF networks, and that the elasticity of these networks is consistent with a theory of semiflexible polymers in an affine thermal (entropic) model (Figure 3f) [56,57]. These results, and the microstructural features predicted by fitting them to models assuming an entropic origin of the elasticity, are in agreement with complementary microrheology experiments Current Opinion in Cell Biology 2015, 32:92–101
[58]. Interestingly, the last 11 residues of vimentin tail domain are crucial in mediating the interaction between vimentin filaments and divalent cations [56]. A comprehensive theoretical model describing network strain-stressed deformations at different applied stresses, reports very good agreement with experimental data and molecular dynamic simulations [59]. The predictions of mechanical properties are based on a bottom-up approach starting with single-chain force-extension. However, NFs persistence length measurements under varying salt conditions and changing NF-H and NF-M ratios present unpredicted trends with respect to both ionic strength and tail compositions [60]. Notably, NF tails may crosslink within the same filament and thus increase the apparent filament flexibility. Such intra-filament attractions are not considered in current theoretical modeling.
Conclusions and perspective The sequential and parallel expression of neuronal IFs, as well as their PTMs, is reflected both in the structure and functionality of the cytoskeleton during development. However, there is still a gap between our understanding of the IF composition variations and their macroscopic organization, dynamics, and pathologies. Driven by the inability to gain atomic structural information, neuronal IFs assembly and structure are studied by simulation, modeling and polymer physics-inspired experiments. Therefore, future efforts will probably extend in vitro studies to a larger subset of IFs, and will include newly developed techniques that have already provided solid results on other IFs [61,62]. Moreover, the relation between cell differentiation and the surrounding matrix mechanical properties has recently drawn immense interest [63,64]. The relation between different IF subunits expression during development and their structure and mechanoelastic function is expected to be intriguing, and to contribute to our understanding of their function.
Acknowledgements We thank Cyrus Safinya, Joanna Deek and Michael Wyrsta for insightful remarks and discussions. This work was supported by the Israel Science Foundation (grant 571/11), the European Community’s 7th Framework Programme (293402), the Sackler Institute for Biophysics and the center from nanoscience and nanotechnology at Tel Aviv University.
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16. Tanaka J, Ogawara M, Ando S, Shibata M, Yatani R, Kusagawa M, Inagaki M: Phosphorylation of a 62 kd porcine alpha-internexin, a newly identified intermediate filament protein. Biochem Biophys Res Commun 1993, 196:115-123. 17. Sihag RK, Jaffe H, Nixon RA, Rong X: Serine-23 is a major protein kinase A phosphorylation site on the amino-terminal head domain of the middle molecular mass subunit of neurofilament proteins. J Neurochem 1999, 72:491-499. 18. Dong DL, Xu ZS, Chevrier MR, Cotter RJ, Cleveland DW, Hart GW: Glycosylation of mammalian neurofilaments. Localization of multiple O-linked N-acetylglucosamine moieties on neurofilament polypeptides L and M. J Biol Chem 1993, 268:16679-16687. 19. Dong DL, Xu ZS, Hart GW, Cleveland DW: Cytoplasmic OGlcNAc modification of the head domain and the KSP repeat motif of the neurofilament protein neurofilament-H. J Biol Chem 1996, 271:20845-20852. www.sciencedirect.com
34. Elder GA: Absence of the mid-sized neurofilament subunit decreases axonal calibers, levels of light neurofilament (NF-L), and neurofilament content. J Cell Biol 1998, 141:727-739. 35. Zhu Q, Couillard-Despre´s S, Julien JP: Delayed maturation of regenerating myelinated axons in mice lacking neurofilaments. Exp Neurol 1997, 148:299-316. 36. Garcia ML, Lobsiger CS, Shah SB, Deerinck TJ, Crum J, Young D, Ward CM, Crawford TO, Gotow T, Uchiyama Y et al.: NF-M is an essential target for the myelin-directed outside-in signaling cascade that mediates radial axonal growth. J Cell Biol 2003, 163:1011-1020. 37. Elder GA: Requirement of heavy neurofilament subunit in the development of axons with large calibers. J Cell Biol 1998, 143:195-205. 38. Garcia ML, Rao MV, Fujimoto J, Garcia VB, Shah SB, Crum J, Gotow T, Uchiyama Y, Ellisman M, Calcutt NA et al.: Phosphorylation of highly conserved neurofilament medium Current Opinion in Cell Biology 2015, 32:92–101
100 Cell architecture
KSP repeats is not required for myelin-dependent radial axonal growth. J Neurosci 2009, 29:1277-1284. 39. Barry DM, Stevenson W, Bober BG, Wiese PJ, Dale JM, Barry GS, Byers NS, Strope JD, Chang R, Schulz DJ et al.: Expansion of neurofilament medium C terminus increases axonal diameter independent of increases in conduction velocity or myelin thickness. J Neurosci 2012, 32:6209-6219. This study demonstrates the dependency of axonal caliber on the length of NF-M C-terminal, by replacing mouse NF-M tail with the longer bovine NF-M tail. Interestingly, this study also shows that this replacement did not alter inter-filament spacing. 40. Fuchs P, Zo¨rer M, Reipert S, Rezniczek GA, Propst F, Walko G, Fischer I, Bauer J, Leschnik MW, Lu¨scher B et al.: Targeted inactivation of a developmentally regulated neural plectin isoform (plectin 1c) in mice leads to reduced motor nerve conduction velocity. J Biol Chem 2009, 284:26502-26509. 41. Krizˇ J, Zhu Q, Julien J-P, Padjen AL: Electrophysiological properties of axons in mice lacking neurofilament subunit genes: disparity between conduction velocity and axon diameter in absence of NF-H. Brain Res 2000, 885:32-44.
stabilizes an isotropic phase at near physiological conditions, which may be preferable in regions where a more flexible NF reorientation is required. 55. Beck R, Deek J, Safinya CR: Structures and interactions in bottlebrush neurofilaments: the role of charged disordered proteins in forming hydrogel networks. Biochem Soc Trans 2012, 40:1027-1031. 56. Lin Y-C, Broedersz CP, Rowat AC, Wedig T, Herrmann H, Mackintosh FC, Weitz DA: Divalent cations crosslink vimentin intermediate filament tail domains to regulate network mechanics. J Mol Biol 2010, 399:637-644. 57. Yao NY, Broedersz CP, Lin Y-C, Kasza KE, Mackintosh FC, Weitz DA: Elasticity in ionically cross-linked neurofilament networks. Biophys J 2010, 98:2147-2153. 58. Ko¨ster S, Lin Y-C, Herrmann H, Weitz DA: Nanomechanics of vimentin intermediate filament networks. Soft Matter 2010, 6:1910.
42. Barry DM, Carpenter C, Yager C, Golik B, Barry KJ, Shen H, Mikse O, Eggert LS, Schulz DJ, Garcia ML: Variation of the neurofilament medium KSP repeat sub-domain across mammalian species: implications for altering axonal structure. J Exp Biol 2010, 213:128-136.
59. Carrillo J.-M.Y., MacKintosh FC, Dobrynin AV: Nonlinear elasticity from single chain to networks and gels. Macromolecules 2013, 46:3679-3692. A theoretical model describing the nonlinear strain-stiffening behavior of biopolymer materials is presented. The model is based on a bottom-up approach starting with the molecular single chain level. It is found to be in good agreement with experimental data on vimentin and NFs.
43. Zhulina EB, Leermakers FAM: The polymer brush model of neurofilament projections: effect of protein composition. Biophys J 2010, 98:462-469.
60. Beck R, Deek J, Choi MC, Ikawa T, Watanabe O, Frey E, Pincus P, Safinya CR: Unconventional salt trend from soft to stiff in single neurofilament biopolymers. Langmuir 2010, 26:18595-18599.
44. Leermakers FAM, Zhulina EB: How the projection domains of NF-L and alpha-internexin determine the conformations of NF-M and NF-H in neurofilaments. Eur Biophys J 2010, 39:13231334.
61. Brennich ME, Bauch S, Vainio U, Wedig T, Herrmann H, Ko¨ster S: Impact of ion valency on the assembly of vimentin studied by quantitative small angle X-ray scattering. Soft Matter 2014, 10:2059.
45. Stevenson W, Chang R, Gebremichael Y: Phosphorylationmediated conformational changes in the mouse neurofilament architecture: insight from a neurofilament brush model. J Mol Biol 2011, 405:1101-1118. 46. Lee J, Kim S, Chang R, Jayanthi L, Gebremichael Y: Effects of molecular model, ionic strength, divalent ions, and hydrophobic interaction on human neurofilament conformation. J Chem Phys 2013, 138:015103.
62. Guo M, Ehrlicher AJ, Mahammad S, Fabich H, Jensen MH, Moore JR, Fredberg JJ, Goldman RD, Weitz DA: The role of vimentin intermediate filaments in cortical and cytoplasmic mechanics. Biophys J 2013, 105:1562-1568. The mechanical role of vimentin is studied using active rheology on wildtype and knock-out mouse embryonic fibroblasts. Several microrheological techniques are used on living cells, allowing for direct and specific measurements of cortical stiffness, intracellular movement and cytoplasmic material mechanics.
47. Chang R, Kwak Y, Gebremichael Y: Structural properties of neurofilament sidearms: sequence-based modeling of neurofilament architecture. J Mol Biol 2009, 391:648-660.
63. Dingal PDP, Discher DE: Material control of stem cell differentiation: challenges in nano-characterization. Curr Opin Biotechnol 2014, 28:46-50.
48. Jeong SM, Zhou X, Zhulina EB, Jho Y: Monte-Carlo Simulation of Neurofilament Brush. Israel J chem 2014 http://dx.doi.org/ 10.1002/ijch.201400139. (in press).
64. Murray ME, Mendez MG, Janmey PA: Substrate stiffness regulates solubility of cellular vimentin. Mol Biol Cell 2014, 25:87-94. Vimentin detergent-solubility and assembly is found to be sensitive to substrate stiffness. The observed biphasic change in solubility coincides with cell spreading and membrane ruffling. Atomic force microscopy images show that the soluble forms are small fragments of one or few unit-length filaments.
49. Stevens MJ, Hoh JH: Interactions between planar grafted neurofilament side-arms. J Phys Chem B 2011, 115:7541-7549. 50. Stevens MJ, Hoh JH: Conformational dynamics of neurofilament side-arms. J Phys Chem B 2010, 114:8879-8886. 51. Jayanthi L, Stevenson W, Kwak Y, Chang R, Gebremichael Y: Conformational properties of interacting neurofilaments: Monte Carlo simulations of cylindrically grafted apposing neurofilament brushes. J Biol Phys 2013, 39:343-362. Monte Carlo simulations of pairs of opposing neurofilaments suggest an entropic interaction under physiological conditions. The study examines the effects of phosphorylation state and ionic conditions on the conformations of the tails of the adjacent NFs. 52. Beck R, Deek J, Jones JB, Safinya CR: Gel-expanded to gelcondensed transition in neurofilament networks revealed by direct force measurements. Nat Mater 2010, 9:40-46. 53. Jones JB, Safinya CR: Interplay between liquid crystalline and isotropic gels in self-assembled neurofilament networks. Biophys J 2008, 95:823-835. 54. Deek J, Chung PJ, Kayser J, Bausch AR, Safinya CR: Neurofilament sidearms modulate parallel and crossed filament orientations inducing nematic to isotropic and reentrant birefringent hydrogels. Nat Commun 2013, 4:2224. An examination of the phase behavior of NF-L, NF-M, and NF-H hydrogels reveals structural regulatory roles of the C-terminal tails. NF-H Current Opinion in Cell Biology 2015, 32:92–101
65. Bennett GS, DiLullo C: Transient expression of a neurofilament protein by replicating neuroepithelial cells of the embryonic chick brain. Dev Biol 1985, 107:107-127. 66. Liu C-H, Chien C-L: Molecular cloning and characterization of chicken neuronal intermediate filament protein a-internexin. J Comp Neurol 2013, 521:2147-2164. 67. Lendahl U, Zimmerman LB, McKay RDG: CNS stem cells express a new class of intermediate filament protein. Cell 1990, 60:585-595. 68. Shaw G, Weber K: Differential expression of neurofilament triplet proteins in brain development. Nature 1982, 298:277-279. 69. Nixon RA, Yuan A: Cytoskeleton of the Nervous System. Springer; 2011. 70. Chien CL, Mason CA, Liem RK: a-Internexin is the only neuronal intermediate filament expressed in developing cerebellar granule neurons. J Neurobiol 1996, 29:304-318. 71. Eriksson KS, Zhang S, Lin L, Larivie`re RC, Julien J-P, Mignot E: The type III neurofilament peripherin is expressed in the www.sciencedirect.com
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tuberomammillary neurons of the mouse. BMC Neurosci 2008, 9:26. 72. Hirokawa N, Glicksman MA, Willard MB: Organization of mammalian neurofilament polypeptides within the neuronal cytoskeleton. J Cell Biol 1984, 98:1523-1536. 73. Herrmann H, Ha¨ner M, Brettel M, Ku NO, Aebi U: Characterization of distinct early assembly units of different intermediate filament proteins. J Mol Biol 1999, 286:1403-1420. 74. Uchida A, C¸olakog˘lu G, Wang L, Monsma PC, Brown A: Severing and end-to-end annealing of neurofilaments in neurons. Proc Natl Acad Sci U S A 2013, 110:E2696-E2705.
www.sciencedirect.com
This study demonstrates the assembly dynamics of NF in neurons. The use of photoconversion and photoactivation techniques enables formation of separate red and green filaments, which later form chimeric filaments and indicate assembly and severing dynamics. 75. Hisanaga S, Hirokawa N: Structure of the peripheral domains of neurofilaments revealed by low angle rotary shadowing. J Mol Biol 1988, 202:297-305. 76. Szaro BG, Strong MJ: Post-transcriptional control of neurofilaments: new roles in development, regeneration and neurodegenerative disease. Trends Neurosci 2010, 33:27-37.
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ScienceDirect Neurofilament assembly and function during neuronal development Adi Laser-Azogui1, Micha Kornreich1, Eti Malka-Gibor2 and Roy Beck1 Studies on the assembly of neuronal intermediate filaments (IFs) date back to the early work of Alzheimer. Developing neurons express a series of IF proteins, sequentially, at distinct stages of mammalian cell differentiation. This correlates with altered morphologies during the neuronal development, including axon outgrowth, guidance and conductivity. Importantly, neuronal IFs that fail to properly assemble into a filamentous network are a hallmark of neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer’s, and Parkinson’s disease. Traditional structural methodologies fail to fully describe neuronal IF assembly, interactions and resulting function due to IFs structural plasticity, particularly in their C-terminal domains. We review here current progress in the field of neuronal-specific IFs, a dominant component affecting the cytoskeletal structure and function of neurons. Addresses 1 The Raymond and Beverly Sackler School of Physics and Astronomy, Tel-Aviv University, Tel-Aviv 69978, Israel 2 Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel Corresponding author: Beck, Roy ([email protected])
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III IF protein of mesenchymal cells, is transiently coexpressed with nestin in neural precursor cells. Its expression declines during differentiation [2]. Neuronal-specific IFs include five principal subunit proteins: the type IV light, medium, and heavy molecular mass neurofilament (NF) triplet proteins (NF-L, NF-M, and NF-H, respectively), a-internexin, and the type III peripherin. Mature filaments are composed of several combinations of these five subunits. a-Internexin is expressed in most differentiated neurons; its expression precedes that of the NF triplet and declines somewhat postnatally, while expression of the NF triplet sharply rises [3]. In mature neurons, the main role recognized for NFs is to increase the axonal caliber of myelinated axons and consequently their conduction velocity [4]. NF proteins represent the main cytoskeletal elements in mature neurons, however, at the initiation of axonal radial growth, NF-H expression is usually delayed relative to NF-L and NF-M [4]. It is speculated that delayed NF-H expression allows the cytoskeleton network to establish in a distal to proximal gradient [5]; although cases of varied sequential expression have been reported (Figure 1).
This review comes from a themed issue on Cell architecture Edited by Sandrine Etienne-Manneville and Elly M Hol
http://dx.doi.org/10.1016/j.ceb.2015.01.003 0955-0674/# 2015 Elsevier Ltd. All rights reserved.
Introduction Differentiated neurons live longer than any other cell type, but unlike other cell types, they do not proliferate. Throughout the development of neurons, their cytoskeleton undergoes modifications to sustain its mechanoelastic properties necessary for proper function. Striking evidence for such remodeling is given by the sequential expression of IF throughout neuronal development and maturation (Figure 1). Nestin, a type IV IF protein, is expressed in neural stem cells at the earliest stage of embryonic development. Nestin is down-regulated after differentiation and replaced by cell type-specific IFs [1]. Vimentin, a type Current Opinion in Cell Biology 2015, 32:92–101
Peripherin, a type III IF, is co-expressed with NF triplet in the adult peripheral nerve system, in a relatively fixed stoichiometry and exhibits a distribution pattern identical to those of triplet NF proteins in sciatic axons [6]. Peripherin is also found in neurons of the central nerve system (CNS) that have projections toward peripheral structures, such as spinal motor neurons [6]. During development, peripherin is differentially regulated in sensory neurons, and plays a role in motor axons’ growth [7]. Synemin, a type IV IF, is not a neuronal-specific protein. Nevertheless, it forms obligate heteropolymers with vimentin or the NF triplet in neurons. The three synemin isoforms are differentially expressed in neuronal subpopulations, and also show developmental stage specificity. Since they are obligate heteropolymers they always co-express with either vimentin (at early developmental stage) or any neuronal IF, that is NF-L, a-internexin or peripherin [8,9]. The sequential IF expression during development raises fundamental challenges in understanding the differential role of each of the IF proteins. Here, we review the recent progress made to understand the assembly, transport, and function of neuronal-specific IFs from a developmental perspective. We begin with a short introduction to neuronal IF self-assembly into filaments, and continue with www.sciencedirect.com
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Figure 1
Central nervous system neurons (c) α -int, NFL, NFM Precursor nerve cells (a) Nestin, Vimentin
(b) α -int
(d)
α -int, NFM
(f) α -int, NFL, NFM, NFH
(g) Tail Phosphorylation: NFM and NFH
(e) α -int, NFL, NFH (h) Distinct CNS neurons
(i)
Peripheral neurons
Pre-natal
Post-natal
α -int/ Synemin/ Peripherin
Peripherin, NFL, NFM, NFH
Mature
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Sequential expression of IF during development of the nervous system. Neuronal IF proteins are sequentially expressed during development to accommodate for the changing mechanoelastic needs of the developing neurons. Several studies have shown that different IF subunits and expression levels are further specified according to spatial location within the nervous system, exact developmental stage, and organism species. The above scheme demonstrates the main combinations with respect to development, but does not reflect all the different pathways. For example, discrepancy between expression levels of NF-M in chicken embryo has been recorded [65,66]. This discrepancy might depend on specific location within the CNS and/or exact developmental stages. References for each stage are as follow: a, [2,67]; b, [3]; c, [68]; d, [69]; e, [66]; f, [13]; g, [4]; h, [8,70,71] (a-internexin, synemin and peripherin respectively); i, [6].
the role of post-translational modifications (PTM). Recent progress in the characterization of NF axonal transport and the resultant axonal growth will then be discussed. Last, we will review current measurements and theories addressing the organization of NFs using methods adopted from polymer physics. We will conclude with some remaining open questions and perspectives on the future of the field.
From subunit proteins to filament assembly Self-assembly of neuronal IF proteins into mature filaments follows the principle paradigm of all cytoplasmic IFs [10]. Currently, atomic structural studies are limited to vimentin [11,12], as presumably the long disordered Cterminal tails of neuronal IFs hamper structural studies. IF proteins share a common tripartite molecular structure; a conserved central a-helical rod domain, flanked by intrinsically disordered N-terminal head, and C-terminal tail domains. The rod domain drives coiled-coil interaction and co-assembly with other subunits to form dimers, protofilaments, and filaments (Box 1). The major differences between the neuronal IF subunits are the length and sequence of their C-terminal tails. These differences are of www.sciencedirect.com
great importance since those intrinsically disordered tails form flexible extensions that link the filaments to each other and to other elements in the cytoplasm (Figure 2). NF-M and NF-H can dimerize to form filaments only in association with either NF-L or a-internexin. NF-L and a-internexin can either self-assemble or co-assemble with any neuronal IF subunit in vitro [13]. In addition, only human NF-L is able to self-assemble in vivo [14], yet in other mammals NF-L does not self-assemble. In vitro, NF-L homopolymers of bovine, porcine, and rodents have been observed. In contrast to the NF triplet, both a-internexin [13] and peripherin [6] are able to homopolymerize, which coincides with the observation that in some neurons they are the sole NF protein (Figure 1). NF assembly depends heavily on their head domains, and especially their PTM. Of special importance are specific serine residues in NF-L; their phosphorylation state and the rapid turnover of these phosphates regulate NF polymer assembly and affect their axonal transport [15]. Similar to NF-L, phosphorylation of the head domain of a-internexin causes filament disassembly [16]. Current Opinion in Cell Biology 2015, 32:92–101
94 Cell architecture
Box 1
Type III IF
(a)
Central α-helical core
Head
Tail
Key: Peripherin N
C Coil 1a
Coil 1b
Coil
Coil 2a/b
KSP repeats
Type IV IFs
E-rich
α-Internexin N
K/E/P-rich
C
K/E-rich
NF triplet
NF-LN
C
NF-MN
C C
NF-HN
(b)
(d)
Myelinated sensory axon
1a
NFs
1b
MTs
2a 2b Unit length filament
Tetramer
Myelinated corticospinal axon
Dimer
MTs
NFs
Filament (c) Unmyelinated sensory axons
NF MTs
NF-M and NF-H tails
250 nm
Current Opinion in Cell Biology
Self-assembly of filaments: (a) In homology to the other IF proteins, neuronal IFs contain three domains: an N-terminal head domain, a central ahelical core which is further divided into coils 1a, 1b and 2a/b, and a C-terminal tail domain. (b) The first step of neuronal IF formation is the dimerization of NF-L or a-internexin with any other neuronal IF proteins, through association of their conserved rod domains to form parallel, sideto-side, coiled-coil dimers. Two coiled-coil dimers line up in a half-staggered manner, forming an anti-parallel tetramer. Then, a rapid lateral aggregation of about eight tetramers occurs to form a unit-length-filament (ULF) of approximately 55 nm in length [73]. An elongation step occurs through the axial aggregation of ULFs to form immature filaments of about 16 nm in diameter and many microns in length. (c) Radial compaction
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Ser23 of NF-M exhibits early phosphorylation and rapid turnover similar to what is observed for NF-L [17], although no substantial evidence has been presented regarding the effect of head domain phosphorylation of NF-M or NF-H on filament assembly. NF-M and NF-H head domains are also glycosylated on sites close to the phosphorylation sites [18,19]. This PTM proximity suggests that glycosylation also affects filament assembly, perhaps by regulating phosphorylation and modulating the early protein–protein interaction of the assembly cascade.
Figure 2
(a)
(b)
The tail domain of a-internexin and NF-L is short, while that of NF-M and NF-H is much longer and contains many PTM sites. In mature axons, NF-M and NF-H are heavily phosphorylated on their tail domains, primarily on serine residues of multiple lys–ser–pro (KSP) repeats. Tail phosphorylation has no effect on filament assembly, but rather contributes to lateral extension and formation of the cytoskeletal lattice [4]. Assembly of axonal cytoarchitecture is a complex process, involving dynamic short and long range interactions between phosphorylated tails of NF-H and NF-M with microtubules, actin, and other NF-associated proteins, such as plectins [20]. Crossbridging between NFs through the tail domains of NF-H and NF-M is influenced by the phosphorylation level of these tails [4] and mediated by divalent metal cations [21]. Phosphorylated NFs are localized mainly in the axon, whereas the dephosphorylated forms are found in the cell body and dendrites [22]. Pant et al. [23] demonstrated that head domain phosphorylation of NF-M inhibits its tail domain phosphorylation, suggesting that KSP phosphorylation of NF-M in axons depends on prior dephosphorylation of head domain sites. These findings support the idea that phosphorylation of head domains regulates both NF assembly and phosphorylation of specific tail domains, thus preventing premature NF assembly and tail phosphorylation in cell bodies. The different phosphorylation patterns in cell bodies versus axons also indicate the role of phosphorylation in NF transport and perhaps in interaction with other cytoskeletal proteins. Glycosylation sites are present near KSP phosphorylation sites in NF-M and NF-H tails [18,19]. O-linked glycosylation is believed to act in competition with phosphorylation in a dynamic cycle. Dysregulation of this relationship,
Current Opinion in Cell Biology
Axonal and dendritic NFs. In axons (a), NFs fill the axoplasm. They are more evenly spaced and their C-terminal tail cross-bridges are longer and more numerous. In dendrites (b), NFs are tightly packed. Although there are cross-bridges between NFs, they appear to be shorter and less frequent than those in axons. Bar, 0.1 mm. Figure is reprinted with permission from Rockefeller University Press, Hirokawa et al.: J Cell Biol 1984, 98:1523–1536 [72].
observed in the brains of a rat model for Alzheimer’s disease, led to NF-M hyper-phosphorylation and accumulation [24]. Similarly, a study on the role of NF-M posttranslational modifications in ALS [25] demonstrated reciprocal changes in phosphorylation and O-linked glycosylation of NF-M tail, which was characterized by aggregated NF proteins. The NF-M glycosylation site in question was localized to the KSP repeat region. NF-H is also extensively modified by O-linked glycosylation in the KSP repeats in the tail domain [19]. The proximity of O-linked glycosylation and phosphorylation sites in both head and tail domains indicate that these modifications may influence one another and play a role in filament assembly and network formation.
Axonal transport of NFs NFs are synthesized in the cell body and travel long distances along axons to reach their sites of function. The mechanisms that underlie their axonal transport are the
( Box 1 Legend Continued ) then takes place resulting in close packing of the molecular filaments to constitute the final 10 nm ‘bottlebrush’ filament [4,10]. Filaments are lengthened by dynamically joining ends of shorter filaments [74]. The core of the filament is composed of the central rod domain, while the flexible C-terminal tails decorate it in a bottlebrush architecture [75]. (d) Electron micrograph cross-sections from different types of axons showing heterogeneous NF cytoarchitecture of myelinated sensory axons (top), unmyelinated axons (middle) and myelinated motor axons (bottom). Reprinted from Szaro BG, Strong MJ: Post-transcriptional control of neurofilaments: new roles in development, regeneration and neurodegenerative disease. Trends Neurosci 2010, 33 (1):27–37, Copyright (2010), with permission from Elsevier [76]. www.sciencedirect.com
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96 Cell architecture
subject of intense study. The state of assembly of NFs during axonal transport has been under debate, with studies that demonstrate movement of non-filamentous assemblies of NF subunits [26], short filaments [27], or both [28]. Yuan et al. showed that at proximal regions of the axon, the transport of subunit assemblies predominates, whereas the transport of short filaments predominates at distal regions of the same axon. It is speculated that NF proteins exist in multiple assembly forms during axonal transport and that the transported NF subunits assemble into filaments during the transport [28]. Yabe et al. suggested that the controversies arise from the use of neurons at different developmental stages, and that the predominant form in which NF subunits undergo axonal transport varies in accordance with the rate of axonal elongation and accumulation of NFs within developing axons [29]. Another long-standing controversy regards NF transport dynamics. The ‘stop-and-go’ transport model suggests that axons contain a single population of NFs that all move in a stochastic, bidirectional and intermittent manner [27,30]. Here, the instantaneous rate of movement is fast, but the overall rate is slow because the movements are interrupted by prolonged pauses [31]. The ‘stop-andgo’ hypothesis is challenged by the stationary hypothesis, suggesting that 10% of all NFs in a given axon are transported in assemblies [28]. These are deposited into a stationary and cross-linked NF cytoskeleton that constitute about 90% of NFs. The stationary NFs remain fixed in place for many months without movement, integrated with other cytoskeletal components [28]. These different perspectives have significant implications for the understanding of NF dynamics and reorganization in axons. The movement of NFs in axons occurs along microtubule tracks, by the retrograde motor dynein and the anterograde motor kinesin-1 [4]. Little is known about the nature of NF interaction with motors. Based on recent observations that NFs move in a fully extended configuration, it is suggested that they may be pulled from their leading end during both anterograde and retrograde motion [32].
Axonal radial growth Following the formation of stable synapses, NFs accumulate and the axonal diameter increases. The axonal diameter influences the rate of axonal impulse propagation. Radial growth is also dependent upon the formation of compact myelin [33]. NF-M and NF-L are required for radial growth, as loss of each results in small caliber axons [34–36]. As NF-H expression increases, radial growth of only the largest axons becomes somewhat dependent on NF-H [37]. The expression of tail-deleted NF-H affects the rate, but not the final magnitude, of radial axonal growth [36]. Current Opinion in Cell Biology 2015, 32:92–101
The mechanism for this radial axonal growth has been thought to involve myelin-dependent phosphorylation of KSP sites in NF-M tail domains; the formation of compact myelin and NF accumulation were both shown to be necessary for radial axonal growth [36]. However, recent studies have shown that KSP phosphorylation within the NF-M tail is not a determinant of radial axonal growth. Axonal diameters of mice expressing full-length, but KSP phospho-incompetent NF-M, are indistinguishable from those of control mice [38,39]. Together, these findings demonstrate the dominant role of NF-M tail domain, but not its phosphorylation, in determining axonal caliber. Nevertheless, NF protein expression by itself has not been found to play a key role for nerve development and function [34–38,39]. A different explanation of NF structural function, phosphorylation and the interplay between those and myelination to determining axonal caliber is required. A possible mechanism suggested by Barry et al. [39] is that the NF-M tail domain serves as a scaffold for cytoskeletal linking proteins, such as plectin. In agreement, deletion of the neuron specific isoform of plectin [40] resulted in a phenotype similar to that observed in NF-Mdeleted mice [41] and mice expressing C-terminally truncated NF-M [36]. Thus, increasing the distance that NF-M C-terminus extends from the filament core may have allowed a longer-range interaction between NF-M tail domain and plectin, resulting in larger axonal diameters. Interestingly, positive correlations were found between the number of KSP repeats of NF-M and the overall animal length, in a subset of mammalian species [42].
Neurofilaments as interacting bottlebrushes The dynamics, structure, and mechanical properties of NF networks are largely determined by their bottlebrush architecture (Box 1c), a well-studied topic in polymer physics. However, due to the complex nature of the NF tails, which include hydrophobic, hydrophilic, and many negative and positive charged residues, a full physical understanding is still missing. This problem was tackled by several groups using polymer physics techniques, including theoretical, computational and controlled in vitro experiments (Figure 3). The theoretical models and simulations currently account for the best available molecular understanding of NF network organization. The theoretical works predict the conformations of the tails during neuronal development, when subunit compositions are different from the stable composition of mature neuron. In addition, those studies examine the role of KSP phosphorylation on the tail expansion. Selfconsistent mean field calculations of NF networks, pioneered by Zhulina and Leermakers [43], studied several NF compositions and phosphorylation levels. These studies predict a dominant role for NF-M in determining www.sciencedirect.com
NF assembly and function during neuronal development Laser-Azogui et al. 97
Figure 3
(a)
(c)
T=0h
24 h
Cs=2.5 mM, 0.9 wt%
Cs=4.2 mM, 1.5 wt%
BG
BG (d)
(e)
(b) 107
Cs (mM) 40
106
150 (f)
105
101
G’,G” (Pa)
P (Pa)
240
104
103
100
2mM Mg 5mM Mg 8mM Mg
0.5 mg/ml 1.5 mg/ml 2.0 mg/ml
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10–1 200
400
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d (Å)
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10–3
10–2
10–1
ω (Hz)
100
10–3
10–2
10–1
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In vitro and simulation studies on NF interactions and self-assembly. (a) MC simulation snapshot of two opposing NFs in physiological condition and 40 nm separation. Higher mutual interpenetration of the C-terminal tails is observed when compared to larger NF separations. Colors indicate different subunit proteins [51]. Color-coding for the different subunit protein: NF-L (green), NF-M (blue), NF-H (red) and backbone (gray). (b) NFs spacing under compaction generated by osmotic pressure and measured by small angle X-ray scattering. The results show a transition from an expanded state at low osmotic pressure to a condensed state at high osmotic pressure where NFs are strongly entangled. Full (empty) symbols stand for nematic (isotropic) hydrogels in various monovalent salt concentrations (Cs). The X symbols represent a second, salt-independent, correlation peak that follows the NF-L homopolymer trend [52]. (c) NFs show water retention ability over many hours. At low salt concentration, within the blue phase, and at high salt concentration of the nematic phase, the hydrogels retain their shape and volume in comparison to faster water release in the isotropic phase [54]. (d) NFs are the softest filament within the cytoskeleton network. NF’s persistence length, measured by AFM, is shown to be tunable by the subunit protein composition which alter intra-filament interactions [60]. Scale bar = 50 nm. (e) Cross polarized microscopy images reveal the macroscopic ordering within the NF hydrogels. For example, after 30 days incubation, NF-L homopolymers hydrogel (top) show more swelling and weaker nematic alignment than NF-L:NF-M hydrogels (bottom) [53]. (f) Rheological measurements of cross-linked NF networks showing variation of the linear viscoelastic moduli at different protein (left) and Mg2+ (right) ion concentrations [57].Plots are reprinted with permission from Jayanthi et al.: J Biol Phys 2013, 39:343–346; Beck et al.: Nat Mater 2010, 9:40–46; Deek et al.: Nat Commun 2013, 4:2224; Beck et al.: Langmuir 2010, 26:18595–18599; Jones and Safinya: Biophys J 2008, 95:823–835; Yao et al.: Biophys J 2010, 98:2147– 2153.
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the inter-filament spacing and non-specific attraction between adjacent filaments. Moreover, recent calculations offer the only theoretical study, to date, which takes a-internexin into account [44]. This study shows that the inclusion of a-internexin subunits regulates the structural properties including tail extensions from the backbone, compressibility and frequency of cross-bridges. The effects of NF phosphorylation are also examined by Monte Carlo (MC) simulations, showing milder effects of NF-H phosphorylation on tail extension compared to the mean field model predictions [45]. Further MC simulations examined the NF triplet under varying ionic strengths, hydrophobicity, grafting locations [46,47], and inclusion of NF-M variant with a different number of KSP repeats [39]. These studies suggest that NF-M tails, regardless of their phosphorylation state, are more extended than NF-H tails, and that the attraction between tails can be regulated by Ca2+ ions and not by hydrophobic interactions. A recent study includes the added effect of backbone charge and explicit salt ions on NF tails conformation. This study demonstrates a tendency to metastability with the tails folding back and forming loops [48], in agreement with previous molecular dynamics simulations [49,50]. Simulations of the interactions of two opposing filaments show that at physiological salt concentration, tails from opposing filaments exhibit coiled conformations with increasing overlap upon compression (Figure 3a) [51]. The structure of reconstituted bovine NF networks, including NF-L homopolymers and co-assemblies with NF-M and/or NF-H, were experimentally studied using small angle X-ray scattering and microscopy (Figure 3b– e) [52,53,54]. These in vitro studies quantify the interfilament distance and macroscopic nematic alignment under controlled conditions, and directly coupled to the above-mentioned theoretical models. Nonetheless, some experimental results still lack a theoretical explanation, such as an unexpected collapsed conformation of NF-L:NF-M networks, liquid crystalline phase transitions and a collapsed condensed state at higher osmotic pressure [55]. Such discrepancies may arise since most simulations do not, for now, fully consider the complex inter-filament interactions and backbone flexibility (Figure 3d). The importance of multivalent ions in cross-linking opposing filaments has been known for a long time. It was demonstrated that divalent ions behave as effective crosslinkers for vimentin and NF networks, and that the elasticity of these networks is consistent with a theory of semiflexible polymers in an affine thermal (entropic) model (Figure 3f) [56,57]. These results, and the microstructural features predicted by fitting them to models assuming an entropic origin of the elasticity, are in agreement with complementary microrheology experiments Current Opinion in Cell Biology 2015, 32:92–101
[58]. Interestingly, the last 11 residues of vimentin tail domain are crucial in mediating the interaction between vimentin filaments and divalent cations [56]. A comprehensive theoretical model describing network strain-stressed deformations at different applied stresses, reports very good agreement with experimental data and molecular dynamic simulations [59]. The predictions of mechanical properties are based on a bottom-up approach starting with single-chain force-extension. However, NFs persistence length measurements under varying salt conditions and changing NF-H and NF-M ratios present unpredicted trends with respect to both ionic strength and tail compositions [60]. Notably, NF tails may crosslink within the same filament and thus increase the apparent filament flexibility. Such intra-filament attractions are not considered in current theoretical modeling.
Conclusions and perspective The sequential and parallel expression of neuronal IFs, as well as their PTMs, is reflected both in the structure and functionality of the cytoskeleton during development. However, there is still a gap between our understanding of the IF composition variations and their macroscopic organization, dynamics, and pathologies. Driven by the inability to gain atomic structural information, neuronal IFs assembly and structure are studied by simulation, modeling and polymer physics-inspired experiments. Therefore, future efforts will probably extend in vitro studies to a larger subset of IFs, and will include newly developed techniques that have already provided solid results on other IFs [61,62]. Moreover, the relation between cell differentiation and the surrounding matrix mechanical properties has recently drawn immense interest [63,64]. The relation between different IF subunits expression during development and their structure and mechanoelastic function is expected to be intriguing, and to contribute to our understanding of their function.
Acknowledgements We thank Cyrus Safinya, Joanna Deek and Michael Wyrsta for insightful remarks and discussions. This work was supported by the Israel Science Foundation (grant 571/11), the European Community’s 7th Framework Programme (293402), the Sackler Institute for Biophysics and the center from nanoscience and nanotechnology at Tel Aviv University.
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