Intermediate Filaments

Intermediate Filament Proteins (IFPs) acquired their name from their filamentous form. The diameter of the filaments (IFs) (8-12 nm) is intermediate between thin actin microfilaments (7 nm) and thick microtubules (25 nm). Cytoskeleton provides recombinant vimentin protein from the Syrian hamster gene, it is > 99.5% homologous to the human isoform. Click on the datasheet below for more information, or click on the FAQs tab for general information about IFPs.

Cytoskeleton's products have been cited hundreds of times over the past 18 years. A select few are described here, for more citations on individual products please use the "Citations" tab on each individual product page.

 

Vimentin protein: recombinant syrian hamster (Cat. #  V01)

Perlson, E., Hanz, S., Ben-Yaakov, K., Segal-Ruder, Y., Seger, R. and Fainzilber, M. (2005). Vimentin-dependent spatial translocation of an activated MAP kinase in injured nerve. Neuron 45, 715-726.
Styers, M. L., Salazar, G., Love, R., Peden, A. A., Kowalczyk, A. P. and Faundez, V. (2004). The endo-lysosomal sorting machinery interacts with the intermediate filament cytoskeleton. Mol. Biol. Cell 15, 5369-5382.

Question 1:  What is unique to intermediate filaments compared to F-actin and microtubules?

Answer 1:  Intermediate filaments (IFs) are proteins that are another fibrous component of the cytoskeleton in addition to microfilaments and microtubules, and which provide structural support for cells.  IFs differ from F-actin and microtubules in some fundamental ways.  Unlike F-actin and microtubules which have a plus and minus end, intermediate filaments lack polarity.  In addition, intermediate filaments do not contain a binding site for a nucleoside triphosphate, and as their name suggests they are intermediate in size (10nm) between microfilaments (7nm) and microfilaments (25nm).

 

Question 2:  Does Cytoskeleton supply any other IFs apart from vimentin?

Answer 2:  Cytoskeleton does not offer other intermediate filament proteins such as GFAP, neurofilament or cytokeratins, we do however offer custom protein purification.  Please contact Technical Support at tservice@cytoskeleton.com for more information and to discuss the IF you are interested in us isolating and purifying. 

 

About Intermediate Filaments

This introduction briefly describes current knowledge concerning the various intermediate filaments. It also introduces the reader to practical assays useful in the study of their activity. For those who simply wish to read about GFAP, Neurofilament Protein, and Vimentin click the appropriate name.

Introduction
Intermediate Filament Proteins (IFPs) acquired their name from their filamentous form. The diameter of the filaments (IFs) (8-12 nm) is intermediate between thin actin microfilaments (7 nm) and thick microtubules (25 nm). Although IFs are ubiquitous in eukaryotic cells, efforts to clearly define their physiological functions have not resulted in any clear answers. It is known for example that cells can grow, divide and differentiate in culture without any known cytoplasmic IFs being present (1). Research efforts are hampered by the possibility of functional redundancy between specific IFs and by the realization that all members of the IF family may not yet be identified (2-4). Certainly some IFPs have a particularly striking role. For example, keratin isotypes function to maintain epidermal integrity, indeed certain keratin mutations result in debilitating diseases of the skin (5).

At Cytoskeleton we are committed to aid IF research progress by providing IFPs that are a) in a highly purified form, b) of high biological activity, c) in a user friendly format and d) are of high interbatch reproducibility.

Classes of Intermediate Filament proteins
Intermediate filament proteins have been subdivided into 6 classes based on homology (see below). Recently another three new IF-like proteins have been identified which may expand the number of types to nine (2,4). IFs do show tissue specific expression patterns. However, the lamin proteins (specifically lamin B) are thought to be ubiquitously expressed in all nucleate cells.

ClassSequence identity

Mol. wt. (kDa)

Cell type

Type I

Type II
Acidic Keratins

Basic Keratins

40-64

52-68

epithelial

epithelial

Type III

Type IV

Type VI
Desmin

GFAP

Peripherin

Vimentin


α-Internexin

β-Internexin

NF-L

NF-M

NF-H

nestin

53

51

54

55


66

70

68

110

130

240

muscle

astroglia

neuronal

mesenchymal, tissue cultured cells.

immature neuronal

neuronal

neuronal

neuronal

neuronal

CNS stem cells

Type VLamin A

Lamin B

Lamin C

70

64

58

most

most

most

UnclassifiedSeptins A,B,C

Minor Lens IFPs

Filensin

Phakinin

Non-a-IFA

50-60

100

variable

nucleated

lens

lens

invertebrates


Structures of Intermediate Filaments
Intermediate filament proteins have a common structural core of 300-330 residues, which are flanked by extra amino- and carboxy terminal domains. The common core is composed of a heptad repeating unit containing a greater than average allowance of hydrophobic residues and sequences that form α-helical conformations. The repeating hydrophobic residues create a coiled-coil dimer. The formation of such a precise structure is central to the nativity and thus function of all intermediate filament proteins in vivo and in vitro. Cytoskeleton takes the effort to provide IFPs with >95% nativity, which allows you unmatched confidence in your results (see later on Working with Intermediate Filament proteins in vitro for a full description of what is necessary to produce the native form). The coiled-coil region may also be split into three subdomains. Below is a schematic diagram of the model intermediate protein structure.

 

image25

Fig. 1Schematic Diagram Of Intermediate Filament Structure.

The amino- and carboxy-termini can be of various lengths and sequence. For example, cytokeratin 19 has less than 10 residues at the carboxy-terminal and the lamins have less than 40 residues at their amino-terminal. In comparison, NF-H and nestin each have a terminus greater than 500 residues. The termini do not contain α-helical rich sequences.

It is clear that the common core domain serves a similar role in all intermediate filaments, probably as a longitudinal spacer and/or a lateral packing module. The termini on the other hand are not homologous and probably therefore confer a specific function on the molecule. For example, the amino terminal of vimentin has been shown to confer cytoplasmic specificity (6). Also, the amino-terminal has been shown to be essential for proper assembly of filaments (7,8).

Most intermediate filament proteins assemble under suitable conditions (see later) to form uniform 8-12 nm wide filaments. However, several types of IFPs are obligate heteropolymers, they require a heterologous IFP to be present in order to assemble in the co-polymer form (for example: NF-M or NF-H). It is generally accepted that the basic form of unpolymerized IFPs is the tetrameric form of the protein. Staggered lateral associations between IF dimers are important for filament formation. It is believed that four dimers intertwine in a staggered fashion to produce a growing filament. Finally, between filaments, longitudinal associations and covalent crossbridges are a major structural component for keratins in the epidermal layers.

In vivo function of intermediate filaments and their association with diseases
IFPs show a strong specificity of expression for particular cell types. For example, GFAP is specifically expressed in the astroglia cell type. Tumors originating from this cell type can be identified by their expression of GFAP by immunocytology. Furthermore, as tumors progress from primary neoplasm to metastatic the cells may revert to embryonic type IFP expression. GFAP can also be used as a marker of cell lineage (see rev. by Osborn and Weber (9) for a more detailed discussion).

A promising line of investigation concerns work performed on the model organism Saccharomyces cerevisiae. The MDM1 gene has homology to intermediate filaments and forms 10 nm filaments in vitro (10). The MDM1 knock out is lethal and cells carrying mutated mdm1 show nuclear and mitochondrial transport defects (10,11).

Cytokeratins have an essential role in the maintenance of epidermal layers. From the intestinal epithelium to the squamous layer of the skin, their role in structural integrity is becoming very clear. Mutations in human cytokeratins that are genetically linked to mild and severe skin diseases have been identified. For example, Epidermolysis Bullosa Simplex (see rev. by E.Fuchs; 5). Hyperkeratosis in Psoriasis is promoted by enhanced inter-molecular crosslinking between IFPs, creating poor epidermal integrity (12).

The importance of IFPs in the central and peripheral nervous system is only beginning to be appreciated. The "Shaky" Quail phenotype was determined to be a natural knock-out of NF-L (see rev. by Yen et al. 1986; 13), which also resulted in 95% reduction of NF-M and NF-H expression. The morphological character was a greater distribution of axonal diameters, which presumably affected neuronal transmission. Thus, NF-L is associated with the development and maintenance of axonal width. Other IFPs have been shown to be associated with glial tangles (GFAP) and Lewy bodies (neurofilaments) in Alzheimer’s disease, multiple sclerosis, amyotrophic lateral sclerosis and Parkinson’s disease (see rev. by Yen et al. 1986; 13). Whether the intermediate filament proteins are the cause or consequence of the diseased state remains to be clarified. However, it is known that a recombinant mouse strain that over expresses NF-H shows signs of amyotrophic lateral sclerosis-like plaques in the brain tissue (14).

Regulation of intermediate filament function
It is now known that the intermediate filament system is a highly regulated, dynamic structure (15, 16, 17). Dynamicity is a property that is common to all 3 eukaryotic cytoskeletal systems (IFs, microfilaments and microtubules) and is crucial to their physiological functions. Exciting progress is being made in this fundamentally important area of research. We would direct the researcher to several excellent reviews on this subject (17-22).

Working with Intermediate Filament Proteins in vitro
IFPs are sensitive to pH and ionic strength. In fact, it is the manipulation of these parameters that can create polymer or tetramer. Commonly, intermediate filament protein is purified into a denaturing buffer containing urea and high concentrations of β-mercaptoethanol. Subsequent sequential steps of dialysis will remove the denaturants and permit renaturation of the protein. Common techniques for working with intermediate filament proteins are listed below:

a) Differential Sedimentation

Differential sedimentation can be used to remove denatured protein or sediment filaments.

b) Electron microscopy:

Uranyl acetate can be used to visualize IFP filaments by negative stain electron microscopy.

c) Optical absorbance:

Optical density measurements can be used to determine the kinetics of polymerization. A wavelength of 300 to 340 nm is generally applicable.

References

1.Sarria A.J., Sankhavaram R.P. and Evans R.M., 1992. A functional role for vimentin intermediate filaments in metabolism of lipoprotein derived cholesterol in human SW13 cells. J. Biol. Chem., 267, 19455-63.

2.Cooper J. and Kiehart D., 1996. Septins may form a ubiquotous family of cytoskeletal filaments. J. C. Biol., 134, 1345-1348.

3.Pruss R.M., Mirsky R.., Raff M.C., Thorpe R., Dowding A.J. and Anderton B.H., 1981. All classes of intermediate filaments share a common antigenic determinat defined by a monoclonal antibody. Cell, 27, 419-428.

4.Reimer et al., 1991. Cloning of the non-neuronal intermediate fialment protein of the gastropod Aplysia Californica); identification of an amino-acid residue essential for the IFA epitope. Eur. J. Cell Biol., 56, 351-357.

5.Fuchs E., 1995. Keratin and the skin. Ann. Rev. Cell Dev. Biol., 11, 123-53.

6.Bader et al., 1991. J.Cell Biol., 115, 1293-1307.

7.Geisler et al., 1982. Cell, 30, 277-286.

8.Coulombe et al., 1990. J.Cell Biol., 111, 3049-3064.

9.Osborn M. and Weber K., 1989. Cytoskeletal proteins in tumor diagnosis. Curr. Comm. in Mol. Biol., Cold Spring Harbor Press, 1989.

10.McConnell S.J. and Yaffe M.P., 1993. Science, 260,687-689.

11.McConnell S.J. and Yaffe M.P., 1992. J. Cell Biol., 118,385.

12.Schroeder et. al., 1992. Type 1 keratinocyte transglutaminase: Expression in human skin and Psoriasis. J. Invest. Dermatol., 99, 27-34.

13.Yen S-H., Dickson D.W., Peterson C. and Goldman J.C. 1986. Cytoskeletal abnormalities in neuropathy. Prog. Neuropathol., 6, 63-90.

14.Cote F., Collard J-F. and Julien J-P., 1993. Progressive neuropathy in transgenic mice expressing the human neurofilament gene: A mouse model of amyotrophic lateral sclerosis. Cell, 73, 35-46.

15.Miller, R.K., Vikstrom, K., & Goldman, R.D., 1991. Keratin incorporation into intermediate filaments is a rapid process. J. Cell Biol., 113, 843-855.

16.Vikstrom K.L., Borisy G.G. and Goldman R.D., 1989. Dynamic aspects of intermediate filament networks in BHK-21 cells. PNAS, 86, 549-553.

17.Skalli, O. & Goldman, R.D. 1991. Recent insights into the assembly, dynamics and function of intermediate filament networks. Cell Motil. Cytoskel., 19, 67-79.

18.Gallicano, G.I. & Capco, D.G. 1995. Remodeling of the intermediate filament network in mammalian eggs and embryos during development: regulation by protein kinase C and protein kinase M. Curr. Topics in Dev. Biol., 31, 277-320.

19.Bonder, E.M. & Fishkind, D.J. 1995. Actin-membrane dynamics in early sea urchin development. Curr. Topics in Dev. Biol., 31, 101-37.

20.Barkalow, K. & Hartwig, J.H. 1995. The role of actin filament barbed end exposure in cytoskeletal dynamics and cell motiltiy. Biochem. Soc. Trans., 23, 451-6.

21.Mitchison, T.J. 1995. Evolution of a dynamic cytoskeleton. Phil. Trans. of the Royal Soc. of London-Series B: Biological Sci., 349, 299-304.

22.Mandelkow, E. & Mandelkow, E.M. 1995. Microtubules and microtubule associated proteins. Curr. Op. in Cell Biol., 7, 72-81.


About Glial Fibrillary Acidic Protein

Introduction

Glial fibrillary acidic protein (GFAP) is a 51 kDa protein that is expressed almost exclusively in astrocytes and cells of astroglial origin. Native GFAP is soluble in low salt buffer at moderately alkaline pH, but the denatured form is not soluble under similar conditions. GFAP can assemble into homopolymeric filaments in vitro. Assembly is sensitive to pH and salt concentration, the optimal conditions being pH 6.9 and 100 mM NaCl.

Function of glial fibrillary acidic protein
Like most other non-cytokeratin IFs, GFAP’s function has remained obscure for many years. Recent evidence suggests that GFAP is required for the formation of stable astrocytic processes that are critical to central nervous system organogenesis (1). There are also hints that GFAP performs diverse functional roles, e.g., it has been reported to bind to the prion protein that is associated with the neurodegenerative diseases Scrapie (in sheep) and Creutzfeldt Jakob disease (in humans) (2). The subunits of GFAP can coassemble with other IFs in the cell or in vitro. GFAP assembly is affected by phosphorylation of the amino-terminal, and it is suspected that this is how polymerization / depolymerization cycles are regulated in the cell (1).

Medical significance
Besides its importance in central nervous system development and its potential involvement in certain neurodegenerative diseases, GFAP is also useful as a tumour diagnosis marker. GFAP is expressed in a specific subset of cells in the mammal, the astroglial type cells. In tumor diagnosis and particular glial cell diseases such as Alzheimer’s it can act as a marker. Alzheimer’s disease is identified by plaques which contain GFAP peptides.

References

1. Oesch, B., et al. 1990. Biochem. 29, 5-15.

2. Weinstein D.E., Shelanski M.L. and Liem R.K.H., 1991. Suppression by antisense mRNA demonstrates a requirement for the glial fibrillary acidic protein in the formation of stable astrocytic process in response to neurons. J. Cell Biol., 112, 1205-1213.

3. Inagaki M., Gonda Y., Nishizawa K., Kitamura S., Sato C., Ando S., Tanabe K., Kikuchi S. and Nishi Y., 1990. J. Biol. Chem., 265, 4722-4729.


About Neurofilament protein

Introduction
Neurofilament protein (NF) is a heterotrimeric protein complex composed of 68, 102 and 112 kDa polypeptides (NF-L, NF-M and NF-H respectively) that are expressed almost exclusively in axonal cells. It should be noted that the molecular weights of NF-M and NF-H proteins in SDS-gels appear much greater, i.e. 160 and 200 kD respectively. This is due to the fact that the carboxy region of these proteins is hyperphosphorylated and contains large amounts of glutamic acid residues. Native NF is soluble in low salt buffer at moderately alkaline pH, but the denatured form is not soluble under similar conditions. NF can form filaments in vitro. Assembly is sensitive to pH and salt concentration, the optimal conditions being pH 6.9 and 100 mM NaCl. Only NF-L is homopolymeric, NF-H and NF-M are obligate heteropolymeric proteins (1).

Structure / Function of Neurofilament Protein
NF-L is known to polymerize on its own, whereas NF-M and NF-H cannot. However, both NF-M and NF-H can coassemble with NF-L in vitro. They are thought to bind laterally to NF-L and protrude their amino- and/or carboxy-termini out to the side of the filament. The filament that results could be interpreted as a space filler brush-like structure. The regulation of the side arms’ adhesive or repulsive properties would appear to be critical to the functional NF.

Like most other non-cytokeratin IFs, NF’s function has remained obscure for many years. However, recently applications of molecular genetics and immunocytology / chemistry have determined a general function in determining axonal width. The shaky Quail phenotype was determined to be a natural knock-out of NF-L (2), which also resulted in 95% reduction of NF-M and NF-H expression. The morphological character was a greater distribution of axonal diameters which presumably affected neuronal transmission. Thus NF-L is associated with the development and maintenance of axonal width. The new interest in maintenance of the CNS increase the importance of NF as a cytoskeletal element that may have broad implications in the maintenance of axonal processes.

Regulation of Neurofilament Function
The dynamic nature of neurofilaments in the axon has been difficult to measure, mainly due to the lack of a system with which to assay dynamics. One point that is clear is that neurofilament proteins whether assembled or not are transported down the axons at the slow rate. As with many other IF proteins, phosphorylation is thought to be involved in regulating protein dynamics (3). NF-H is one of the most highly phosphorylated IFPs, this protein may have up to fifty phosphates bound to the carboxy-terminal. Differential phosphorylation may create aberrant structures containing IFPs which are associated with many pathological states (see below).

Medical Significance
As with other IFs, NF is expressed in a specific subset of cells in the mammal, the axonal cells. In tumor diagnosis it can act as a marker. Lewy bodies, which contain neurofilament protein, are pathological determinants of Parkinson’s disease. Whether the neurofilament proteins are the cause or consequence of the diseased state remains to be clarified. Amyotrophic lateral sclerosis is also associated with neurofilament tangles. Recent evidence suggests that the protein is denatured by general oxidative damage, which leads to accumulation of neurofilament aggregates (4). Also, it is known that a recombinant mouse strain that over expresses NF-H shows signs of amyotrophic lateral sclerosis-like plaques in the brain tissue (4).

References

1. Geisler, N. & Weber, K. 1981. J.Mol. Biol., 151, 565-571.

2. Ohara, O. et al. 1993. J. Cell Biol., 121, 387-395.

3. Gotow, T. et al. 1994. J. Cell Sci. 107, 1949-1957.

4. Brown R.H. Jr., 1995. Cell, 80, 687-692.

ABOUT VIMENTIN
Introduction
Vimentin is a type III intermediate filament of molecular weight 54 kDa (pI 5.3). Vimentin purified from native sources usually appears in several isoforms, this is thought to be the result of differential phosphorylation. Vimentin purified from overexpression of a recombinant clone in a bacterial system appears homogeneous as there is no phosphorylation. In vitro vimentin is soluble in the native form and insoluble in the denatured form. Polymerization to form IFs can proceed by the addition of NaCl to 150 mM final concentration.

Dynamics of vimentin
The dynamic nature of IFPs has been shown by injection of biotin labeled vimentin into tissue cultured cells and following their fate by immunofluoresence. The vimentin distributed from a starting point in the perinuclear region, to eventually migrate to the periphery of the cells (1). These studies underscore the importance of IF dynamicity in cells, which brings the IFs in line with actin and tubulin which also are dynamic.

Function of vimentin
Vimentin has been depleted from cells in tissue culture (2) and knocked-out from the mouse genome (3). Both systems support the fact that vimentin is not essential for growth, division or development. However, the vimentin depleted cells have a defect in lipoprotein / cholesterol metabolism.

Medical significance
One of the most common practical applications of vimentin research is the identification of neoplasms. There is an enormous amount of data concerning metastatic origins and IF markers that are currently used to identify many different tumors (see 4).

References

1. Vikstrom K.L., Borisy G.G. and Goldman R.D., 1989. Dynamic aspects of intermediate filament networks in BHK-21 cells. PNAS, 86, 549-553.

2. Sarria A.J., Sankhavaram R.P. and Evans R.M., 1992. A functional role for vimentin intermediate filaments in metabolism of lipoprotein derived cholesterol in human SW13 cells. J. Biol. Chem., 267, 19455-63.

3. Colucci-Guyon E., Portier M-M., Dunia I., Paulin D., Pournin S. and Babinet Ch., 1994. Mice lacking vimentin develop and reproduce without an obvious phenotype. Cell, 79, 679-94.

4. Osborn M. and Weber K., 1989. Cytoskeletal Proteins in Tumor Diagnosis, in Curr. Comm. in Mol. Biol. 1989. Cold Spring Harbor Press.

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