Cytoskeleton and Intermediate Filaments

• The cytoskeleton is a network of structural proteins in all cell types.
• It facilitates cell signaling, vesicular transport, and cell motility.
• The cytoskeleton defines cell shape and the distribution of cellular contents.

Cytoskeleton Protein Functions

• Cytoskeletal proteins bind to targets (other proteins) to form polymers.
• Binding causes conformational changes in cytoskeletal proteins.
• Protein function depends on the number and type of bound cytoskeletal proteins.

Proteins in the Cytoskeleton

• Intermediate Filaments: Provide mechanical strength.
• Actin: Supports large-scale movements (motility and contractility).
• Microtubules: Support trafficking within cells.

Introduction to Intermediate Filaments

• Intermediate filaments are the strongest filaments, providing mechanical strength to resist shape changes.

Major Classes of Intermediate Filaments

• Acidic Keratins: In epithelial cells, provide tissue strength and integrity.
• Basic Keratins: In epithelial cells, provide tissue strength and integrity.
• Desmin, GFAP, vimentin, peripherin: In muscle, glial cells, mesenchymal cells, and peripherin neurons; aid in sarcomere organization and integrity.
• Neurofilaments: In neurons, aid in axon organization.
• Lamins: In the nucleus, aid in nuclear structure and organization.
• Nestin: In neurons, aids in axon growth.

Primary Structure of Intermediate Filaments

• Newly synthesized protein is a polymer of amino acids linked by peptide bonds.

Secondary Structure of Intermediate Filaments

• Intermediate filaments are rich in alpha-helices
• Alpha-helices are responsible for the long, coiled structure of filaments
• Hydrogen bonds stabilize the structure, resisting stretching and preventing collapse.

Tertiary and Quaternary Structure of Intermediate Filaments

• Monomer: Coiled monomer represents the tertiary structural level.
• Dimer: Two coiled monomers form a dimer, wrapping around each other in a coiled coil.
  • Coiled coil allows for maximum hydrogen bonding, giving the dimer strength.
  • Described as a quaternary structure.
• Tetramer: Two dimers assemble in an antiparallel staggered manner.
  • Increases hydrogen bonding and strength.
  • This tetramer is the fundamental building block of intermediate filaments.

Assembly of Intermediate Filaments

• Stage 1: Formation of a unit-length filament.
  • Formed by 8 tetramers coming together.
• Stage 2: Unit-length filaments form an immature filament.
  • These interact loosely end-to-end.
• Stage 3: Immature filament compacts to form a mature filament.
  • This is the final step to create a fully assembled intermediate filament.

Intermediate Filaments: Post-Translational Modifications

• Post-translational modifications control shape and function, usually on the head and tail domains.
• Phosphorylation: Dissolves intermediate filaments into unit-length filaments.
  • Removal by phosphatases causes the filaments to reform.
  • Assembly and disassembly is important during cell division.

Specialized Intermediate Filaments

• Lamins: Found solely in the nucleus; form the nuclear matrix to protect chromatin.
• Desmins: Connect different cellular structures; important for muscle structural integrity.
• Keratins: Bind to desmosomes, forming a complex; found in hair, skin, and nails.

Introduction to Microtubules

• Microtubules facilitate cellular trafficking.
• This includes the movement of proteins, vesicles, and some cellular organelles within the cytoplasm.

Microtubule-Organizing Centre

• Microtubule assembly occurs in microtubule-organizing centres (MTOCs).
• MTOCs are cellular structures from which microtubules arise.
• The centrosome, near the nucleus, is an example of an MTOC.
• During cell division it is copied so that the two resulting centrosomes can form the poles of the mitotic spindles.

Protein Structure of Microtubules

• Microtubules are made of tubulins, which are composed of dimerized proteins.
• Alpha-tubulin and beta-tubulin are globular proteins.
  • Bind tightly in a head-to-tail fashion to form a dimer.
• Both tubulin proteins bind to GTP molecules.
• Beta-tubulin can cleave GTP to GDP
• When bound to GDP, beta-tubulin has a shape change.

Microtubule Polymerization

• Dimers form Polymers: Dimers will spontaneously assemble into unstable polymers that quickly fall apart
• Polymer Growth: Once a polymer of at least six dimer subunits forms, it is more stable, and it may grow laterally and longitudinally. This is a protofilament.
• Protofilament Tubes: Eventually, protofilaments will form a sheet and will assemble into a tube of 13 protofilaments. This is the nucleation site for microtubule elongation.
• Assembly/Disassembly: At the ends of a microtubule, dimers continue to come and go.
• If the rate of assembly is greater than disassembly, the microtubule grows.

Microtubule Assembly, Disassembly, and Polarity

• Assembly:
  • Alpha-tubulin always has GTP bound to it.
  • Beta-tubulin may have either GTP or GDP.
  • GTP bound to beta-tubulin favors dimer polymerization.
  • Dimers will attach to each other.
• Disassembly:
  • When beta-tubulin’s GTP is hydrolyzed to GDP, the dimer undergoes a conformational change
  • Promotes depolymerization.
• Polarity:
  • Microtubules have different ends due to end-to-end polymerization of dimers.
  • Dimer binding is preferred at the plus end.
  • Microtubules extend from the plus end faster than the minus end.

Microtubule Dynamic Instability

• GTP Cap: A growing microtubule has a cap of GTP subunits at its tip.
• Hydrolysis: GTP hydrolysis occasionally exposes GDP-bound subunits at the tip.
• Depolymerization: Following hydrolysis, rapid catastrophic depolymerization occurs.
• Recap: Enough GTP subunits bind at once to recap the microtubule and stop depolymerization.
• Growth: Microtubule resumes growing when GTP-bound dimers are available.

Microtubule Dynamic Instability: Catastrophe

• Conversion of GTP to GDP on tubulin dimers causes rapid dimer detachment.
• This can initiate a catastrophe, rapid depolymerization of tubulin dimers at the plus end, shortening the microtubule.

Catastrophe Aversion

• Capping: Capping proteins bind to the plus end, stabilizing microtubules even in the GDP-bound form.
• Rescue: Spontaneous rescue can occur with enough GTP-bound dimers or with other proteins.

Motor Proteins

• Kinesins move along microtubules towards the plus end.
• Dyneins move to the minus end.
• Motor protein heads contain microtubule-binding domains.
• Tails bind to cargo that needs to be trafficked.
• Kinesin and dynein have two heads that "walk" along the microtubule, consuming ATP.

Walking of Motor Proteins

• Step 1: Head 1 is bound to the microtubule; head 2 is bound to ADP.
• Step 2: ATP binds to head 1, initiating walking-
• Causes a conformational change that includes head 2 swinging around.
• Step 3: Head 2 binds to the microtubule and releases ADP.
• Step 4: ATP at head 1 is hydrolyzed to ADP, causing head 1 to release from the microtubule.
• Step 5: The process repeats with ATP binding to head 2, causing head 1 to swing around.

Actin Filaments and Microtubules

• Composition: Both are composed of globular proteins.
• Movement: Motor proteins initiate movement along both.
• Network Formation:
  • Microtubules move cargo along a dynamic network.
• Actin forms a stronger network.
  • Contributes to both structure and large scale movements such as muscle contraction.

Actin Filaments

• Structure: Similar to a double coil.
  • Actin monomers bind both longitudinally and laterally.
• The combination of both longitudinal and lateral bonding gives high tensile strength
• Can withstand pulling forces that microtubules cannot.
• Polarization: Ends are not of the same charge
  • Positive end is the barbed end.
  • Negative end is the pointed end.
  • Barbed appearance is due to the association of other proteins such as myosin.

Actin Polymerization

• Actin monomers bind to nucleotide phosphates (ATP/ADP).
• ATP binding promotes assembly or polymerization.
• ADP binding discourages polymerization which may lead to filament disassembly.
• Preference for ATP replaces ADPs if there is a constant supply.

Stages of Actin Polymerization

• Stage 1: Nucleation
  • Two actin monomers can dimerize, but nucleation occurs when a third actin monomer binds to the dimer to form a nucleus trimer.
  • The trimer forms the core for the rest of the actin filament.
  • Structurally simpler than an MTOC, but serves the same purpose.
• Stage 2: Elongation
  • Actin monomers are added to the nucleus.
  • The actin filament is capable of elongating in both directions; polymerization is favoured at the plus end.
  • It is also a dynamic process which actin monomers being added and removed.
• Stage 3: Steady State
  • The rate of assembly equals the rate of disassembly and the net actin filament elongation ceases

Actin Treadmilling

• Favoured addition of monomers at one end with the same rate of monomer removal at the other.
• Keeps the actin filament the same length but can result in the filament moving within the cell.
• Regulation: Regulated by the ATP-actin concentration compared to the ADP-bound action.
  • The critical concentration of ATP-actin to polymerize is lower at the plus end than it is at the minus end.
  • If the ATP-actin concentration is just right, monomers are added to the plus end and removed from the minus end to cause treadmilling.
  • If the ATP-actin concentration increases above the critical concentration for the minus end, then actin monomers can again be added to that end.
• Treadmilling allows cells to rapidly adjust the actin cytoskeleton, much faster than intermediate filaments (require phosphorylation for disassembly).

Actin-Binding Proteins

• Monomer-Binding Proteins: Bind directly to actin monomers and influence polymerization.
• Nucleating Proteins: Bind to actin polymers to increase their stability and can allow for growth of a new branch.
• Capping Proteins: Bind to the plus or minus end and can stabilize the polymer to prevent disassembly and further assembly.
• Severing and Depolymerization Proteins: Bind to the actin polymer and sever or induce disassembly.
• Cross-Linking Proteins: Allow the side-to-side linkage of actin polymers to form bundles of actin filaments.
• Membrane Anchors: Link actin filaments to nonactin structural proteins.
• Actin-Binding Motor Proteins: Bind to the actin filament and allow movement.

Actin-Binding Motor Proteins

• All myosins are multi-subunit proteins.
• Subunits are light chains or heavy chains.
• Motor Domain: Formed by the heavy chain and binds to the actin filament and ATP.
• Regulatory Domain: Formed by one heavy chain and two light chains.
  • Moves back and forth as the myosin moves along an actin filament.
• Tail Domain: Each tail domain binds to other cellular proteins or other myosins.

Movement of Actin-Binding Motor Proteins

• Hydrolysis: With ATP bound to the motor domain, the myosin is unbound to the actin filament.
  • Hydrolysis of the ATP to ADP and inorganic phosphatase causes a conformational shift in the regulatory domain, swinging it like a lever.
• Actin Binds: The motor domain then binds to the actin filament.
  • The inorganic phosphate is released from the myosin, causing another conformational change and pulling the myosin along the actin filament.
• The ADP is then released and the binding of new ATP causes the myosin to unbind from the actin filament.
• Movement: In most instances, myosin moves towards the barbed (positive) end of the actin filament.