Intermediate Filaments Notes

Intermediate Filaments

Overview

  • Intermediate filaments (IFs) are strong, flexible polymers that provide mechanical support for metazoan cells.
  • They have a diameter of approximately 10 nm, which is intermediate between the diameters of actin (thin) and myosin (thick) filaments found in striated muscles.
  • Composed of various homologous proteins, but are not found in plants, fungi, or prokaryotes, with the exception of coiled-coil proteins in some bacterial species e.g. Caulobacter crescentus.

Structure and Composition

  • Building Blocks: Composed of intermediate filament proteins, forming a large and heterogeneous family.
  • Location:
    • Some IFs form a meshwork called the nuclear lamina beneath the inner nuclear membrane.
    • Other types extend across the cytoplasm.
  • Function: Provide mechanical strength to cells and strengthen epithelial tissues by spanning the cytoplasm from one cell-cell junction to another.
  • Arrangement:
    • Cytoplasmic IFs, particularly keratin filaments, cluster into wavy bundles, forming a branching network between the plasma membrane and the nucleus.

Intercellular Connections

  • Desmosomes: Anchor intermediate filaments to the plasma membrane, transmitting mechanical forces between adjacent cells.
  • Hemidesmosomes: Connect intermediate filaments across the plasma membrane to the extracellular matrix.
  • Continuum: The network of intermediate filaments and junctions prevents excessive stretching of cells and maintains the mechanical integrity of tissues like epithelia and heart muscle.

Diversity

  • Skin appendages, such as hair and whale baleen, demonstrate the flexibility and high tensile strength of crosslinked keratin intermediate filaments.
  • Defects in cytoplasmic IFs or associated junctions lead to rupture of skin cells and blistering diseases.
  • Defects in lamins, associated with the nuclear envelope, cause a range of diseases.

Molecular Structure of IF Proteins

  • Central Rod Domain: α-helical coiled-coil structure, approximately 310 residues long (46 nm).
    • Lamins have an additional 42 residues.
    • Contains heptad repeat patterns with hydrophobic residues at the first and fourth positions to facilitate interactions between α-helices.
    • Two highly conserved sites, L1 and L12, with interruptions in the coiled-coil.
    • Zones of positive and negative charge that provide electrostatic bonds for assembly into filaments. Approximately 20 conserved residues at each end are crucial for filament elongation via head-to-tail interactions of dimeric molecules.
  • End Domains: Variable N- and C-terminal domains ranging from 6 to 1200 residues. The N-terminal end (“head”) domains are essential for assembly, while the C-terminal end (“tail”) domains control filament diameter and interact with other cellular components.

Classification of Intermediate Filament Proteins

  • Based on rod domain sequences, IF proteins are grouped into five classes.

    • Class I & II: Acidic and basic keratins, found in epithelial cells and their appendages. Important for skin integrity, mutations lead to blistering skin, corneal dystrophy, and brittle hair and nails.
    • Class III:
      • Desmin: Found in muscle cells; mutations cause cardiac and skeletal myopathies.
      • GFAP (Glial Fibrillary Acidic Protein): Found in glial cells; associated with Alexander disease.
      • Peripherin: Found in peripheral neurons.
      • Vimentin: Found in mesenchymal cells. Null mutations are viable in mice.
      • Synemin: Interacts with other Class III IFs in muscle cells.
    • Class IV:
      • Neurofilaments (NFL, NFM, NFH): Obligate heteropolymers found in neurons. Mutations are a risk factor in amyotrophic lateral sclerosis.
      • Nestin and α-Internexin: Found in embryonic neurons.
    • Class V: Lamins: Found in metazoan nuclei; mutations result in cardiomyopathy, lipodystrophy, Emery-Dreifuss muscular dystrophy, progeria, and more.

Gene Evolution

  • IF protein genes are present in a wide range of organisms descended from the last common eukaryotic ancestor.
  • Animal IF genes evolved from nuclear lamin genes in early metazoan cells.
  • Cytoplasmic IF genes arose from a duplicated lamin gene in an invertebrate organism leading to chordates.
  • Orthologs are more similar than paralogs due to strong selective pressure.
  • Caulobacter crescentus has a coiled-coil protein, crescentin, with some IF protein features, but lacks conserved residues essential for filament elongation.

Filament Structure and Assembly

  • Filaments are about 10 nm in diameter with wavy profiles.
  • Head and tail domains project radially from the filament core (e.g., neurofilaments).
  • Assembly:
    • The building blocks are antiparallel complexes of two coiled-coil molecular dimers.
    • Assembly occurs via the lateral association of eight antiparallel dimers, forming unit-length filaments (ULF), which then longitudinally anneal into IFs.
    • Molecular dimers lack polarity, making IFs apolar.
    • Unit-length filaments are approximately 60 nm long.
  • IFs are more heterogeneous than actin filaments or microtubules due to the variable number of protofilaments.

Stability and Dynamics

  • IFs are insoluble under physiological conditions but can be dissociated in low ionic strength and high pH buffers.
  • Isolated subunits spontaneously repolymerize in minutes.
  • Growth occurs by longitudinal annealing of ULF at both ends, unlike actin filaments and microtubules that add single subunits at their ends.
  • No nucleotides or cofactors are needed for assembly.
  • Head domains are required for assembly, while tail domains are dispensable but influence lateral packing.
  • IFs are chemically stable, resisting extremes of temperature, salt, and detergents although subunits can exchange within minutes to hours during interphase.

Post-translational Modifications

  • Phosphorylation:
    • Affects polymer assembly and dynamics.
    • Multiple kinases phosphorylate different sites, with rapid turnover of phosphates.
    • The impact depends on the specific residue modified.
    • Mitotic kinase Cdk1(Cyclin-Dependent Kinase)-cyclin B phosphorylates sites flanking the rod domains of lamins, disrupting head-to-tail overlap needed for filament elongation and lateral association.
    • Vimentin filaments disassemble during mitosis, requiring coassembly with nestin. Keratin organization has subtle changes during mitosis.
    • Neurofilaments are stabilized by phosphorylation of their C-terminal tail end domain.
  • Other Modifications:
    • Keratin IFs in hair are crosslinked via disulfide and amide bonds, creating a tough composite material.

Expression in Specialized Cells

  • Animal cells express at least one of the three major nuclear lamins and vary in their expression of cytoplasmic IF proteins.
  • Most cells express predominantly one or two classes of cytoplasmic IF proteins.
  • Epithelial cells express class 1 and class 2 keratins, muscle cells express desmin, and mesenchymal cells express vimentin.
  • Some cells may express two types of IF proteins that sort into separate filaments. During tissue maturation, cells express a succession of IF isoforms.

Intermediate Filaments and Cancer

  • Tumors often express the IF protein characteristic of their cell of origin, aiding in the diagnosis of poorly differentiated or metastatic cancers. For example, tumors of muscle cells express desmin, while epithelial tumors express keratin, and mesenchymal tumors express vimentin.

Proteins Associated with Intermediate Filaments

  • Various proteins bind IFs, linking them to membranes and other cytoskeletal polymers.
  • Nuclear Envelope Transmembrane Proteins: Anchor nuclear lamins to the nuclear membrane.
  • Filaggrin: Mediates aggregation of keratin filaments in the upper layers of skin.
  • Plakins: Link cytoskeletal polymers to each other and to membranes.
    • Plectin: Crosslinks IFs to each other, actin filaments, and microtubules.
  • Desmoplakin: anchors keratin filaments to cadherins at desmosomes.
  • Lamin Associated Proteins: E.g. LAPI, LAP2, LBR, and Emerin link lamins to the nuclear envelope.

Desmosomes and Hemidesmosomes

  • Desmosomes
    • Transmembrane molecules of the desmosomes are cadherins and are calcium-dependent adhesion molecules.
    • Anchors keratin filaments to cadherins at desmosomes.
  • Hemidesmosomes: The transmembrane molecules include the integrin class of cell matrix receptors.

Functions of Intermediate Filaments in Cells

  • IFs act as flexible intracellular tendons, preventing excessive stretching of cells under physical forces.
  • Interactions with microtubules, actin filaments, and membranes complement this function.
  • Muscle Cells: Desmin filaments form a network between cytoplasmic dense bodies and the plasma membrane, transforming into a continuous strap upon stretching.
  • Z-disks: are surrounded by desmin filaments and are are involved in the transmission of force developed during muscle contraction.
  • Epithelial Cells: Keratin IFs form a dense network connected to desmosomes and hemidesmosomes, imparting mechanical stability. Failure of junctions or filaments leads to cell rupture and blistering.

Diseases and Mutations

  • Mutations affecting keratin IF assembly or junctions cause skin diseases like epidermolysis bullosa simplex.

  • Dominant Negative Mutations: Lead to imperfect assembly of defective keratin subunits with normal subunits, compromising filament integrity.

  • Defective keratin expression affects specific epithelial cells, such as basal cells (keratin 14 or keratin 5 mutations) or higher cell layers (keratin 10 or keratin 1 mutations).

  • Corneal mutations: Mutations in keratin 12 or keratin 3 cause sores on the cornea.

  • In contrast to dominant negative keratin mutations, loss of a keratin subunit by a null mutation can be less severe.

  • Desmin mutations may cause mildly disorganized muscle architecture, severe generalized muscle failure, or dilated cardiomyopathy.

Role in Nerve Cells

  • Neurofilaments expand axon diameter, enhancing electrical communication by increasing the velocity of action potentials.

Nuclear Lamins

  • Mutations in lamins disrupt lamin assembly, interfering with DNA replication and affecting interphase nucleus organization.
  • Mutations in the lamin A/C gene LMNA cause diverse diseases including progeria, Emery-Dreifuss muscular dystrophy, and disorders of fat tissue and nerves.

General Functions

  • Intermediate filaments provide mechanical support and maintain cell shape.

Classification

  1. Keratin filaments in epithelial cells
  2. Vimentin and vimentin-related filaments in connective-tissue cells, muscle cells and supporting cells of the nervous system (glial cells)
  3. Neurofilaments in nerve cells
  4. Nuclear lamina, which strengthen the nuclear membrane of all animal cells