Cytoskeletal Filaments and Polymerization

• BIOL 362 Cellular Dynamics, Module 1-2: Cytoskeletal Dynamics II was held on January 14, 2025.
• The lecture includes topics about:
  • The cytoskeleton in general
  • Properties of F-actin and microtubule dynamics
  • Actin filament dynamics in vitro: treadmilling (review)
  • Microtubule dynamics in vitro: dynamic instability
  • Accessory proteins that control filament dynamics in vivo
  • Drugs that inhibit cytoskeletal dynamics
• Relevant reading includes MBoC (7th edition): Chapter 16

Learning Goals

• Employ a simple equation to predict cytoskeletal dynamics
• Understand the concept of dynamic instability during microtubule assembly

Actin Filament Dynamics

• Actin polymerization follows a sigmoidal curve, including nucleation (lag phase), elongation (growth phase), and steady state (equilibrium phase).
• Adding actin oligomers accelerates polymerization by bypassing the lag phase.
• Elongation rate is defined by equation: dn/dt = kon[free subunit] – koff
  • kon: rate constant of association
  • koff: rate constant of dissociation
  • n refers to the filament mass.
  • Incorporation depends on free subunit due to interaction frequency between filament ends.
  • Elongation rate differs from polymerization rate: elongation rate can be negative.
  • Incorporation rate equals the polymerization rate.
  • Dissociation rate equals the depolymerization rate.
• Critical concentration refers to the subunit concentration at a steady state where net incorporation and dissociation are balanced.
• At steady state, dn/dt = 0, resulting in kon[subunit] = koff.
• [Subunit] = koff/kon = Critical Concentration
• Only the addition fo certain proteins effects the critical concentration
• It is possible to predict polymer dynamics when the critical concentration (Cc) is known.
  • If [subunit] = Cc, the system is at equilibrium.
  • If [subunit] > Cc, the polymer will grow.
  • If [subunit] < Cc, the polymer will shrink.
• More intuitive mathematical explanation
  • The rate of change is also equal to to kon[subunit] - koff which is a linear equation

Critical Concentrations at Filament Ends and Treadmilling

• Critical concentration can be defined for understanding polymer dynamics in detail for each filament end.
• Elongation rate at the minus end: dn-/dt = kon-[subunit] – koff-
  • At equilibrium: Cc- = koff-/kon-
• Elongation rate at the plus end: dn+/dt = kon+[subunit] – koff+
  • At equilibrium: Cc+ = koff+/kon+
• Differences in Cc between plus and minus ends create a new state: treadmilling, a state of dynamic equilibrium where one end grows while the other shrinks.
  • If [G-actin] < all Ccs, both ends shrink.
  • If [G-actin] > all Ccs, both ends grow.
  • If Cc+ < [G-actin] < Cc-, the plus ends grow and the minus ends shrink.

Microtubule Dynamics

• Microtubule dynamics in vitro are also predictable when Cc is known.
  • If [free subunit] = Cc, there is no net growth.
  • If [free subunit] > Cc, polymers grow.
  • If [free subunit] < Cc, polymers shrink.
• Bulk assembly theories have existed for a long time
  • In order to infer the bulk assembly of filaments
  • Mass equals number x length
  • Individual microtubules, however, exhibit unexpected behaviors

Individual Microtubule Dynamics

• MT dilution assay involves diluting tubulin.
• In 1984, Mitchison and Kirschner performed a MT dilution assay to determine the number of MTs after dilution.
• Surprisingly, microtubules kept growing after the subunit dilution below Cc.
• Individual MTs are either growing or shrinking (occurs randomly)
• A microtubule keeps growing until it randomly transitions into the shrinking phase.
• Time-lapse imaging shows individual MT dynamics at a steady state
• GTP hydrolysis serves as a switch between growing and shrinking phases.

Dynamic Instability

• In dynamic instability:
  • Rapid growth occurs with a GTP-capped end.
  • Random loss of the GTP cap leads to catastrophe, promoting rapid shrinkage.
  • Regaining the GTP cap induces "rescue"
• Catastrophe is triggered by a microtubule lattice conformational change.

Summary

• Understanding bulk assembly and individual filament assembly are important to understand filament dynamics.
• Microtubules repeat between growth and shrinkage
• This transition is called catastrophe and rescue

Practice Questions

• Indicate if each of the following structures is based on actin filaments (A), microtubules (M), or intermediate filaments (I). Put your answer in four letters composed of A, M, and I.
  • Cell cortex (Actin filaments)
  • Mitotic spindle (Microtubules)
  • Nuclear lamina (Intermediate filaments)
  • Thin filaments (Actin filaments)
• Indicate true (T) and false (F) statements below regarding the cell cytoskeleton.
• The major building blocks of cytoskeletal filaments can bind to and hydrolyze nucleotides. (False)
• The building blocks of actin filaments and microtubules are globular proteins, whereas those of intermediate filaments are themselves filamentous proteins. (True)
• Intermediate filaments provide mechanical support to the cell. (True)
• Plant cells lack microtubules. (False)
• Which of the following events corresponds to the "lag phase" of the filament growth curve during in vitro polymerization? Nucleation
• Which of the time courses of seeded actin polymerization under two different conditions corresponds to a higher C for polymerization? (2), higher koff rate constant? (2)
• If the concentration of free subunits is C, under which condition does the growth of a cytoskeletal filament proceed spontaneously? C>koff/kon
• Question 6: Slope of the line on question 6 is kon
• According to the provided graph, at which concentration (A to E) does the total length of the filament remain more or less constant with time? B

Explore the dynamics of cytoskeletal filaments, focusing on elongation rates and factors influencing polymerization. Understand the role of actin oligomers and the impact of technological advancements on microtubule discovery. Learn about key scientists and their contributions to cell biology.