Lecture 17: Intermediate filaments and force resistance

Question 1

Describe an experiment to observe keratin assembly

Answer 1

• Monomeric type I keratin is purified n chemically labelled w biotin n microinjected into living epithelial cells
  ○ These cells hv a pre-existing IF network
  
  • After 20 min
    ○ Blobs of labelled keratin are observed
  • After 4h
    ○ Label incorporates into existing filaments
    CONCLUSION: Keratins can spontaneously assemble in vitro and can assemble into pre-existing intermediate filaments over a number of hours in vivo.

Question 2

What is the structural organization of intermediate filament (IF) proteins, and how do they assemble into mature IF structures?

Answer 2

• Defining feature of intermediate filament (IF) proteins is a 310-355 residue coiled-coil domain in each IF molecule which is flanked by N- and C-terminal blobs (characteristic of each class)
  
  • The base unit of the IF is a dimer where the 2 N-termini and C-termini are in close proximity and the coiled-coil region is wrapped around the other.
    ○ These dimers associate laterally with each other in an anti-parallel fashion and make end-on-end contacts with other dimers where the N- and C-termini of adjacent dimers interact.
    ○ This results in the formation of a proto-filaments. Tetramers of proto-filaments are twisted around each other to form the mature IF.
    Note that formation of the IF is due to the intrinsic properties of the IF proteins and do not require any nucleators or adaptors.

Question 3

What are the characteristics and functions of intermediate filaments in cells?

Answer 3

• Resist force
• Do not require a fuel source for assembly
• Form polymers that are extremely long
• Not regulated in space and time for assembly
• Less dynamic compared to microfilaments and microtubules
  Provide structural integrity to cell tissues