ANAT3231 Cell Biology Course - 2012 Lectures- currently under revision for 2013.

Cytoskeleton - Intermediate Filaments

Introduction

Intermediate filament organization

IF keratin in keratinocytes (tem)

This lecture introduces the intermediate filaments, their structure and function within the cell. Unlike the microfilament and microtubule systems, the filaments themselves consist of a wide variety of different proteins. These intermediate filaments have important structural roles in cell integrity both internally and through specialized cellular junctions that occur between cell-cell and cell-matrix which surrounds them. This topic will be addressed again when we look at the cell cytoskeleton and the extracellular matrix.

Objectives

Broad understanding of the cell cytoskeleton
Understanding of intermediate filament function
Understanding of intermediate filament structure
Understanding of intermediate filament proteins
Understanding of intermediate filament location in nucleus and cytoplasm
Understanding of intermediate filament adhesion junctions
Broad understanding of intermediate filament abnormalities

Structure

Physical Characteristics

10 nm diameter
Named by size relative to other cytoskeletal filaments
intermediate filaments have no structural polarity
Monomer - central α-helical domain
Dimer - 2 monomers form parallel coiled coil
Tetramer - pair of parallel dimers associates in an antiparallel staggered fashion
- tetramer is the soluble subunit (analogous to MT αβ-tubulin dimer, or MF actin monomer)
Provide rope-like resistance to mechanical stress
In muscle- link Z discs of adjacent myofibrils
Organization can be altered by phosphorylation

IF Types

Type I (n = 28)
- Acidic keratins (pI < 5.7) 40–64 kDa
  - K9-28 (epithelia)
  - K31-40 (hair/nail)
Type II (n = 26)
- Basic keratins (pI ≥ 6.0) 53–67 kDa
  - K1-8, K71-80 (epithelia)
  - K81-86 (hair/nail)

Keratins form heterodimers that assemble into heteropolymeric keratin filaments

Type III
- Desmin (cardiac, skeletal and smooth muscle)
- Vimentin (widespread distribution: leukocytes, blood vessels, endothelial, some epithelial and mesenchymal cells) 56 kDa
- Peripherin (neurons) 57 kDa
- Glial fibrillary acidic protein (GFAP) (astrocytes/glia) 50 kDa

Type III intermediate filament proteins can form both homo- and heteropolymeric filaments

Type IV
- Neurofilament Low NF-L (neurons) 62 kDa
- Neurofilament Medium NF-M (neurons) 110 kDa
- Neurofilament High NF-H (neurons) 130 kDa

Neurofilaments form heteropolymers

- α-internexin (CNS neurons)
- Synemins (muscle)
- Syncoilin (muscle)
- Nestin (stem cell marker) 220 kDa
Type V
- Lamin A/C (ubiquitous) 62–72 kDa
- Lamin B1/2 (ubiquitous) 65–68 kDa
Orphan
- Phakinin (lens)
- Filensin (lens)

Intermediate Filament Associated Protein (IFAP)

Cross-link intermediate filaments with one another
- forming a bundle (also called a tonofilament) or a network
IFAPs
- Plectin 500 kDa Striated muscle, epithelia Nuclear envelop
- Syncoilin 64 kDa Striated muscle
- Nesprin-3 117 kDa Kerotinocytes
- Paranemin 280 kDa
- Desmuslin 230 kDa

Function

found in some metazoans (vertebrates, nematodes, and molluscs)
Desmin interacts with nebulin linking intermediate filament network and sarcomeres at the Z-discs
Keratin filament formation originates mainly from sites close to the actin-rich cell cortex
2 alternate theories as to IF load/strain transmission
- entropic gels - where no individual intermediate filaments experiences direct loading in tension
- single intermediate filaments and bundles - extensible and elastic in vitro, and therefore well-suited to bearing tensional loads

Applications

Take advantage of the unique cell type expression pattern of IF proteins.

Nestin as a stem cell marker
GFAP can be used as an astrocyte marker in the analysis of neuronal tissue
Vimentin antibody can be used as a neural stem cell marker (ab45939)
Vimentin is highly expressed in fibroblasts and some expression in T- and B-lymphocytes. Expressed in many hormone-independent mammary carcinoma cell lines.

Abnormalities

Hutchinson-Gilford Progeria Syndrome

Cytoplasmic inclusions in several diseases Link
Hutchinson–Gilford progeria syndrome - Lamin A IF OMIM
Amyotrophic Lateral Sclerosis (ALS) - NF-H, peripherin OMIM
Alexander Disease - GFAP Genetics Home Reference OMIM
Charcot-Marie-Tooth disease - NF-L OMIM
Parkinson's Disease - NF-L, NF-M OMIM
Pachyonychia Congenita Genetics Home Reference
Giant Axonal Neuropathy Genetics Home Reference
Desminopathies desmin-related cardio-skeletal myopathy (CSM), also known as desmin-related myopathy (DRM)
- 3 cardiomyopathy forms – dilated, hypertrophic and restrictive cardiomyopathies PMCID: 2592258

Inherited Epidermolysis Bullosa (EB) Medline Plus EB classification

Is characterized by a marked mechanical fragility of epithelial tissues and the formation of blisters, erosions and poorly healing skin ulcers.

Represents four major groups of skin diseases:

Epidermolysis Bullosa Simplex (EBS) - mutations in KRT14 and KRT5 genes Genetics Home Reference
Junctional epidermolysis bullosa (JEB) (gene products involved: laminin V, integrin, collagen, not IF related) GeneReviews
Dominant dystrophic epidermolysis bullosa (DDEB) (a collagen mutation not IF related)
Recessive dystrophic epidermolysis bullosa (RDEB) (a collagen mutation not IF related)

Links: Human Intermediate Filament Mutation Database | Genes and Diseases | OMIM - Online Mendelian Inheritance in Man

History

Below are some example historical research finding related to cell junctions from the JCB Archive.

1968 [1] [2] Not actin, not myosin, but intermediate. They are neither thick nor thin: Howard Holtzer identifies intermediate filaments as a completely new kind of filament.

References

Textbooks

Essential Cell Biology

Chapter 19 Tissues Epithelial sheets and Cell-Cell Junctions p606

Molecular Biology of the Cell

Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter New York and London: Garland Science; c2002

Molecular Cell Biology

Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E. New York: W. H. Freeman & Co.; c1999

Molecular Cell Biology - Chapter 19 Cell Motility and Shape II: Microtubules and Intermediate Filaments
Intermediate Filaments
Figure 19-51. Levels of organization and assembly of intermediate filaments

The Cell- A Molecular Approach

Cooper, Geoffrey M. Sunderland (MA): Sinauer Associates, Inc.; c2000

The Cell - A Molecular Approach - III. Cell Structure and Function Chapter 11 The Cytoskeleton and Cell Movement
Intermediate Filaments
Signal Transduction and the Cytoskeleton

Search Online Textbooks

"intermediate filament" Molecular Biology of the Cell | Molecular Cell Biology | The Cell- A molecular Approach | Bookshelf

IF Database

Human Intermediate Filament Database

Books

PubMed

PubMed is a service of the U.S. National Library of Medicine that includes over 18 million citations from MEDLINE and other life science journals for biomedical articles back to 1948. PubMed includes links to full text articles and other related resources. PubMed

PubMed Central (PMC) is a free digital archive of biomedical and life sciences journal literature at the U.S. National Institutes of Health (NIH) in the National Library of Medicine (NLM) allowing all users free access to the material in PubMed Central. PMC

Online Mendelian Inheritance in Man (OMIM) is a comprehensive compendium of human genes and genetic phenotypes. The full-text, referenced overviews in OMIM contain information on all known mendelian disorders and over 12,000 genes. OMIM

Entrez is the integrated, text-based search and retrieval system used at NCBI for the major databases, including PubMed, Nucleotide and Protein Sequences, Protein Structures, Complete Genomes, Taxonomy, and others Entrez

Search Pubmed

"intermediate filament" Entrez all databases
"lamin" Entrez all databases
"desmosome" Entrez all databases

Reviews

Intermediate filaments mediate cytoskeletal crosstalk. Chang L, Goldman RD. Nat Rev Mol Cell Biol. 2004 Aug;5(8):601-13. Review. PMID: 15366704

Intermediate filament scaffolds fulfill mechanical, organizational, and signaling functions in the cytoplasm. Kim S, Coulombe PA. Genes Dev. 2007 Jul 1;21(13):1581-97. Review. PMID: 17606637

The desmosome and pemphigus. Waschke J. Histochem Cell Biol. 2008 Jul;130(1):21-54. Epub 2008 Apr 3. Review. PMID: 18386043

Intermediate filaments: versatile building blocks of cell structure Goldman RD, Grin B, Mendez MG, Kuczmarski ER. Curr Opin Cell Biol. 2008 Feb;20(1):28-34. Epub 2008 Jan 4. Review. PMID: 18178072

Electron microscopy of intermediate filaments: teaming up with atomic force and confocal laser scanning microscopy. Kreplak L, Richter K, Aebi U, Herrmann H. Methods Cell Biol. 2008;88:273-97. Review. PMID: 18617039

Oshima RG. Intermediate filaments: a historical perspective. Exp Cell Res. 2007 Jun 10;313(10):1981-94. Epub 2007 Apr 11. Review. PMID: 17493611

Fuchs E, Cleveland DW. A structural scaffolding of intermediate filaments in health and disease. Science. 1998 Jan 23;279(5350):514-9. Review. PMID: 9438837

Articles

Visualization of a system of filaments 7-10 nm thick in cultured cells of an epithelioid line (Pt K2) by immunofluorescence microscopy. Osborn M, Franke WW, Weber K. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2490-4. PMID: 329288

Different intermediate-sized filaments distinguished by immunofluorescence microscopy. Franke WW, Schmid E, Osborn M, Weber K. Proc Natl Acad Sci U S A. 1978 Oct;75(10):5034-8. PMID: 368806 PNAS