Cytoskeletal Filaments: Microtubules and Actin Filaments

Cytoskeletal Filaments: Microtubules

• Microtubules transport materials within the cell and are crucial for the cell's structure and function.

• They maintain cell shape and are dynamic, unlike intermediate filaments.

Microtubule Structure

• Microtubules consist of tubulin dimers, each with an alpha and a beta subunit.

• Alpha and beta subunits are globular proteins.

• Subunits interact to form a protofilament (alpha, beta, alpha, beta stacking).

• Polarity: Microtubules have distinct ends.

  • The positive end is associated with the beta monomer.

  • The negative end is associated with the alpha monomer.

• Interactions are noncovalent, but numerous interactions make them strong.

• A complete microtubule comprises 13 protofilaments arranged around a central core.

• Noncovalent interactions occur vertically (between alpha and beta subunits) and horizontally (between protofilaments).

• This creates a tube-like structure.

Microtubule Organization

• Microtubules originate from specific cell regions, notably the centrosome.

• The centrosome contains two pairs of centrioles and is vital for cell division.

• The beta end is positive, and the alpha end is negative due to polarity, not charge differences.

Microtubule Directionality

• Directionality is essential for intracellular movement.

Microtubule Nucleation

• Microtubule assembly requires nucleation, which is the starting point for microtubule growth.

• Nucleation occurs at the centrosome.

• Microtubules begin with a gamma tubulin ring complex, comprising 13 alpha and beta monomers arranged in a circle.

• Dimers are added to gamma tubulin to extend the microtubule.

• Gamma tubulin rings speed up nucleation inside the cell.

• Microtubules grow from the negative end towards the positive end.

• The positive ends extend into the cell, while the negative ends remain within the centrosome.

• Centrosomes are surrounded by a protein-rich, gel-like matrix containing gamma tubulin structures.

Other Nucleation Centers

• Cells can have multiple nucleation centers, contingent on cell type and function.

• In polarized cells (e.g., intestinal epithelial cells), nucleation centers are near the apical side.

• In these cells, positive ends of microtubules extend toward the basolateral side.

• Neurons have nucleation centers throughout the cell.

• In yeast, nucleation occurs at the nuclear envelope, from which microtubules extend.

Microtubule Dynamics

• Microtubules are dynamic; they continuously grow from and degrade into the nucleation center.

• Growth and degradation occur independently.

• Microtubules explore the cell to fulfill biological functions or extend as the cell expands.

Microtubule Instability

• Tubulin dimers are GTP-binding proteins; GTP binding stabilizes the structure.

• Rapidly growing microtubules develop a GTP cap, which prevents degradation because dimers quickly add to the structure and have a lot of GTP bound dimers that are present.

• When GTP is hydrolyzed to GDP, the structure becomes unstable.

• The rate of growth determines stability.

• Faster growth maintains the GTP cap, preventing hydrolysis.

• Slower growth leads to GTP hydrolysis, destabilizing the structure and causing disassembly.

• This process is spontaneous but is regulated by microtubule-associated proteins inside the cell. And in a test tube, this occurs perfectly, inside a cell, there are other proteins involved.

• GTP-binding proteins eventually hydrolyze GTP.

Microtubule-Associated Proteins

• A pool of alpha-beta dimers exists within the cell, available for microtubule assembly.

• Proteins facilitate nucleation by forming the gamma tubulin ring.

  • Branching proteins: direct microtubule branching (e.g., Augmin) creating tree-like structures.

  • Positive end-binding proteins: enable microtubules to interact with other cell structures.

  • Polymerizing proteins: accelerate microtubule elongation, acting predominantly at the positive end.

  • Note: Tubulin dimers can add to either end, but they add to the positive end more quickly.

  • Stabilizing proteins: reduce GTP hydrolysis, promoting microtubule stability.

  • Severing proteins: break microtubules.

Microtubules as Cellular Highways

• Microtubules act as cellular highways for transporting materials, such as in neurons.

• The negative end faces the nucleus, and the positive end faces the plasma membrane.

• Transport occurs from the negative to the positive end and vice versa.

• Motor proteins facilitate movement along microtubules.

• This transport system is necessary because the cytoplasm is densely packed, hindering free diffusion.

• All eukaryotic cells use this system.

Motor Proteins

• Motor proteins, like kinesins and dyneins, share a similar structure with globular heads and tails.

• Kinesins move towards the plus end (away from the nucleus).

• Dyneins move towards the minus end (towards the nucleus).

• Tails bind to different cellular structures, dictating cargo.

• They transport organelles and macromolecules.

• Motor proteins move by hydrolyzing ATP in their globular head domains.

• ATP binding strengthens the interaction with microtubules, while ADP binding weakens it.

• The process involves a "walking" motion as the protein hydrolyzes ATP and one globular head binds tightly and one loosely with the microtubule, causing movement between them.

• Hydrolysis of ATP causes conformational changes that induce association and dissociation from the microtubule.
  Transport Along Microtubules

• Movement occurs bidirectionally along microtubules.

• Kinesins transport from the minus to the plus end. (don't have to start at the - end itself).

• Dyneins transport from the plus end to the minus end.

• Tails dictate the cargo through direct or adapter proteins.

• Transport vesicles carry endocytosed materials or newly produced substances.

Cilia and Flagella

• Cilia are on cell surfaces, moving materials (e.g., in the respiratory system).

• Flagella are tail-like appendages that enable cellular movement (e.g., sperm).

• Both contain microtubules connected to basal bodies.

• Movement of the cilia is caused by a power stroke.

• Cilia and flagella are moved by microtubules.

• They depend on motor proteins to move.

Cilia vs. Flagella

• Flagella: Long appendages used to move the cell through liquids.

• Cilia: Stay connected to the cell membrane, moving items along the cell surface (mostly found in tissues).

Eukaryotic vs. Bacterial Flagella

• Eukaryotic and bacterial flagella are examples of convergent evolution.

• They have similar functions, but different origins.

• In animals, the most common example is sperm.

Cilia and Flagella Structure

• Both have a "9 + 2" arrangement: nine microtubule dimers surrounding two central microtubules.

• Proteins, including motor proteins (dynein), link these structures.

• Dynein facilitates movement by hydrolyzing ATP.

So there are motor protein problems that occur, and the most famous one is on sperm. If there's a problem with Dynein, tails of flagellated cells can't move so the cell fails to move.
Other problems include developing pneumonia and not moving things very well in the GI track. In reproduction in animals. Wont' get viable sperm.
Additionally, cigarette smoke paralyzes the cilia.

Movement Mechanism

• Linking proteins connect microtubule dimers, preventing sliding.

• Dynein hydrolyzes ATP, causing the structure to bend.

• The cell harnesses the energy of bending in all nine dimers to move the cell or cilia.

Microfilaments (Actin Filaments)

• Microfilaments, or actin filaments, consist of actin protein.

• Actin Filaments are important for cell shape and function.

• They are involved in cell division, cytokinesis, and cell motility.

• Actin filaments are also associated with microtubules.

Microfilament Structure

• Actin filaments are composed of actin monomers with plus and minus ends, similar to microtubules.

• They are generally thinner, shorter, and more flexible than microtubules and intermediate filaments.

• They are shorter in structure overall.

• The overall direction is overlapping each other.

Actin and ATP

• Actin is an ATP-binding protein.

• Actin monomers are added to either end in test tubes.

• Actin is added to the plus end but released from the minus end inside cells.

• Actin bound to ATP is added, and actin bound to ADP is released.

• As well as being more unstable, actin filaments and microtubules are also more dynamic than intermediate filaments.

Treadmilling

• Actin filaments maintain constant length through treadmilling.

• ATP-bound monomers add to the plus end, while ADP-bound monomers release from the minus end.

• Individual monomers move from the plus to the minus end before being released.

Actin-Binding Proteins

• Monomer-sequestering proteins: Prevent actin monomers from polymerization.

• Nucleating proteins: Initiate filament formation (e.g., ARP complex).

• Severing proteins: Cut actin filaments.

• Cross-linking proteins: Form a mesh-like network, creating a gel structure.

• Capping proteins: Bind to the plus end, preventing monomer addition.

• Proteins also bind along the sides and have different functions.

Myosin

• Myosins are motor proteins associated with actin filaments.

• The two subfamilies are myosin one and myosin two.

Myosin One

• Myosin one has one head domain and one tail.

• In comparison, the motor proteins for the microtubules have two globular head domains and two tails.

• They can move to the plus or minus end, transporting vesicles or influencing plasma membrane shape.

• Plasma membrane shape is moved and taken via these motor proteins because, without them, and by itself, the plasma membrane would deform very easily.

• Tail amino acid sequences determine cargo.

Due to only having one head domain, and one tail, to move along with the motion, these have to slightly fall off and reattach in order to propel and move, instead of the typical moving of a microtubule motor protein.