MCB 252 - Topic 8: Intermediate Filaments

Intermediate Filaments (IFs)

Learning Objectives

  • Understand the stability of intermediate filaments relative to actin filaments.

  • Describe the different IF classes and their localization.

  • Discuss the evolution of the IF superfamily.

  • Understand the size of the IF superfamily relative to cell types in the human body.

  • Describe IF structure.

  • Explain the process and goal of immune-gold electron microscopy (EM).

  • Discuss IF dynamics and turnover in vivo.

  • Describe the regulation of IF assembly in vivo and in vitro.

  • Explain dominant negative mutations and their use in understanding IF function.

  • Describe the poison polymer model and its relation to IF function.

  • Discuss the role of desmin in skeletal muscles.

  • Discuss the role of neurofilaments in neuronal biology.

  • Explain the evolution of keratins relative to animal evolution.

  • Discuss the role of IFs in human evolution and migration, including genes acquired from Neanderthals.

Reading Material

  • Lodish section 18.7, pp. 861-867.

  • Genetic Analysis Lodish, pp. 228-229.

Overview

  • Intermediate filaments (IFs) are a key component of the cytoskeleton.

  • Topics covered:

    • Classes of IFs

    • Structure of monomers and polymers

    • Dynamics

    • Functions

IF Classes and Distribution

Class

IF Type

Distribution

I

Acidic keratin

Epithelial cells

II

Basic keratin

Epithelial cells

III

Desmin, Vimentin, GFAP

Muscle, mesenchymal cells, glia

IV

Neurofilaments (NF-L, NF-M, NF-H)

Neurons

V

Lamins

Animal and plant nuclei

VI

Nestin

Embryonic neurons

  • Cytoplasmic vs. Nuclear IFs: IFs can be found in both the cytoplasm and the nucleus (Lamins).

  • Vertebrates have approximately 70 genes encoding 6 classes of IF proteins.

  • IF proteins are differentially expressed in nearly all cells of the body.

    • Desmin: muscle

    • GFAP: glia

    • NFs: neurons

    • Keratins: epithelial cells

  • IFs generally constitute ~1% of cellular proteins, but can be up to ~85% in certain cell types like neurons and epidermal keratinocytes.

  • IF expression is highly specific.

Intermediate Filament Structure

  • IFs do not bind nucleotides.

  • They contain a coiled-coil region.

Evolution of IF Superfamily

  • Lamins are considered the progenitors of IFs.

  • Cytoplasmic IFs arose in animals after branching off from plants.

  • Lamins lost the Nuclear Localization Signal (NLS) and prenylation region (required for membrane attachment), leading to cytoplasmic localization.

  • Cytoplasmic lamins formed several families, each with specific functions (e.g., skin, muscles, nerves).

  • Alpha-Helical Coiled Coil: Characteristic structural motif.

Determining Monomer Orientation

  • Method 1 (if N- and C-termini are quite different in size):

    • This approach relies on size differences between the N- and C-termini to determine monomer orientation in dimers and tetramers.

  • Method 2 (if N- and C-termini are similar in size):

    • Use immunogold electron microscopy to determine the orientation of monomers in dimers and tetramers.

Polymer Structure

  • Proto-filament: many tetramers stacked end-to-end (head-to-tail).

  • 4 Protofilaments = 1 proto-fibril.

  • 4 Proto-fibrils = 1 Intermediate filament.

IF Dynamics

  • Fluorescence Recovery After Photobleaching (FRAP):

    • Technique used to study IF dynamics.

    • GFP-vimentin is photobleached and recovery is observed.

  • Microinject biotin-labeled keratin into cells to observe dynamics.

Regulation of IF Assembly In Vivo

  • It is not known if there are accessory proteins.

  • No additional proteins are needed in vitro for IF assembly.

  • Most cells produce only 1-2 types of IFs.

  • If a cell produces 2 types, they can independently assemble in vitro showing self-assembly properties.

Functional Studies

  • Microinject antibodies that block assembly into tissue culture cells.

  • Disruption of filaments doesn't kill cells.

  • Some organisms (e.g., insects) don’t have cytoplasmic IFs which suggests IFs aren't absolutely essential for cell survival under all conditions.

Keratin Expression in Skin Epithelium

  • Skin Layers:

    • Stratum corneum (surface)

    • Granular

    • Spinous (K1/K10)

    • Basal (K5/K14)

    • Dermis

Thinking About Genetics

  • What effect would a Loss-of-Function allele have in an otherwise wild-type cell?

Transgenic Organisms & Dominant Negative Mutations

  • How can we make a transgenic organism that doesn’t make Keratin filaments?

  • How can we make a transgenic organism (with a dominant allele) that results in a loss of function phenotype?

  • Dominant Negative Mutations:

    • A relatively rare type of mutation.

    • Only one copy is enough to display the mutant phenotype (dominant).

    • The phenotype is similar to the null phenotype rather than being opposite to it.

    • Produce mutant phenotype in cells carrying a wild-type copy (allele) of the gene.

    • The dominant negative phenotype is similar to the loss-of-function mutant phenotype.

    • The phenotype results from the dominant negative form of the protein interfering with the wild-type version, e.g., by preventing assembly of multimers.

  • Poisoned polymer model:

    • Wild-type protein is rendered non-functional due to the presence of a dominant negative mutant.

Dominant Negative Keratin Mutation

  • K14 mutant blocks filament assembly in the basal layer.

  • This results in skin blisters due to weakening of basal cells.

Cytoplasmic Intermediate Filaments: Keratins

  • Keratins give skin cells mechanical integrity and continuity, serving as a barrier.

Effects of Keratin Mutations on Skin

  • Autosomal dominant mutations and null mutations can both lead to mechanical stress and lysis of cells

  • Hyperkeratosis: Thickening of the stratum corneum (e.g., K10 mutant).

Keratin Mutations in Human Disease

Disorder

Mutant gene

Cells involved

Epidermolysis bullosa simplex

K5, K14

basal epidermis

Epidermolytic hyperkeratosis

K1, K10

spinous layer

Epidermolytic palmoplantar keratoderma

K9

spinous layer, thick skin

Pachyonychia congenita

K6, 16, 17

nails, hair

White sponge nevus

K4, K13

esophagus, oral epithelia

Meesmann's corneal dystrophy

K3, K12

corneal epithelia

Monilethrix

K Hb6

hair

Desmin Localization in Skeletal Muscle

  • Desmin mutations in humans lead to disorganization of myofibrils and generalized muscle failure.

IFs in Axons = Neurofilaments

  • Neurofilaments increase the diameter of the axon and the rate of electrical signal transmission.

Evolution of Keratins

  • The keratin family seems to have arisen around the time animals with soft exteriors appeared (animals lacking an exoskeleton).

Roles of Intermediate Filaments

  • Distribute tensile forces across cells in tissues.

  • Integrate cells into tissues.

Intermediate Filaments and Human History

  • Related to human evolution and migration.

Neanderthal-Human Interactions

  • Neanderthal-Human divergence occurred ~600,000 years ago.

  • Modern Humans migrated out of Africa ~100,000 years ago.

  • Humans interbred with Neanderthals ~50,000-60,000 years ago in the Middle East.

  • Modern Humans migrated to Europe and Northern Asia ~40,000-45,000 years ago.

  • Neanderthals died out ~30,000 years ago.

Sequencing Neanderthal Genomes

  • The complete genome sequence of a Neanderthal was obtained from the Altai Mountains (Nature, 2014).

  • Research focuses on resurrecting surviving Neanderthal lineages from modern human genomes (Science, 2014).

  • Svante Pääbo won the 2022 Nobel Prize in Medicine or Physiology for his work in this area.

Human DNA Derived from Neanderthals in Non-Africans

  1. BNC2: a zinc finger protein associated with skin pigmentation.

  2. POUF2F3: a transcription factor expressed in the epidermis that mediates keratinocyte proliferation and development.

  3. A cluster of keratin genes.