Intermediate filaments

Intermediate filaments (IFs) make up one of three cytoskeletal systems in human cells. This family of proteins include cytoplasmic intermediate filaments, which form an extensive network through the cytosol, as well as nuclear intermediate filaments, which form the thin nuclear lamina underlying the nuclear membrane. The major role for the cytoplasmic filaments is in providing structural organization and mechanical support to the cell, as well as contributing to cell shape, cell migration and cell adhesion (Leduc C et al. (2015)). Nuclear lamins are involved in organization of chromatin. In the Human Protein Atlas, staining of proteins that localize to nuclear lamins appears similar to a staining of the nuclear membrane and are therefore not included in the annotation of intermediate filaments. Example images of proteins localizing to cytoplasmic intermediate filaments can be seen in Figure 1.

In the subcellular resource, 151 genes (1% of all protein-coding human genes) have been shown to encode proteins that localize to intermediate filaments (Figure 2). A Gene Ontology (GO)-based functional enrichment analysis of genes encoding proteins that localize to intermediate filaments shows enrichment of genes related to the organization, assembly and functions of the intermediate filament cytoskeleton, and cell growth. About 71% (n=107) of the proteins localized to intermediate filaments are also detected in additional cellular compartments, the most common one being the cytosol.


GFAP - U2OS

GFAP - HEK293

NES - U2OS


KRT13 - A-431

KRT19 - RT-4

DES - Rh30

Figure 1. Examples of proteins localized to the intermediate filaments. GFAP (detected in U2OS and HEK 293 cells) and NES (detected in U2OS cells). KRT13 (detected in A-431cells), KRT19 (detected in RT-4 cells) and DES (detected in RH-30 cells).

0%10%20%30%40%50%60%70%80%90%100%% genesNot mappedSecretedOther organellesIntermediate filaments

  • 1% (151 proteins) of all human proteins have been experimentally detected in the intermediate filaments by the Human Protein Atlas.
  • 30 proteins in the intermediate filaments are supported by experimental evidence and out of these 11 proteins are enhanced by the Human Protein Atlas.
  • 107 proteins in the intermediate filaments have multiple locations.
  • 69 proteins in the intermediate filaments show single cell variation.

  • Proteins that localize to intermediate filaments are mainly involved in organization of the intermediate filament cytoskeleton and cell groowth.

Figure 2. 1% of all human protein-coding genes encode proteins localized to the intermediate filaments. Each bar is clickable and gives a search result of proteins that belong to the selected category.

Structure of intermediate filaments

Intermediate filaments are assembled from a diverse family of proteins, which are unified by a characteristic tripartite structure and an ability to self-assemble into homo- or heteropolymeric filaments with a diameter of approximately 10 nm. This diversity, and the fact that intermediate filaments are non-polar, clearly distinguish them from the other cytoskeletal filaments in human cells (microtubules and actin filaments). Moreover, intermediate filaments are characterized by a high degree of stability and mechanical strength, while yet remaining dynamic.

There are more than 70 genes known to encode proteins that form intermediate filaments (Lowery J et al. (2015)). These proteins are classified into five or six subgroups, mainly based on sequence homology. The acidic and basic keratins are categorized into the type I and type II groups, respectively.The type III group include vimentin (VIM), desmin (DES), glial fibrillary acidic protein (GFAP) and peripherin (PRPH). Neurofilament (NF) triplet proteins and α-internexin are categorized as type IV intermediate filament proteins, while the nuclear lamins are categorized as type V (7979242)). The sixth subgroup would contain nestin (NES) and synemin (SYNM) (Leduc C et al. (2015)), but they are often also categorized as type IV (Robert A et al. (2016); Fuchs E et al. (1994)). Figure 3 shows immunofluorescent staining of keratins in different cell types.

While intermediate filament proteins have an intrinsic ability to self-assemble, their in vivo assembly is a regulated process. Moreover, it has been demonstrated that intermediate filaments are continuosly undergoing assembly and disassembly, as well as exchange of subunits within existing filaments. This enables dynamic remodeling of the intermediate filament networks, which is necessary to adjust to changing needs in terms of mechanical support, flexibility and attachment to the surrounding matrix during differentiation and cellular processes such as migration and cell division (Robert A et al. (2016)). A selection of proteins suitable as markers for intermediate filaments can be found in Table 1. A list of highly expressed proteins that localize to intermediate filaments can be found in Table 2.

Table 1. Selection of proteins suitable as markers for the intermediate filaments or its substructures.

Gene
Description
Substructure
KRT19 Keratin 19 Intermediate filaments
KRT4 Keratin 4 Intermediate filaments
DES Desmin Intermediate filaments
NES Nestin Intermediate filaments
KRT17 Keratin 17 Intermediate filaments
KRT13 Keratin 13 Intermediate filaments

Table 2. Highly expressed single localizing intermediate filaments proteins across different cell lines.

Gene
Description
Average nTPM
VIM Vimentin 2456
KRT8 Keratin 8 903
KRT17 Keratin 17 806
KRT19 Keratin 19 382
KRT14 Keratin 14 222
NEFM Neurofilament medium chain 89
KRT13 Keratin 13 76
NES Nestin 73
GFAP Glial fibrillary acidic protein 42
KRT80 Keratin 80 41


KRT17 - U2OS

KRT19 - MCF-7

KRT14 - HaCaT

Figure 3. Examples of the morphology of intermediate filaments in different cell lines, represented by immunofluorescent staining of keratins: KRT17 in U2OS cells, KRT19 in MCF-7 cells and KRT14 in HaCaT cells.

Function of intermediate filaments

Intermediate filaments are essential for providing physical support and stability to cells and tissues, thereby enabling them to withstand mechanical stress and tension. A subgroup of intermediate filaments charaterized by the presence of vimentin (VIM) subunits, has been shown to exhibit different properties when exposed to increasing levels of strain in vitro. When increasing the strain, the filaments were shown to stiffen and resist breakage (Janmey PA et al. (1991); Köster S et al. (2015)). Intermediate filaments have also been implicated in cell adhesion and motility (Leduc C et al. (2015)). Indeed, there seems to be a dynamic rearrangement of intermediate filaments in response to changes in cell motility, as it has been shown that they are organized closer to the nuclear membrane in immobile cells, whereas in migrating cells they are aligned with lamella in the cell's leading edge. Both mutations in genes encoding cytoplasmic intermediate filaments, as well as genes coding for nuclear lamins, have been linked to a number of severe diseases (Herrmann H et al. (2007)).

A Gene Ontology (GO)-based analysis of genes encoding proteins localized to intermediate filaments shows enrichment of terms for both Biological Processes (Figure 4a) and Molecular Functions (Figure 4b) that are well in-line with the known functions of the intermediate filaments. The enrichment for genes involved in cell differentiation during development of the placenta is in agreement with the fact that thos process seems to involve differential expression of keratins (Gauster M et al. (2013)).

010203040506070Fold EnrichmentIntermediate filament cytosk...Intermediate filament-based ...Hemidesmosome assemblyCornificationPositive regulation of cell ...

Figure 4a. Gene Ontology-based enrichment analysis for the intermediate filaments proteome showing the significantly enriched terms for the GO domain Biological Process. Each bar is clickable and gives a search result of proteins that belong to the selected category.

0102030405060708090100Fold EnrichmentIntermediate filament bindingStructural constituent of cy...Scaffold protein binding

Figure 4b. Gene Ontology-based enrichment analysis for the intermediate filaments proteome showing the significantly enriched terms for the GO domain Molecular Function. Each bar is clickable and gives a search result of proteins that belong to the selected category.

Intermediate filament proteins with multiple locations

Approximately 71% (n=107) of the intermediate filament proteome detected in the subcellular resource also localize to other compartments in the cell. The cytoscape network plot (Figure 5) shows that the most common location shared with intermediate filaments is cytosol and a hypergeometric test demonstrates that the number of proteins localizing to both of these compartments is significantly higher than expected. This could reflect cytosolic staining of soluble intermediate filament subunits, that have not yet assembled into filaments, or staining of proteins that interact with the soluble as well as the filamentous forms of intermediate filament proteins, or of proteins that connect intermediate filaments to other cellular structures.

Figure 5. Interactive network plot of intermediate filament proteins with multiple localizations. The numbers in the connecting nodes show the proteins that are localized to intermediate filaments and to one or more additional locations. Only connecting nodes containing at least 1.0% of proteins in the intermediate filament proteome are shown. The circle sizes are related to the number of proteins. The cyan colored nodes show combinations that are significantly overrepresented, while magenta colored nodes show combinations that are significantly underrepresented as compared to the probability of observing that combination based on the frequency of each annotation and a hypergeometric test (p≤0.05). Note that this calculation is only done for proteins with dual localizations. Each node is clickable and results in a list of all proteins that are found in the connected organelles.

Expression levels of intermediate filament proteins in tissue

Transcriptome analysis and classification of genes into tissue distribution categories (Figure 6) shows that a larger portion of genes encoding proteins that localize to intermediate filaments are detected in many tissues, while a smaller portion are detected in all tissues, compared to all genes presented in the subcellular resource. This is well in-line with the known cell type- and tissue type-dependent expression patterns of intermediate filament proteins (Herrmann H et al. (2007)).

**Detected in singleDetected in someDetected in manyDetected in allNot detected0.0102030405060708090100%Intermediate filamentsAll localized genes

Figure 6. Bar plot showing the percentage of genes in different tissue distribution categories for intermediate filament-associated protein-coding genes compared to all genes in the subcelluar resource. Asterisk marks a statistically significant deviation (p≤0.05) in the number of genes in a category based on a binomial statistical test. Each bar is clickable and gives a search result of proteins that belong to the selected category.

Relevant links and publications

Leduc C et al., Intermediate filaments in cell migration and invasion: the unusual suspects. Curr Opin Cell Biol. (2015)
PubMed: 25660489 DOI: 10.1016/j.ceb.2015.01.005

Lowery J et al., Intermediate Filaments Play a Pivotal Role in Regulating Cell Architecture and Function. J Biol Chem. (2015)
PubMed: 25957409 DOI: 10.1074/jbc.R115.640359

Robert A et al., Intermediate filament dynamics: What we can see now and why it matters. Bioessays. (2016)
PubMed: 26763143 DOI: 10.1002/bies.201500142

Fuchs E et al., Intermediate filaments: structure, dynamics, function, and disease. Annu Rev Biochem. (1994)
PubMed: 7979242 DOI: 10.1146/annurev.bi.63.070194.002021

Janmey PA et al., Viscoelastic properties of vimentin compared with other filamentous biopolymer networks. J Cell Biol. (1991)
PubMed: 2007620 

Köster S et al., Intermediate filament mechanics in vitro and in the cell: from coiled coils to filaments, fibers and networks. Curr Opin Cell Biol. (2015)
PubMed: 25621895 DOI: 10.1016/j.ceb.2015.01.001

Herrmann H et al., Intermediate filaments: from cell architecture to nanomechanics. Nat Rev Mol Cell Biol. (2007)
PubMed: 17551517 DOI: 10.1038/nrm2197

Gauster M et al., Keratins in the human trophoblast. Histol Histopathol. (2013)
PubMed: 23450430 DOI: 10.14670/HH-28.817