Intermediate filament assembly: dynamics to disease

Lisa M. Godsel, Ryan P. Hobbs and Kathleen J. Green

Department of Pathology, Northwestern University Feinberg School of Medicine, 303 E. Chicago Avenue, Chicago IL 60611, USA

Intermediate filament (IF) belong to a large and providing mechanical integrity that is cru- diverse family with broad representation in cially important for tissue function. This is highlighted tissues. Although considered the ‘toughest’ by a growing list of >75 human genetic diseases caused cytoskeletal fibers, studies in cultured cells have revealed by deficiencies in this network, including skin fragility that IF can be surprisingly dynamic and highly regulated. and epidermolytic disorders, , myopathies, This review examines the diversity of IF assembly beha- neuropathies, cataracts and premature aging [6,7] viors, and considers the ideas that IF proteins are co- or (Human Mutation Database, post-translationally assembled into oligomeric precur- Notably, an emerging set of sors, which can be delivered to different subcellular com- mutations in nuclear (reviewed elsewhere) com- partments by or actomyosin and associated prise a large proportion of human diseases attributable to motor proteins. Their interaction with other cellular IFs [8,9]. This article focuses on cytoplasmic IFs, which elements via IF associated proteins (IFAPs) affects IF are now recognized as players in signaling, growth, dynamics and also results in cellular networks with prop- epithelial polarity, and apoptosis in erties that transcend those of individual components. We addition to providing the cell with resilience to environ- end by discussing how mutations leading to defects in IF mental stress [2,10]. assembly, network formation or IF–IFAP association com- These broad-ranging functions derive from the diversity promise in vivo functions of IF as protectors against of IFs coupled with their unique mechanical and bio- environmental stress. chemical properties. IFs are the most flexible of the bio- logical filaments. Furthermore, unlike MFs and MTs, a Introduction single IF can withstand stretching to more than three Intermediate filaments (IF) are flexible, rod-shaped fibers times its resting length before breaking [11]. Although averaging 10 nm in diameter, a size that is ‘intermediate’ integration with other filament systems is necessary to between microfilaments (MF; 7–8 nm) and microtubules create the final viscoelastic properties of the cytoplasm, it (MT; 25 nm) [1,2]. Of the three non-muscle cytoskeletal is thought that IFs contribute the tensile strength necess- fibers, IFs are the most diverse and are encoded by an ary for maintaining cell integrity (Box 1) [1,11,12]. IFs are estimated 70 IF in the (Human also biologically stable structures. However, a recent con- Intermediate Filament Mutation Database; http://www. vergence of in vitro and in vivo explorations has shown the IFs are classified into five major families IF to be a malleable and dynamic system that expressed in cell-, tissue-, differentiation- and developmen- can be structurally and functionally tailored to suit cells’ tal-specific patterns (Table 1). Families I–IV are localized changing needs. In this review we explore how recent to the cell cytoplasm whereas the type V nuclear lamins are trends have shaped our understanding of IF function, important organizers of the and karyo- organization and assembly properties in the test tube plasm. IF family members share a common blueprint built and in living cells. We have yet to fully understand how from a central a-helical coiled-coil rod flanked by flexible, these properties are translated into physiologically highly variable N- and C-termini that lead to exceptional relevant in vivo situations. However, the work discussed structural diversity among IFs [3]. This diversity presents here provides insight into how human disease phenotypes many opportunities for tailoring IF networks to cell type- might arise from fundamental defects in IF assembly and specific functions in contrast to the broadly conserved integration with IF-associated proteins (IFAPs) into func- functions of MT and MF. tionally competent networks. In most vertebrate cells cytoplasmic IFs are tethered to the nucleus and extend into the cytoplasm where they IF structure and in vitro assembly properties provide a scaffold for mitochondria, the Golgi complex, IF proteins exhibit an extended secondary structure built organizing centers (MTOCs) and other cyto- from a conserved a-helical rod domain of 310–350 amino skeletal elements (Figure 1) [1,2,4,5]. In the periphery acids flanked by divergent non-helical N- and C-termini IFs associate with plasma membrane specializations (Figure 2) [1]. The rod domain drives the formation of such as , and focal adhe- parallel a-helical coiled-coil dimers through long-range sions. The resulting network integrates and organizes the heptad repeats organized as shown in Figure 2, each with a characteristic pattern of apolar residues in the first (a) Corresponding author: Godsel, L.M. ([email protected]). and fourth (d) positions. These dimers constitute the

28 0962-8924/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2007.11.004 Review TRENDS in Vol.18 No.1

Table 1. Members of the IF superfamily Type Cell types Localization Proteins Disease associations I Epithelia Cytoplasm, Acidic (pI < 5.7) K14 – epidermolysis bullosa simplex diseases 17 human epithelial keratins; K9–K28 K10, K16, K14 – disorders Hair 11 human hair keratins; K31–40 (Ha1–8) K12 – Meesmann corneal dystrophy K13 – white nevus of cannon K16 – type I K17 – pachyonychia congenita type II II Epithelia Cytoplasm, hair Basic keratins (pI  6.0) K5 – simplex diseases 20 human epithelial keratins; K1–8, K71–80 K1, K9, K2 – keratoderma disorders 6 human hair keratins: K81–86 (Hb1–6) K3 – Meesmann corneal dystrophy K4 – of cannon K6a – pachyonychia congenita type I K6b – pachyonychia congenita type II Hair K81 (Hb1), K83 (Hb3), K86 (Hb6) – K85 (Hb5) – pure hair- type ectodermal dysplasia III Muscle Cytoplasm Desmin – desmin related myopathy, 1I, familial restrictive cardiomyopathy 2 Mesenchymal Peripherin – amyotrophic lateral sclerosis and GFAP GFAP – IV Neurons Cytoplasm NF-L NF-L, M and H – amyotrophic lateral sclerosis Neurons NF-M NF-L – Charcot-Marie-Tooth diseases Neurons NF-H NF-M – Parkinson disease Neurons a- NF-H – neuronal IF disease Muscle a Muscle Synemin b (desmuslin) Muscle Neuroepithelia V Ubiquitous Lamins A/C Lamins – large number of disorders, including lipodystrophies, muscular dystrophies, neurological disorders and premature aging B1 B2 Orphan Eye lens Cytoplasm Phakinin (CP49) CP49 – autosomal dominant cataract disease Filensin (CP115) CP115 – autosomal recessive cataract disease elemental building blocks of IFs and depending on the IF predicted that the head domains of type I and type II type these can be hetero- (e.g. type I and II keratins and epidermal keratins and type III IFs exhibit a flexible type IV neurofilament chains) or homodimeric (e.g. type III structure and could interact with sites in the rod domain. vimentin and desmin). In both cases these N-termini are required for in vitro A hypothetical model based on the in vitro behavior of the filament assembly [13,15–17]. Deletion of the vimentin type III vimentin provides a useful platform for C-terminus did not block filament formation, but did result understanding how individual polypeptides might be in an increase in their mass-per-length [3]. However, assembled into an apolar filament (Figure 2). According mutations affecting the K5 tail found in patients with to this model, filament assembly comprises several major epidermolysis bullosa simplex (EBS) dramatically steps starting with the formation of parallel, in-register impaired IF assembly and network formation [18]. Impor- dimers. Dimers then associate into tetramers, thought to tantly, exposure of the N- and C-termini on the filament be organized primarily in a mode termed A11, in which the surface also leaves them free to associate with other fila- 1B subunits of the rods overlap in an anti-parallel manner ments and cellular structures. [13,14]. Tetramers aggregate into higher order oligomers to We are far from understanding the specific mechanism of form unit length filaments (ULF) 60 nm long, which assembly for all IF family members, but it is clear that undergo reorganization and elongation by longitudinal differences exist, underscored by the observed rapid kinetics annealing to form immature IF. Within the , other of polymerization and the completely distinct anti-parallel arrangements A12,A22 and ACN can also occur, assembly behavior of nuclear lamins, which form long corresponding to associations between the 1B and 2B sub- head-to-tail arrays that associate laterally [16]. However, domains, between the 2B subdomains, or between the IF family members all share certain attributes that dis- C- and N-termini. The final step is radial compaction of tinguish them from MTs and MFs, including their apolar the filament from 16 nm to a diameter of 10–12 nm. This structure, their inability to bind or hydrolyze nucleotides step has been proposed to occur via lateral rearrangements and their capacity for undergoing cycles of assembly and of protein subunits such that compaction occurs without disassembly simply by altering the ionic strength and pH of losing mass or increasing the length of the filament [1,3,13]. solution [1,16]. By contrast, MFs and MTs are polar fila- The rod domains form the IF core and the N- and ments that use cofactors to facilitate polymerization and C-termini are displayed on the filament surface. It is conformational changes, as well as nucleotide hydrolysis to

29 Review TRENDS in Cell Biology Vol.18 No.1

Figure 1. IFs are integrators of cyto- and tissue architecture. IFs emanate from the nucleus, extending throughout the cytoplasm and out to contact points at the plasma membrane. (a) Immunofluorescence microscopy reveals the keratin network in epithelial cells (red) linking to the (colocalization shown in yellow) via interactions with the desmosomal protein, (green). In this way the IF networks of adjacent cells are linked together giving the tissue mechanical

30 Review TRENDS in Cell Biology Vol.18 No.1

Figure 2. Schematic model of filament formation from dimer to mature filament, based on in vitro assembly data for the type III IF protein vimentin. Intermediate filaments are made up of an a-helical coiled-coil domain flanked by two non-helical N- and C-termini (which vary considerably among IF types). IF assembly begins with the formation of parallel a-helical coiled-coil dimers mediated by four heptad repeat-containing regions designated 1A, 1B, 2A and 2B, separated by short, flexible non-coiled linkers, L1, L12 and L2 [1,3,17]. 1B and 2B harbor motifs important for ‘triggering’ and stabilizing the coiled-coil structure and for keratin IF heterodimerization [91]. Local variations in the heptad repeat regions, such as a three residue deletion in 2B, also contribute to IF structure and assembly [17]. Dimers associate laterally into anti-parallel, half- staggered tetramers or protofilaments in which the 1B subdomains are aligned – shown as a cylinder in this Figure [13]. ULFs subsequently anneal longitudinally to form filaments that are loosely arranged and larger in caliber than mature filaments. During a subsequent compaction phase, IF are compressed radially from 16 nm to 10 nm resulting in formation of a mature filament [1,3,13]. One hypothesis based on data acquired from vimentin studies suggests that the strands form a cylinder with a visible open or low-density central core when observed in cross-section [3,13]. This figure presents a general model; however the number of tetramers varies when filaments are viewed in cross-section and the extent of variation is dependent on IF type and assembly conditions [3,16]. regulate polymer dynamics. IF have not traditionally been regulate polymerization of IF in vitro, but how these rules thought to require co-factors. However, recently it has been apply to IF assembly and remodeling in cells remains shown that a neurofilament light chain (NF-L)-binding largely unknown. protein called NUDEL (nuclear distribution element-like), One characteristic that is maintained in cells is the which regulates neuronal migration, facilitates assembly of apolar nature of IFs and it has been speculated that IFs IF. This observation raises the possibility that there might might over or remodel in part through a process of be a more important role for co-factors in IF function than intrafilament exchange. The possibility that IF turnover originally thought [19]. can occur by exchange within a network has been con- sidered for a number of years, based in part on the observed IF dynamics in cells: initiation and remodeling of fluorescence recovery after photobleaching (FRAP) along IF networks vimentin IF networks in cultured cells [29]. Further stu- Although IF polypeptides typically have long half-lives and dies will be necessary to determine whether IF subunits or are biochemically stable, IF networks routinely undergo oligomers can exchange along individual filaments in cells rearrangements involving disassembly and reassembly and tissues, or if exchange occurs at filament ends, or during processes such as cell spreading, wound healing whether both scenarios occur. Mechanisms that govern and , and in response to environmental stres- in vivo assembly and remodeling are also beginning to ses such as stretching and shear flow [20–28]. As described emerge. It has been suggested that of here, progress is being made in elucidating the rules that the vimentin end domains might regulate IF assembly strength. Scale bar, 20 mm. (b) The details of IF interactions with the nucleus, intracellular and cell-surface junctional structures are highlighted. IF link through and plakin family members to the nucleus, and extend into the cytoplasm where they associate with mitochondria, Golgi membranes [e.g. formiminotransferase cyclodeaminase (FTCD)], and the endolysosomal system [e.g. adaptor -3 (AP-3)] [1,5,89]. IF might also position microtubule organizing centers (MTOCs), perhaps through interactions with the g- ring complex (gTuRC) protein GCP6 [90], thus influencing MT organization and proper trafficking and distribution of membrane proteins in polarized epithelial cells [5].

31 Review TRENDS in Cell Biology Vol.18 No.1

pendent fashion, leaving the stationary particle behind Box 1. Intermediate filament mechanical properties [42]. Once separated from the mRNA, these particles are MTs, MFs and IFs all contribute to cell structure, however IFs exhibit also capable of rapid MT-dependent translocation. Evi- properties that make them uniquely suited for maintaining cell dence for post-translational precursor formation in fila- integrity in the face of mechanical and environmental stresses. IFs are more flexible than MTs or MFs. This flexibility might be because ment turnover also exists. One example in support of a of the short persistence length of IFs compared with or MTs; pre-existing pool of IF subunits comes from the observation that is, IFs curve along their length more than MTs and MFs [12]. that type II keratins are produced earlier in embryogenesis Compared with MTs and MFs, which rupture with small amounts of than their type I partners, and are stored in aggregates strain, , desmin and some keratins have been until heterodimerization takes place in the correct observed to extend to two to three times their original length, becoming thinner and increasing in stiffness in response to the temporal sequence [50]. Thus it is possible that both co- strain placed on the filament. This extensibility might be due in part and post-translational mechanisms for precursor assembly to intrafilament sliding, a property unique to IFs because of the are used in cells, providing multiple modes for regulating longitudinal annealing of filament subunits [1,11,12,92,93]. Remark- IF network formation. ably, unlike the other networks, IFs can recover from large amounts Although various subcellular locations throughout the of strain, thus the intrafilament sliding and resultant thinning is to some extent reversible. Interactions among the IF, MT and MF cell have been proposed as IF organizing centers, the cell can increase the resultant stiffness of the combined periphery has been identified as a ‘hot spot’ for initiation of networks. For example, the stiffness reported for vimentin and actin IF assembly. Fluorescent-labeled keratin probes have been mixtures is greater than that observed for either cytoskeleton alone shown to incorporate into small particles in the actin-rich when subjected to the same force [94]. periphery of epithelial cells and move towards the cell center, converting into rodlets and eventually integrating in vivo by altering the equilibrium constant of subunit into the existing IF network [45]. The idea that the IF exchange towards a higher off-rate [25,30]. Post-transla- network can be replenished primarily by addition of pre- tional modifications such as O-linked glycosylation and cursors at the network periphery was supported by FRAP interactions with IFAPs might also have an impact on analysis. Together, these observations suggest another the dynamics of IF remodeling [2,25,31,32]. IFs thus have way in which existing networks might be remodeled in the potential to be highly variable in structure and readily cells. Recently, both vimentin and keratin precursors have remodeled in a dynamic, spatially regulated manner in been shown to form in close proximity to focal complexes at vivo. sites of close apposition to the cell substrate [43,47,51] and The possible exchange of IF polypeptides into existing they seem to move along associated actin fibers [47]. networks could help explain how remodeling of networks Intriguingly, -deficient focal adhesions do not support might occur in vivo, but it doesn’t tell us how an IF network cortical keratin precursor formation and dynamics. forms de novo inside a cell. Recent studies using fluor- Although it seems possible that indirect effects owing to escent-tagged reporter proteins have identified non-fila- alterations in focal contact-mediated adhesion or signaling mentous ‘particles’ and small, filamentous forms of IFs might contribute to impaired IF behavior, collectively the called ‘squiggles’ in cells, which can incorporate into the data support an important role for the cell periphery in IF polymerizing network [20,22,23,33,34]. Although the dynamics [47]. The ability to initiate precursor formation possibility that these ectopically expressed probes could from multiple cellular locations might be important for influence normal IF dynamics should be kept in mind, customizing the IF network with high spatial resolution to these live-cell imaging studies have nevertheless been allow structural and signaling functions to be targeted to instructive in exposing potential mechanisms of assembly specific cell compartments. inside a cell. The formation of putative IF precursors seems to be a general property of IF because this has been IF precursors: on the move observed for neurofilament proteins (NFs) [35–40], the Unlike MTs and MFs, IFs do not seem to serve as tracks for type III proteins peripherin [41,42] and vimentin movement of membrane vesicles and other cellular traffic. [22,23,33,43] and some keratins [20,21,24,34,44–47]. IF particles are themselves cargo that can be moved Particles seem to merge to form squiggles, however the around the cell by various motor proteins. The direction, precise structure of the precursors observed in cells is rate of movement and pause frequency vary as a function of unknown [23]. It is hypothesized that the particles and IF type, cell type and size of IF precursor (Figure 3). Type squiggles contain aggregates of intermediates, such as IV NFs and type III vimentin and peripherin have several ULFs, and short filaments comprising polymerized ULFs, dynamic behaviors in common. They have each been respectively [14,44,48,49]. observed to move 60% of the time in the anterograde Several ideas have been put forward to explain how and and 40% of the time in the retrograde direction where IF precursors arise in cells. Precursors might form [35,36,40,41]. MT motors in the family have been de novo from newly synthesized protein, or they might implicated in anterograde translocation of vimentin assemble from a pre-existing soluble pool of protein, or [22,33,48,52,53] and NF [37,54–58] whereas and both. Support for a co-translational mechanism comes from are part of a retrograde complex, which in recent experiments carried out in rat PC12 cells, in which neurons might be mediated by an interaction between type III peripherin mRNA and its pr