Lecture 20: Intermediate Filaments Part I

Are intermediate filaments stable?

yes, they persist for long periods of time

Vertebrates have how many genes encoding 6 classes of IF proteins?

70 genes

Which IF is expressed in muscle?

desmin

Which IF is expressed in GFAP?

glia

Which IF is expressed in neurons?

NFs

Which IFs are expressed in epithelial cells?

keratins

True or False: IFs typically constitute 1% of cellular proteins, but there are some types of cells that can IFs can be 85% of total proteins.

true

What is the structure of IFs?

• Head region

• Tail region

• Middle region that connects head and tail

What else is important to know about IFs?

• Don’t bind nucleotides

• Doesn’t hydrolyze any nucleotide/use energy

• Is an equilibrium reaction

What is important to know about the evolution of the IF superfamily?

• Lamins were the progenitors

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

• Lamins lost NLS and prenylation region (membrane attachment) resulting in cytoplasmic localization

• Cytoplasmic lamins formed several families each with specific functions: skin, muscles, nerves

What is important to know about alpha-helical coiled coils?

• Alpha helices wrap around each other and form stable dimers

• Every 7 amino acids, there are 2 amino acids from the different helices that face each other and often hydrophobic

  • 2 sections bind together tightly

How can one determine the orientation of monomers in dimers and tetramers?

through immunogold electron microscopy

What occurs during immunogold electron microscopy?

• Antibody binds to head regions of intermediate filaments

• Protein A binds to tails of antibodies and attaches gold bead to the antibody

• This approach only works if N- and C-termini are quite different in size

What the structure of a polymer?

• Protofilaments are many tetramers stacked end-to-end

• Proto-fibrils are 4 protofilaments

• Intermediates are 4 proto-fibrils

Are intermediates/polymers polar?

no, they don’t have any directionality

Do filaments recover after photobleaching?

yes, they do

How are intermediate filaments assembly regulated in vivo?

• Not known if there are accessory proteins

• No additional proteins needed in vitro

• Most cells produce only 1-2 types

• In cells with 2 types, the 2 types can independently assemble in vitro

What happens if antibodies are microinjected into tissue culture cells?

the filaments become disrupted but the cell is alright

What effect would a LOF allele have in an otherwise wild-type cell?

no LOF phenotype would be seen because LOF is typically recessive

What is important to know about dominant negative mutations?

• Rare type of mutation

• Dominant so that only one copy is enough to display mutant phenotype

• Dominant negatives phenotypes are similar to null phenotypes

• Negative means LOF mutation

• Almost all DNMs act by a “poisoned polymer” model

What is the poisoned polymer model?

dominant negative mutations are chain terminating subunits—meaning adding a dominant negative mutation will not allow any more intermediate filament subunits to be added

What happens if there’s a K14 dominant negative mutation in a transgenic animal?

• Filament assembly is blocked in basal layer

• Skin blisters due to weakening of the basal cells

What do cytoplasmic intermediate filaments do?

attach to transmembrane proteins—allowing cells to attach to each other

Why are cytoplasmic IFs so important in the skin?

Keratins give skin cells mechanical integrity and continuity to serve as a barrier

What are some diseases caused by keratin mutations?

• Epidermolysis bullosa simplex

• Epidermolytic hyperkeratosis

• Epidermolutic palmoplantar keratoderma

• Pachyonychia congenita

• White sponge nevus

• Meesmann’s corneal dystrophy

• Monilethrix 

What does a desmin mutation in humans cause?

disorganization of myofibrils and generalized muscle failure

What does desmin do?

ties Z-disks from one myofibril to another myofibril

What do neurofilaments do?

increase the diameter of the axon and the rate of the electrical signal