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Absence of cytoplasmic Intermediate Filaments in Arthropods

Absence of cytoplasmic Intermediate Filaments in Arthropods


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Cytoplasmic intermediate filaments such as vimentins support the architecture of the cell and have been known to aid signaling processes. However, as in this article, it is stated that out of all eumetazoans in Kingdom Animalia, only arthropods do not contain such structures. How is this possible? Are there any replacements to the conventional intermediate filaments present in cells? Do arthropods have special cellular behaviors or signaling networks to correspond with this absence?


It is stated that out of all eumetazoans in Kingdom Animalia, only arthropods do not contain such structures.

I found this paper(1), eliciting an exception (within an exception)- Isotomurus maculatus:

Here, we report the first evidence for the expression of a cytoplasmic IF protein in an arthropod - the basal hexapod Isotomurus maculatus. This new protein, we named it isomin, is a component of the intestinal terminal web and shares with IFs typical biochemical properties, molecular features and reassembly capability.

I would suggest you to read the paper, for more information.

How is this possible? Are there any replacements to the conventional intermediate filaments present in cells?

Both electron microscopy and molecular cloning studies have been unable to detect any cytoplasmic IF protein in these organisms, although they do express a nuclear IF system made of authentic lamins. Other cytoskeletal components have been proposed to have assumed, in arthropods, the mechanical functions that are usually played by cytoplasmic IFs in other organisms. For example, wing epithelial cells are stabilized in insects by a cytoskeletal array consisting of parallel bundles of 15-protofilament microtubules and actin filaments.

Do arthropods have special cellular behaviors or signaling networks to correspond with this absence?

I am not sure if this is what you might be looking for, but the Delta/Notch signalling, which may be involved in posterior elongation in arthropods, does not require IF's.(2)

The Notch cascade consists of Notch and Notch ligands, as well as intracellular proteins transmitting the notch signal to the cell's nucleus. The Notch/Lin-12/Glp-1 receptor family was found to be involved in the specification of cell fates during development in Drosophila and C. elegans.

1- https://bmcbiol.biomedcentral.com/articles/10.1186/1741-7007-9-17

2-https://en.wikipedia.org/wiki/Notch_signaling_pathway#Mechanism_of_action


From hair to nuclear organization

Biological specimens enriched in intermediate filament (IF) proteins were among the first to be placed into an x-ray beam for structural analysis, back in the 1930s by William Astbury. He used hair, in its non-stretched and stretched form, rightly deducing that such extended, stable and flexible rods are made from highly ordered proteins. However, it took the thesis work of a graduate student several years later to explain fully these first, surprisingly simple diffraction patterns. The student was Francis Crick. He realized that the keratin α-helices in hair are packed as “simple coiled coils”, remarking later, in his 1988 autobiography, that at that time “helices were in the air”. This excitement was partly due to the discovery of the α-helical fold by Linus Pauling and his group as a fundamental structural principle embodied in the muscle proteins myosin and tropomyosin and in the years that followed, more α-helix-rich proteins were discovered and grouped together as fibrous proteins. Years later the excitement of biochemists was gone. The second edition of classical textbook Biological Chemistry by Mahler and Cordes (1971) lists them simply under scleroproteins together with collagen and gelatin, without further mention.

This scenario changed when in 1968 the group of Howard Holtzer discovered IFs as a further independent filament system in cells obtained from chicken muscle in addition to the well established actin and myosin filaments, highly abundant in myocytes. By conventional electron microscopy, the diameter of these new filaments was determined to be intermediate between that of actin and myosin filaments, hence intermediate filaments or 10-nm filaments (see [1]). Within the next 10 years these new filaments were found in all vertebrate tissues and cultured cell lines investigated, and in many other animals too and subsequently, the massive DNA sequencing efforts of the 1980s produced two major insights into this protein class. First, IF proteins from various tissues all exhibit a conserved central α-helical rod domain, organized so that two chains can form a parallel in-register coiled coil (Figure 1 [1]]), and which is flanked by non-α-helical domains of very different character and size. Second, they are only found in metazoan species and appear to be absent from plants and fungi.

All intermediate filaments have essential structural features in common. A schematic molecular model of a coiled-coil dimer is shown for human lamin A (upper part) and human keratins 5 and 14, which heterodimerize to assemble into the keratin filament (lower part). The two molecular complexes are aligned with respect to coil 2. NLS, nuclear localization signal pb, paired bundle L1, linker L1 L12, linker L12 st, stutter (adapted from [10]).

It came as a great surprise when the cell nucleus was found to contain fibrillar substructures - the nuclear lamina - composed of specialized IF proteins, the lamins. As so often in science, these entities, discovered last, turned out to be the evolutionary ancestors of the whole intermediate filament multigene family. Simple metazoans such as Hydra attenuata were found to express at least nuclear IF proteins, and a comparative analysis of their lamin sequences and the other known lamin and IF protein sequences led to the conclusion that IFs originated in an ur-lamin [2]. The simple invertebrate Caenorhabditis elegans, which has a single nuclear lamin, also harbors eleven genes coding for cytoplasmic IF proteins, four of which have been demonstrated to be essential for viability [3] but the fruit fly Drosophila melanogaster, which expresses the two nuclear lamins - lamin A and lamin B - characteristic of mammalian species, does not exhibit any cytoplasmic IF protein. This led to the conclusion that insects lack cytoplasmic IFs - a conclusion that is now challenged by Mencarelli et al. [4], who detected abundant cytoplasmic structures in the mid-gut cells of the hexapod Isotomurus maculatus (commonly known as the springtail), and have isolated the protein, cloned the DNA from the deduced sequence, compared the sequence with those of known IFs, reassembled filaments from the expressed protein in vitro, and conclude that the protein, which they call isomin, is an intermediate filament protein.

Before discussing the implications of this discovery, we need to ask what are the defining features of an intermediate filament protein, and how does isomin fit the definition?

All intermediate filament proteins are coiled-coil proteins, but not all coiled-coil proteins are intermediate filament proteins. The conclusion that IF proteins are absent from plants and fungi for example rests on the basis of the fully sequenced genomes of the thale cress (Arabidopsis thaliana) and of bakers’ yeast (Saccharomyces cerevisiae) but coiled coil proteins are quite abundant in these organisms, in myosins, kinesins and tropomyosins as well as in transcription factors and in the structural maintenance of chromosomes proteins (SMCs), which contain extended coiled coils and are found in all cells. Even bacterial cells are assumed to contain some extended coiled-coil proteins [5], and notably, a bacterial protein essential for the organization of cell curvature, crescentin, has been described to exhibit many features that are found in IF proteins, and hence has been termed IF-like [6]. So how are coiled-coil proteins recognized, and how are IF proteins distinct?


Filaments and phenotypes: cellular roles and orphan effects associated with mutations in cytoplasmic intermediate filament proteins

Cytoplasmic intermediate filaments (IFs) surround the nucleus and are often anchored at membrane sites to form effectively transcellular networks. Mutations in IF proteins (IFps) have revealed mechanical roles in epidermis, muscle, liver, and neurons. At the same time, there have been phenotypic surprises, illustrated by the ability to generate viable and fertile mice null for a number of IFp-encoding genes, including vimentin. Yet in humans, the vimentin ( VIM) gene displays a high probability of intolerance to loss-of-function mutations, indicating an essential role. A number of subtle and not so subtle IF-associated phenotypes have been identified, often linked to mechanical or metabolic stresses, some of which have been found to be ameliorated by the over-expression of molecular chaperones, suggesting that such phenotypes arise from what might be termed "orphan" effects as opposed to the absence of the IF network per se, an idea originally suggested by Toivola et al. and Pekny and Lane.

Keywords: background effects chaperones intermediate filament proteins mutation phenotypes stress response.

Copyright: © 2019 Klymkowsky MW.

Conflict of interest statement

No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.

Figures

Figure 1.. Interaction networks (derived from the…

Figure 1.. Interaction networks (derived from the STRING-DB website) for vimentin and desmin.


Tools

Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments

-internexin was able to selfassemble into extensive filamentous networks. In contrast, the neurofilament triplet proteins were incapable of homopolymeric assembly into filamentous arrays in vivo. NF-L coassembled with either NF-M or NF-H into filamentous structures in the transfected cells, but NF-M could not form filaments with NF-H. ot-internexin could coassemble with each of the neurofilament triplet proteins in the transfected cells to form filaments. When all but 2 and 10 amino acid residues were removed from the tail domains of NF-L and NF-M, respectively, the resulting NF-L and NF-M deletion mutants retained the ability to coassemble with ot-internexin into filamentous networks. These mutants were also capable of forming filaments with other wild-type neurofilament triplet protein subunits. These results suggest that the tail domains of NF-L and NF-M are dispensable for normal coassembly of each of these proteins with other type IV intermediate filament proteins to form filaments. Ct-internexin and the neurofilament triplet proteins (designated NF-L, NF-M, and NF-H for low, middle, and high molecular weight subunits, respectively) are members of the type IV intermediate filament (IF)

family of proteins (for reviews see Steinert and Roop, 1988 Fliegner and Liem, 1991 Shaw, 1991). ot-internexin is expressed abundantly in young, postmitotic neurons of the developing peripheral and central nervous systems, but in the adult it is found primarily

. roteins (designated NF-L, NF-M, and NF-H for low, middle, and high molecular weight subunits, respectively) are members of the type IV intermediate filament (IF)

family of proteins (for reviews see =-=Steinert and Roop, 1988-=- Fliegner and Liem, 1991 Shaw, 1991). ot-internexin is expressed abundantly in young, postmitotic neurons of the developing peripheral and central nervous systems, but in the adult it is found prima.

Disruption of muscle architecture and myocardial degeneration in mice lacking desmin

. re built from subunits that are ubiquitously expressed, the proteins which form IFs display a very tissue specific and developmentally regulated pattern of expression (for review see Lazarides, 1980 =-=Steinert and Roop, 1988-=- Fuchs and Weber, 1994). Numerous plausible functions for IFs have been proposed over the years, including functioning as strength supporters and mechanical integrators of intracellular space, influe.

Caspase cleavage of keratin 18 and reorganization of intermediate filaments during epithelial cell apoptosis

. I, vimentin, desmin, GFAP, and peripherin type IV, neurofilaments, nestin, and internexin and type V, lamins. Higher eukaryotes express IF proteins in a tissue- and differentiation-specific manner (=-=Steinert and Roop, 1988-=-). Keratins are obligate heteropolymers that constitute the IFs of epithelial cells by the association of at least one type I and one type II keratin (Steinert et al., 1976 Hatzfeld and Franke, 1985).

Pheromone-regulated genes required for yeast mating differentiation

. uggesting it either associates with itself or another protein to function in nuclear membrane fusion. Since many structural proteins such as intermediate filaments contain large coiled-coil segments (=-=Steinert and Roop, 1988-=-), Kar5p may play some structural role during nuclear fusion. Kar5p might act in conjunction with the previously described KAR2/BiP gene product, which is also involved in nuclear fusion and has mutan.

Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro

. lectric point. This classification is functionally significant since epithelial cells express type I and II keratins in specific pairs (Eichner et al., 1984 Hanukoglu and Fuchs, 1983 for review see =-=Steinert and Roop, 1988-=-). The predominant pair expressed in mouse basal epidermal cells is a 60-kD type II keratin (K5) and a 55-kD type I keratin (K14) (Breitkreutz et al., 1984 Roop et D. R. Roop's present address is Dep.

Functional analysis of desmoplakin domains: specification of the interaction with keratin versus vimentin intermediate filament networks

. F networks. This differential interaction probably reflects structural differences between keratin versus vimentin IF. The globular end domains of keratin and vimentin IF are significantly different (=-=Steinert and Roop, 1988-=-) these differences may be responsible far differential interactions with DP. However, differences between the rod domains of vimentin and keratin must be considered as well. Although the rod domains.

High resolution scanning electron microscopy of the nuclear envelope: demonstration of a new, regular, fibrous lattice auaehed to the baskets of the nucleoplasmic face of the nuclear pores

. consist of the same or similar molecules. (e) Composition of NEL. This is unknown. As mentioned above the structure could possibly be assembled from tetramers of intermediate type filament proteins (=-=Steinert and Roop, 1988-=-), possibly lamin or laminlike proteins. As discussed above, the NEL is unlikely to be the lamina as such. The structure of the NEL has a 36-nm repeat in one direction and 72 nm in the other. The lami.

The coiled coil of in vitro assembled keratin filaments is a beterodimer of type I and II keratins: use of site-specific mutagenesis and recombinant protein expression

. l of the dimer or of the tetramer i.e. it is not clear whether the tetramer consists of two identical heterodimers or of a homodimer of a type I and a homodimer of a type II keratin (for review, see =-=Steinert and Roop, 1988-=-). Although cross-linking studies of tetramers have been interpreted to suggest the presence of heterodimers (Quinlan et al., 1984 Ward et al., 1985), these experiments could not discriminate between.

Nuclear and mitochondrial inheritance in yeast depends on novel cytoplasmic structures defined by the MDM1 protein

. ted with antibodies against a second intermediate filament protein, cytokeratin (data not shown). This result is consistent with the colocalization of vimentin and cytokeratin in many cultured cells (=-=Steinert and Roop, 1988-=- Lazarides, 1980). These results, taken together, demonstrate that antibodies against MDM1 recognize the intermediate filament network in a variety of animal ceils. MDM1 Distribution Is Greatly Alter.

Phosphorylation on carboxyl terminus domains of neurofilament proteins in retinal ganglion cell neurons in vivo: influences on regional neurofilament accumulation, interfilament spacing and axonal caliber

. units are distinguished from NF-L and subunits of other intermediate filament classes by the exceptional length of their carboxylterminal tail domains (Shaw, 1991 Geisler et al., 1983 Franke, 1987 =-=Steinert and Roop, 1988-=-). These carboxyl-tall domains are localized peripherally along the filament core (Willard and Simon, 1981 Sharpe et al., 1982) and are believed to form radially projecting sidearms that maintain a m.


Intermediate Filaments and the Establishment of a Cellular Architectural Framework

Throughout development, cells both exert and are subject to an array of forces. These physical interactions are initiated not only by the extra-organismal environment, but also by neighboring cells and extracellular matrix. To maintain the integrity of multicellular tissues, cells must (1) avoid rupturing due to mechanical strain and (2) remain adherent to one another. Intermediate filaments have unique features that not only distinguish them from the other two cytoskeletal elements, actin and microtubules, but also make them major contributors in providing mechanical resistance to the cells (Table 3). The persistence length of intermediate filaments is much shorter than both actin microfilaments and microtubules, thus classifying them as flexible polymers (Gittes et al., 1993 M࿌ke et al., 2004 Schopferer et al., 2009 Lichtenstern et al., 2012 N཭ing et al., 2014 Pawelzyk et al., 2014). Cytoplasmic filaments along with being flexible and elastic are also highly extensible and can be stretched ߢ.8-fold without rupturing (Kreplak et al., 2005). Microfilaments and microtubules are more fragile and tend to rupture at strains 㱐% (Janmey et al., 1991). Furthermore, intermediate filaments exhibit strain-induced strengthening without catastrophic failure, making them very suitable as intracellular load bearing springs (Ackbarow et al., 2009 Pawelzyk et al., 2014). Both in vitro and in vivo analyses corroborate this conceptual model of intermediate filaments as important contributors to cells' elasticity and tensile strength (Janmey et al., 1991 Ma et al., 1999 Fudge et al., 2008 Nolting et al., 2015). The dominant function of intermediate filaments in defining cell stiffness is emphasized in keratinocytes devoid of the entire keratin cytoskeleton (Ramms et al., 2013 Seltmann et al., 2013a). Indirect perturbation of cytoplasmic intermediate filaments likewise has detrimental effects on cell stiffness. Cells exposed to lipids such as sphingosylphosphorylcholine (SPC), induce perinuclear reorganization of keratins through site-specific phosphorylation, leading to a marked decrease in the elastic modulus (Beil et al., 2003). Studies using keratin mutants that either mimic or abrogate phosphorylation of keratins at specific sites further underscore the importance of phosphorylation on the mechanical properties of intermediate filaments (Fois et al., 2013 Homberg et al., 2015). Although tensile strength is most often attributed to the keratin filaments present in epithelial cells, vimentin also contributes to structural integrity, such that cell stiffness is reduced in vimentin depleted or disrupted cells (Wang and Stamenović, 2000 Gladilin et al., 2014 Sharma et al., 2017) and stiffness is increased in cells overexpressing vimentin (Liu et al., 2015). Vimentin further protects fibroblasts against compressive strain (Mendez et al., 2014).

Table 3. Comparison of the mechanical properties of cytoskeletal elements.

Along with maintaining the general mechanical integrity of the cytoplasmic volume, cytoplasmic intermediate filaments are also vital determinants of intracellular organelle organization. Vimentin plays a critical role in influencing actin and Rac1 driven (Dupin et al., 2011 Matveeva et al., 2015) localization of cytoplasmic organelles such as endoplasmic reticulum, Golgi complex, nucleus, and mitochondria (Gao and Sztul, 2001 Nekrasova et al., 2011 Guo et al., 2013). In Xenopus laevis, vimentin intermediate filaments form a cage around melanophores and are involved in their transport and localization at distinct sites within the cells (Chang et al., 2009). Vimentin intermediate filaments are also involved in endoplasm spreading (Lynch et al., 2013). Nuclear position and shape have emerged as important downstream outcomes of mechanical stimuli. Changes in nuclear position relative to other organelles can determine cell polarity, and modulation of nuclear shape influences gene expression and stability. Cytoplasmic intermediate filaments physically link to the nuclear envelope via plectin (an intermediate filament-interacting protein) and SUN/nesprin complexes, also known as Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes (Wilhelmsen et al., 2005 Ketema et al., 2007 Burgstaller et al., 2010). Targeted deletion of nesprin-3 or expression of dominant negative nesprin alters perinuclear intermediate filament organization, cell polarization, and migration (Lombardi et al., 2011 Morgan et al., 2011 Postel et al., 2011). Plectin knockout or plectin mutations related to severe skin blistering disease (epidermolysis bullosa simplex) impair perinuclear keratin architecture, but not the linkages to the nucleus, nonetheless leading to misshapen nuclei and abnormal nuclear deformability (Almeida et al., 2015).

At the cell periphery, adhesions to neighboring cells and the surrounding extracellular matrix provide the interface with which cells interact. Intermediate filaments are most commonly known to be anchored to adjacent cells by desmosomes and to the extracellular matrix by hemidesmosomes. The classic view is that intermediate filaments simply provide internal scaffolding attachments to desmosome and hemidesmosome complexes, adhesive junctions that convey relatively long term associations between a cell and its environment. However, the association of keratin intermediate filaments with desmosomes and hemidesmosomes is also mechanistically supportive of the molecular adhesive complex. That is, keratin filaments can promote the formation and maintenance of desmosomes and hemidesmosomes (Kröger et al., 2013 Seltmann et al., 2015 Loschke et al., 2016). Reciprocally, desmosomal adhesions act as organizing centers for de novo keratin network formation in native state tissues (Jackson et al., 1980 Schwarz et al., 2015).

In addition to this classical view of intermediate filaments associating with hemidesmosomes and desmosomes, intermediate filaments interact with cell adhesions often inaccurately believed to be exclusively actin-linked, including junctions mediated by classical cadherins (Kim et al., 2005 Leonard et al., 2008 Weber et al., 2012) and focal adhesions (Tsuruta and Jones, 2003 Windoffer et al., 2006). Association of the intermediate filaments with these otherwise actin-linked adhesions is bolstered in response to physical forces applied to these adhesions (Tsuruta and Jones, 2003 Weber et al., 2012). As with desmosomes and hemidesmosomes, intermediate filaments modify the stability of actin-linked focal adhesions and classical cadherin adhesions. In endothelial cells, vimentin regulates the size and adhesive strength of focal adhesions (Tsuruta and Jones, 2003 Bhattacharya et al., 2009). Vimentin is also implicated in the regulation of vesicular transport of integrin toward the cell membrane (Ivaska et al., 2005).

Intermediate filaments are physically linked to these various adhesion complexes. Vimentin intermediate filaments interact with integrins either directly with binding to 㬣 integrin tail (Kim et al., 2016) or indirectly via linker proteins including plectin (Bhattacharya et al., 2009 Burgstaller et al., 2010 Bouameur et al., 2014) and BPAG (bullous pemphigoid antigens 1 and 2) by forming dynamic linkages with plakin repeat domains (Fogl et al., 2016). In X. laevis mesendoderm (also known as anterior head mesoderm) cells, plakoglobin acts as recruitment signal for keratin intermediate filament association with cadherins (Weber et al., 2012). Similarly, in endothelial cells p120 catenin recruits vimentin intermediate filaments to cadherins (Kim et al., 2005). Both vimentin and keratin precursor assembly show dependence on focal adhesions as recruitment sites for motile precursors (Burgstaller et al., 2010). Tethering of adhesion complexes to intermediate filaments presents an ideal circumstance wherein intermediate filaments can serve as mediators of both tension and the coincident signaling that we now know occurs as a function of these adhesions.

Although intermediate filaments, microtubules and actin cytoskeletal networks are often viewed as three separate entities, these filamentous arrays cooperatively interact in more ways than not. Actin filaments and microtubules both have impacts on intermediate filament organization through multiple direct, indirect and steric interactions. Bidirectional motility of both mature filaments and their non-filamentous precursors of intermediate filaments can occur on either microtubules or actin microfilaments (Prahlad et al., 1998 Helfand et al., 2002 Liovic et al., 2003 Wöll et al., 2005 Kölsch et al., 2009 Hookway et al., 2015). The filament network used to transport intermediate filament precursors is entirely dependent on context. Neither keratin nor vimentin seems to be exclusively limited to microtubules or actin. Motility of intermediate filament precursors can be both fast and slow, in retrograde and anterograde. Although the mechanism of transport seems to be well-defined in some cases (e.g., entirely dependent on actin or microtubules), defining trends have yet to emerge that reliably predict a mechanism of transport for intermediate filaments. Actin filaments are essential to retrograde motility of keratin precursors in some epithelial cells (Kölsch et al., 2009), yet actin cytoskeleton can restrict microtubule-dependent vimentin precursor movement, establishing a complex three-way cytoskeletal communication (Robert et al., 2014). Perturbing either microtubules, microfilaments, or their associated molecular motors can lead to intermediate filament collapse (Knapp et al., 1983 Wöll et al., 2005). Absence of vimentin intermediate filaments alters the microtubule network orientation, thus suggesting a function of vimentin in organizing the cytoskeletal architecture necessary for cell polarity (Shabbir et al., 2014 Liu et al., 2015). In addition, vimentin filaments, but not “non-filamentous” vimentin negatively regulate actin stress fiber assembly and contractility (Jiu et al., 2017). In vitro and in vivo studies have highlighted direct interactions between the tail domain of vimentin and actin (Cary et al., 1994 Esue et al., 2006). Furthermore, numerous cytoskeletal linkers have been identified that allow for indirect interaction among the cytoskeletal polymers. These proteins include plectin (Svitkina et al., 1996 Osmanagic-Myers et al., 2015), myosin (Kölsch et al., 2009 Robert et al., 2014), fimbrin (Correia et al., 1999), filamin A (Kim et al., 2010), kinesin (Prahlad et al., 1998 Kreitzer et al., 1999), adenomatous polyposis coli (APC Sakamoto et al., 2013), dynein, and dynactin (Helfand et al., 2002). Along with distinct cytoskeletal entities interacting with one another, vimentin and keratin intermediate filament networks have been observed to interact at the helical 2B domain of vimentin, and mutations in this region negatively impact collective cell migration (Velez-delValle et al., 2016).

Some data suggest specific roles for each of the cytoskeletal networks in how a cell normally manages different physical forces and stresses. And yet when systems are disrupted, cells often find ways to compensate using the available “protein toolkit.” Mechanical probing of fibroblasts shows that actin contributes to cortical stiffness, whereas vimentin dominates cytoplasmic stiffness (Guo et al., 2013). Disruption of vimentin intermediate filaments mandates that cells find other ways of dealing with imposed forces. In some cases, cells compensate to accommodate self-generated forces by increasing actin stress fibers and myosin activity to facilitate ECM substrate traction while exhibiting disruption of cell-cell adherens junctions (Osmanagic-Myers et al., 2015 Jiu et al., 2017). In response to increased externally-derived physical strain and mechanosensing of these forces, keratins can promote stress fiber formation and cell stiffness by activation of ROCK signaling pathway (Bordeleau et al., 2012). These data illustrate great versatility in how cells use the cytoskeletal networks available to facilitate adhesion, cohesion, and balance intracellular tension and externally-derived stresses. In the context of the complex multicellular animal, keratin, and vimentin establish an important scaffolding framework inside the cell. Cytoplasmic intermediate filaments enable the cells to resist deformation, localize organelles, change shape, and are integrally coupled to adhesion complexes (Figure 1).

Figure 1. Interdependent network model of cytoplasmic intermediate filaments as a centerpiece between mechanical stimuli and directional cell migration. External forces act on (arrows) adhesion molecules on the cell surface to impact a complex network of bidirectional interactions within the cell (lines). Adhesions are linked to the three major cytoskeletal networks. Of these, actin with its myosin motors is the primary force-generating apparatus. Intermediate filaments can be pre-stressed by actomyosin generated tension. Intermediate filaments also act to resist strains imposed on the cell. Through modulation of cell signaling pathways, direct and indirect, intermediate filaments effect cell polarity and protrusive behavior. Stabilization of distinct subcellular locales promotes persistent directional migration.


What is the function of intermediate filaments?

The tight association between protofilaments provide intermediate filaments with a high tensile strength. This makes them the most stable component of the cytoskeleton. Intermediate filaments are therefore found in particularly durable structures such as hair, scales and fingernails.

The primary function of intermediate filaments is to create cell cohesion and prevent the acute fracture of epithelial cell sheets under tension. This is made possible by extensive interactions between the constituent protofilaments of an intermediate filament , which enhance its resistance to compression, twisting, stretching and bending forces. These properties also allow intermediate filaments to help stabilize the extended axons of nerve cells, as well as line the inner face of the nuclear envelope where they help harness and protect the cell&rsquos DNA.


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Unanswered questions

Which of the phenotypic effects associated with mutations in IFp-encoding genes are direct, that is, due to the absence of an intact IF network, and which are indirect, due to the redistribution of proteins normally associated with IFs, remains to be resolved. That IFps interact with cellular factors was indicated to us by the observation that Xenopus vimentin protein failed to assemble a filament network in Xenopus oocytes 32 . The role of host cell factors has been further illustrated by studies in which human IFps were expressed in Drosophila, which has no cytoplasmic IFs of its own. In Drosophila S2 cells and mesenchymal tissues (the types of tissues that would normally express VIM in humans), human vimentin was unable to form filament networks on the other hand, it formed cage-like filament networks around the nuclei of internal epithelial cells 8 .

There are a number of tools available to visualize protein–protein interaction networks 33 . (It is worth noting the formal distinction between a polypeptide gene product and a functional protein, which may be composed of multiple different gene products and multiple subunit polypeptides. See https://bioliteracy.blog/2018/05/15/when-is-a-gene-product-a-protein-when-is-it-a-polypeptide.) An often-used tool is STRING 77 , which displays a range of interactions graphically. Here, I have used STRING to present a crude snapshot of interactions involving VIM and DES proteins (Figure 1). One immediately notes that a number of known DES-interacting proteins 78 derived from the BioGRID database 79 are absent (Table 1 and Figure 1). I refer to interacting proteins that may be influenced by the absence of an IFp as orphan proteins. In the absence of an intact IF network, such orphans may adopt wayward (toxis) structures and interact inappropriately with other cellular structures, leading to secondary phenotypes, an idea originally suggested by Toivola et al. 80 and Pekny and Lane 81 (see also Capetanaki et al. 82 ). It is likely that many functionally significant interactions have yet to be identified. An example is the molecular chaperone αB-crystallin (CRYAB), whose STRING interaction network (Figure 1) does not include any IFps. In this case, the orphan effect involves defects in the assembly of IF networks in astrocytes associated with mutations in the gene encoding glial fibrillary acidic protein (GFAP). Such mutations lead to increased levels of soluble oligomers that act to inhibit proteosome activity in Alexander disease 83 . In mouse models of the disease, inhibition of CRYAB expression led to increased mortality whereas increased CRYAB expression “rescued animals from terminal seizures” 83,84 . In a sense, the chaperone provides a home or safe haven for the non-filamentous GFAP oligomers, an idea suggested by the chaperone network described by Taipale et al. 85 and others (see below).

Figure 1. Interaction networks (derived from the STRING-DB website) for vimentin and desmin.

We list the desmin-interacting proteins—from Costa et al. 78 (2004)—that are absent from either map. As an example, chaperone αB-crystallin (CRYAB) is absent. Its interaction map is displayed in the upper right hand corner.

The gigaxonin (GAN) gene encodes a E3-ubiquitin adaptor protein involved in IF network organization and degradation 86–88 . GAN is mutated in the fatal human disease giant axonal neuropathy. Our studies revealed the conditional nature of the GAN-associated VIM organization phenotype in two patient-derived primary fibroblast cell lines 89 . Of note, the GAN protein does not appear in lists of IF associated proteins or in the STRING data base. In other cell types, the absence of glial IF networks was found to lead to an increase in neuronal and glial cell division and improvements in post-trauma regeneration 90–92 as well as effects on gene expression in neighboring microglia 93 . The mechanism(s) underlying these effects have yet to be resolved.

Traub et al. described the interaction between a number of IFps and nucleic acids 94–97 . (In our own lab, we routinely purified VIM on single-stranded DNA columns.) It is worth noting that the VIM –/– mouse generated by Colucci-Guyon et al. 37 may leave the N-terminal DNA binding domain intact. Soluble (tetrameric) forms of IFps have been identified 98 and found in the nuclei of cells 99 . VIM has been reported to influence transforming growth factor beta (TGFβ)-Slug (Snai2) 100 and nuclear factor kappa B (NF-κB) 101 signaling as well as the NLRP3 inflammasone 102 , all of which are known to influence gene expression. Similarly, desmin has been reported to enter the nucleus, associated with chromatin, and influence gene expression 103 . These observations raise the obvious question, answerable by RNA-seq (RNA-sequencing) and proteomic studies, how does the expression (or absence) of a particular IFp influence the overall pattern of gene expression? This is a question that, to my knowledge, has not been directly answered, even though VIM-free human SW13 cells and the ability to control expression of various IFps (including VIM) have been available for some time 19,75,104–108 . Steps in this direction have been made, however. These include a microarray analysis of control and Alzheimer’s disease model mice null for both GFAP and VIM these authors reported that the expression of hundreds of genes was altered 93 . A similar response has been found in DES –/– mice 109,110 . Levels of inflammation, interleukin 1 beta (IL-1β) expression, and endothelial and alveolar epithelial barrier permeability, together with tissue remodeling and fibrosis, are attenuated in the lungs of VIM –/– mice 102 . The absence of KRT expression influenced epidermal barrier formation and mitochondrial lipid composition and activity in the cornified epithelia of transgenic mice 111 . In some cases, IFp concentrations have been found to increase dramatically in the context of cell stress, suggesting that IFps themselves may act as stress proteins, part of a stress response network 112 .

There are multiple reports of interactions between IFs and mitochondria 111,113–123 , as well as with endoplasmic reticulum, which interacts with mitochondria 124,125 , and the microtubule-anchoring centrosome 126 . The disruption of these interactions could lead to a range of effects, including mitochondrial dysfunction, which has been reported in a number of IFp-null mice. Given the central role of mitochondrial activity in a wide range of tissues and cellular processes 127–130 , such effects may be more impactful than the “primary” defects arising from the absence of the IF network itself. As an example, mitochondrial effects have been linked to the behavior of primary cilia, an organelle closely involved in a number of intra- and intercellular signaling systems active during embryonic development and within mature tissues 131 . Abnormal mitochondrial structure, function, and activity may be involved in a wide range of IF-associated phenotypes, such as increased oxidative stress in macrophages, leading to vascular inflammation and attenuated atherosclerosis in mice 132 , the accumulation of body fat 133 , and differences in the growth behavior of wild-type and VIM-null cells 115 .

Perhaps the most obvious example of IF–stress interactions and organismic phenotypes is the cardiomyopathy phenotypes observed in DES –/– mice and associated with human DES mutations 134 . DES –/– mice display “progressive degeneration and necrosis of the myocardium” and defects in mitochondrial distribution, morphology, and function 135,136 . Weisleder et al. 136 observed that the most severe aspects of the DES –/– phenotype in mice were suppressed by the over-expression of Bcl2, a mitochondrial outer membrane protein involved in the regulation of apoptosis 137 . In our own studies, expression of the related anti-apoptotic protein Bcl-xL suppressed neural crest defects associated with the loss of the transcription factor Slug (Snai2) through the activation of NF-κB signaling 138 , suggesting the possible involvement of complex “downstream” effects. Diokmetzidou et al. 139 followed up on the rescue ability of the mouse DES –/– phenotype by adopting a strategy first applied by the Goldman 83 , Messing 84 , and Quinlan 140,141 groups, who found that the expression of the molecular chaperone CRYAB 142 suppressed the toxicity of GFAP mutants in mouse models of Alexander disease (see above). In the case of DES –/– -null mice, the Capetanaki group found that expression of αB-crystallin ameliorated many of the mitochondrial defects displayed in heart muscle, leading to “almost wild-type levels” of mitochondrial activity 143 . In a related study, this group found that over-expression of tumor necrosis factor alpha (TNFα) led to expression of the simple epithelial keratins Krt8 and Krt18 in the heart these keratins assumed many of the structural roles normally carried out by DES and rescued mitochondrial defects 144 . In the absence of these keratins (and DES), critical desmosomal and adherens junction proteins, all known to influence intracellular signaling systems and gene expression networks, were displaced 61,145–147 . These observations reinforce the idea that the loss of wild-type DES in particular, and IFps in general, can lead to the mislocalization of proteins known to play important roles in the regulation of mitochondrial function and gene expression.

Simple epithelial keratins provide a classic example of both genetic background effects and the role of IFps under conditions of cellular and tissue stress. The first reported knockout of any IFp was Krt8. In C57BL/6 mice, Krt8 –/– animals displayed about 94% embryonic lethality 62 . However, when crossed into the FVB/N genetic background, embryonic lethality was suppressed, although Krt8 –/– mice displayed colonic hyperplasia and inflammatory phenotypes in desmin null mice 148 . In 20-week-old Krt8 –/– (FVB/N) mice, analysis of liver structure revealed no overt phenotypes associated with the absence of KRT filaments. KRT filaments do not form in this simple epithelial tissue in the absence of Krt8. On the other hand, a rapid increase in blood flow and the cellular stresses associated with partial hepatechomy led to 100% lethality in Krt8 –/– (FVB/N) mice compared with significant levels of survival in heterozygous and wild-type mice 149 . A similar increase in hepatechomy-associated lethality was observed in Krt18 –/– mice 150 as well as in humans with KRT mutations/variants 13,151 . Clearly, genetic background effects, the presence of particular stresses, and cellular responses to those stresses play important roles in the various disease phenotypes associated with IFp variants 152 .

There have been a number of reports on roles for VIM in cell migration and epithelial-mesenchymal transition (for example, 153–157), a key developmental event associated most dramatically with the formation and migration of neural crest cells and their roles in a number of tissues, particularly the vertebrate craniofacial skeleton 156,158–160 . Yet to my knowledge, no craniofacial or cell migration-dependent defects have been described in VIM –/– mice or VIM mutations/variants in humans. It remains unclear whether the phenotypes associated with aberrant VIM expression are due to the absence of VIM per se or to secondary effects involving orphaned VIM-associated proteins. An obvious experiment would be to ask whether increased expression of molecular chaperones, such as αB-crystallin, rescued any or all of such cell migratory phenotypes.

The size of the IFp gene family raises another recently identified potential complication in the link between mutation and phenotype. As reviewed by Wilkinson (161 and references therein), non-sense mutations can provoke a non-sense–mediated, RNA decay–based gene regulatory feedback system that can lead to the activation of (often) sequence-related genes. More generally, the viability of biological systems in the face of molecular level noise (including mutations) is enhanced by a range of adaptive molecular chaperones and feedback networks 85,162,163 . Given the effects of expressing chaperones on mutant IFp phenotypes (see above), a more complete understanding of the molecular mechanisms responsible for the phenotypes associated with mutant IFp genes is likely to suggest more effective therapeutic strategies, such as the use of small molecule “chemical chaperones” 164 , as well as a deeper understanding of the responsive interaction networks that underlie biological behaviors.


DNA constructs

Mutations in plasticin ( 1 ) were generated byin vitro mutagenesis using the method described by Kunkel ( 32 ). In brief, plasmids were transformed into the Escherichia coli strain CJ236 to yield single-stranded, uracil-containing circular DNA using the M13K07 helper phage. Single-stranded plasmid was purified from the helper phage by preparative gel electrophoresis. Phosphorylated oligonucleotides containing internal mutations (synthesized by Genosys Bio-technologies, The Woodlands, TX, U.S.A.) were hybridized to the single-stranded plasmid and extended with T7 DNA polymerase. The resulting double-stranded plasmid was transformed into E. coli strain XL1-Blue MRF' (Stratagene, La Jolla, CA, U.S.A.). Appropriate mutations were identified by sequencing DNA obtained from mini-preps (RPM kit Bio 101, Vista, CA, U.S.A.) as described previously ( 1 ). All plasticin cDNAs were subsequently cloned as HindIII-EcoRI fragments into pBluescript P/X HA3 ( 38 ) using standard PCR techniques. These plasticin-hemagglutinin (HA) tag fusion cDNAs were then cloned as HindIII-XbaI fragments into the mammalian expression vector pcDNA3.1 (Invitrogen, San Diego, CA, U.S.A.). Untagged plasticin cDNAs were cloned as EcoRI-XbaI fragments into pCS2+ ( 44 51 ) using standard PCR techniques. Plasmids used for transfection were purified using the Plasmid Maxi Kit (Qiagen, Hilden, Germany). The pRSVi-NF-L, pRSVi-NF-M, and pRSVi-vimentin expression constructs ( 9 49 ) were generously provided by Dr. Ronald H. K. Liem (Columbia University College of Physicians and Surgeons, New York, NY, U.S.A.). The VimGG+D expression construct ( 4 ) was kindly provided by Drs. Peter Traub and Robert Shoeman (Max Planck Institute for Cell Biology, Ladenburg, Germany).


An Atypical Tropomyosin in Drosophila with Intermediate Filament-like Properties.

<p>A longstanding mystery has been the absence of cytoplasmic intermediate filaments (IFs) from Drosophila despite their importance in other organisms. In the course of characterizing the in vivo expression and functions of Drosophila Tropomyosin (Tm) isoforms, we discovered an essential but unusual product of the Tm1 locus, Tm1-I/C, which resembles an IF protein in some respects. Like IFs, Tm1-I/C spontaneously forms filaments in vitro that are intermediate in diameter between F-actin and microtubules. Like IFs but unlike canonical Tms, Tm1-I/C contains N- and C-terminal low-complexity domains flanking a central coiled coil. In vivo, Tm1-I/C forms cytoplasmic filaments that do not associate with F-actin or canonical Tms. Tm1-I/C is essential for collective border cell migration, in epithelial cells for proper cytoarchitecture, and in the germline for the formation of germ plasm. These results suggest that flies have evolved a distinctive type of cytoskeletal filament from Tm.</p>


Watch the video: THE CYTOSKELETON - MICROTUBULES, INTERMEDIATE FILAMENTS, MICROFILAMENTS (May 2022).