Lec 7-8 Flashcards
(38 cards)
Q: What is α-actinin-4 (ACTN4)?
A: An actin cross-linking protein critical for cytoskeletal structure and mechanics; implicated in podocyte function and kidney disease.
What are the three actin-binding sites on ACTN4?
ABS1, ABS2, and ABS3 within the CH1 and CH2 domains.
What is the normal conformation of ABS1 in ACTN4?
Typically buried in a “closed” conformation and becomes active in the “open” state.
What is ωr (relaxation frequency)?
The frequency at which G″ (loss modulus) peaks — reflects cross-linker unbinding rate and network relaxation.
lower = slower unbinding
What does the K255E mutation do?
Replaces lysine with glutamic acid → opens CH1-CH2 domains → exposes ABS1 → increases actin-binding affinity.
How does K255E affect actin networks?
Forms coarse, tightly bound networks with smaller mesh size; more brittle and rigid.
What does the QT>AA mutation do?
Disables ABS1, reducing actin-binding affinity and removing strain-stiffening response.
What happens when both K255E and QT>AA are present?
The hyper-affinity effect of K255E is neutralized; forms thick, unbranched bundles with little branching.
What happens to ωr in K255E networks?
Decreases — indicating prolonged cross-link binding and slower relaxation.
What happens to ωr in QT>AA networks?
Increases, indicating faster unbinding and weaker cross-linking.
How does cross-linker concentration affect ωr?
It does not change ωr. Relaxation frequency is a property of the linker, not its concentration.
Which ACTN4 isoform shows no stress-stiffening?
QT>AA — since ABS1 is necessary for strain-stiffening.
How does K255E affect stress-strain behavior?
Increases stress and work (~2.5x and ~5x respectively), decreases cell speed, increases stiffness.
What is the significance of ABS1 exposure in K255E?
It enables stronger, prolonged cross-linking — contributing to stiffness and pathological aggregates.
Why does WT ACTN4 stiffen under stress?
A: Stress may open CH domains, exposing ABS1 and enhancing binding affinity temporarily.
What does strain-stiffening require?
A: An active ABS1 domain — lost in QT>AA mutants.
What cellular effects are observed in K255E-expressing cells?
A: Larger cell area, slower motility, higher contractile stress and mechanical work.
How does vimentin affect cell mechanics?
A: Increases cytoplasmic stiffness and G′/G″, creating a more solid-like cytoplasm without significantly affecting the cortex.
What happens to particle movement in vimentin-deficient cells?
A: Cytoplasmic particles diffuse more freely — suggesting a softer interior.
(“Cells spread more on…”
“Stiffer substrates.”)
(“Cells exert larger contractile stress on…”
“Stiffer substrates.”)
(“On softer substrates cells… deform substrate”
more, less themselves
(“Focal adhesion size and density increase with…”
“Contractile stress.”)
Cell spread area and aspect ratio increases
(“Traction stress
