Microfilaments and F-actin

Microfilaments

• Microfilaments are made of actin
• Microfilaments exist as filamentous actin (F-actin)
• F-actin is a helical, linear protein
• F-actin consists of G-actin molecules
• G-actin molecules’ orientation differs between the positive and negative ends
• The positive end has ATP-bound G-actin
• The negative end has ADP-bound G-actin

Microfilament Synthesis in Vitro

• Synthesized via nucleation, elongation, and steady state
• Synthesis requires high concentration of ATP-G-actin
• Synthesis requires critical concentration

Critical Concentration of Microfilaments

• The concentration of ATP-G-actin for equal F-actin assembly and disassembly rates
• F-actin length remains constant at this concentration
• F-actin elongates when the ATP-G-actin concentration is above the critical concentration
• F-actin shortens when the ATP-G-actin concentration is below the critical concentration

F-actin Ends

• The critical concentration is lower at the positive end than at the negative end
• With certain conditions, the concentration of ATP-G-actin can fall between each end's critical concentrations, leading to treadmilling

Treadmilling

• Microfilaments appear to be in a steady state with actin monomers adding to the plus end and removing at the minus end
• Treadmilling is a phenomenon where the filament gives the illusion of movement, even though the filament itself doesn't change in length
• The cycling of G-actin molecules from the negative end of F-actin to the positive end
• Occurs when ATP-G-actin concentration falls between the critical concentrations of the positive and negative ends
• Depolymerization occurs at the negative end
• Polymerization occurs at the positive end
• Profilin and cofilin affect treadmilling in vivo
• Capping proteins affect treadmilling in vivo

Nucleation

• Nucleation forms a small stable cluster (the nucleus) of actin monomers
• Nucleation is the starting point for filament growth
• Without nucleation, no filament growth can occur because there would be no “seed” for elongation to build upon

Formin

• A protein that enables the synthesis of linear actin filaments

Arp2/3

• A protein that enables the synthesis of branched actin networks

Microtubules

• Hollow, cylindrical structures found throughout the cell
• Stiffer than microfilaments and intermediate filaments
• Resist compression, but become flexible when present as long tubules
• Support cell structure
• Act as tracks for movement of cellular content

Tubulin

• Microtubules are made of α-tubulin and β-tubulin heterodimers
• One molecule of GTP can bind to each molecule of α-tubulin
• GTP cannot be hydrolyzed once bound to α-tubulin
• One molecule of GTP can bind to each molecule of β-tubulin
• This GTP can only be hydrolyzed once the αβ-tubulin subunit is incorporated into the microtubule
• αβ-tubulin can only be incorporated into the microtubule if both subunits are bound to GTP
• αβ-tubulin forms linear polymers called protofilaments
• Protofilaments associate laterally to form cylindrical, hollow tubes called microtubules
• The GTP bound to the α-subunit is stable
• The GTP bound to the β-subunit can be hydrolyzed to GDP
• α- and β-tubulin form heterodimers
• These α/β-tubulin dimers associate with each other to form linear filaments, known as protofilaments
• The α/β-tubulin dimers are oriented in the same direction
• There are alternating α- and β-subunits throughout each protofilament
• There is polarity to this protein, with an α-subunit on one end and a β-subunit on the opposite end
• Each microtubule consists of 13 protofilaments
• 13 protofilaments associate with one another along the longitudinal axis of the tubule
• The α- and β-subunits are roughly aligned with those of their neighbors
• The alignment of the protofilaments provides polarity to the microtubule
• The positive end contains β-subunits, and the negative end contains α-subunits

Microtubule Synthesis in Vitro

• Microtubules spontaneously assemble in vitro from αβ-tubulin dimers, similarly to microfilaments
• Synthesis happens with GTP-bound αβ-tubulin dimers are greater than the critical concentration
• Polymerization occurs faster at the positive end than the negative end
• Microtubules go through growth, disassembly and re-growth
• In dynamic instability, some microtubules grow while others shrink via catastrophe
• The process of alternating between growing and shrinking is dynamic instability
• Microtubules undergoing catastrophe can also be rescued and begin to polymerize again

Conditions for Dynamic Instability

• Dynamic instability occurs even when the GTP-tubulin concentration is greater than the critical concentration of each microtubule end
• The positive end terminates in a GTP-α/GTP-β tubulin cap.
• Underneath the cap, the GTPase within β-tubulin is active and generates GTP-α/GDP-β tubulin dimers.
• Loss of the GTP cap results in catastrophe.
• The GTP cap keeps the microtubule stable and promotes growth at the positive end.
• Loss of the cap = instability and rapid shrinkage at the positive end or catastrophe
• αβ-tubulin dimers must contain GTP bound to both subunits to be added to the microtubule
• Terminal β-subunits have essentially negligible rates of GTP hydrolysis
• A new αβ-tubulin dimer added to a growing microtubule allows the GTP to be hydrolyzed to GDP within the formerly terminal β-tubulin subunit
• The result of GTP hydrolysis is in a cap of GTP-bound β-subunits end of each microtubule
• The central region consists of GDP-bound β-subunits
• GDP bound β-tubulin subunits are unstable
• If the terminal β-subunits’ GTP cap is removed, catastrophe and rapid disassembly occur
• Regions of GTP bound β-tubulin subunits are located within the central region
• Catastrophe ceases once the GTP bound region is reached, allowing rescue

MTOC

• Microtubule Organizing Centres
• Microtubules extend outward from MTOC, extend and retract until they reach the appropriate target
• Attachment between microtubule and target stabilizes microtubule
• Contain proteins that facilitate nucleation
• Nucleation occurs in the centrosome
• The y-tubulin ring complex primarily achieves nucleation in the centrosome
• The y-tubulin ring complex serves as a template for microtubule synthesis
• Microtubules elongate at the positive end
• The negative end is typically anchored to the MTOC
• Microtubule-organizing enters (MTOCs) mediate nucleation
• In animal cells, the centrosome serves as the primary MTOC
• Cilia and flagella use basal bodies as their MTOCs

Microtubules vs. Microfilaments – Similarities

• Both microfilaments and microtubules are polarized structures
• Growth occurs faster on the positive end than the negative end
• Each end has a critical concentration
• Treadmilling can occur in both structures
• Nucleoside triphosphates must be present on ‘fundamental subunits’ for polymerization to occur (ATP/GTP)
• The nucleation step is rate-limiting in the synthesis of both
• Both provide structural support and tracks for movements

Microtubules vs Microfilaments – Differences

• GTP binds to αβ-tubulin, and ATP binds to actin
• Microfilaments polymerize through the addition of actin monomers
• Microtubules polymerize through the addition of αβ-tubulin dimers
• Microtubules are hollow, and microfilaments are solid
• Only microtubules go through dynamic instability

Microtubule Associated Proteins

• Interact with microtubules to regulate stability and growth
• Stabilizing proteins enhance stability
• Stabilizing proteins reduce frequency of catastrophe
• Stabilizing proteins link microtubules, microfilaments, chromosomes, and increase growth rate
• Destabilizing proteins enhance disassembly
• Destabilizing proteins enhance frequency of catastrophe
• Alter stability, ability to associate with motor proteins
• Motor proteins rely on microtubules to transport cellular cargo
• Kinesins and dyneins are two types of motor proteins for microtubules
• Kinesins move cargo toward the plus end
• Dyneins move cargo toward the minus end

Motor Proteins: Kinesins and Dyneins – Similarities

• Both transport cargo using microtubules as a track
• Both use ATP to power movements

Motor Proteins: Kinesins and Dyneins – Differences

• Kinesins move cargo toward the positive end, and dyneins move towards the negative end
• Dyneins require an intermediate protein to interact with cargo, whereas kinesins can directly interact with cargo

Kinesin

• Made of heavy and light chains
• Heavy chains contain a head that binds to ATP and the microtubules and contains ATPase
• Heavy chains contain a tail that binds to light chains and cargo
• Kinesin moves along a single protofilament within the microtubule
• ATP hydrolysis fuels this process

Dynein

• Requires linker proteins
• Dynein goes toward the negative end while kinesin moves toward the positive end
• An important aspect is that this allows for contents to be transported to different regions of a cell from our away from the centrosome, based on motor protein type
• Dynein cannot interact directly with cargo while kinesins can