Cell Biology Lecture 7 - The Cytoskeleton

Overview of the Cytoskeleton

• The cytoskeleton is a complex network of fibers that serves several fundamental roles within the cell:

  • It supports the cell and maintains its structural integrity and shape.

  • It fixes the position of organelles within the cytoplasmic space.

  • It facilitates the movement of organelles and vesicles throughout the cell.

  • It connects to extracellular structures, assisting them in anchoring to the plasma membrane.

  • It is essential for cellular motility and movement.

Cellular Energy Currency: ATP and GTP

• Adenosine Triphosphate (ATP) acts as the primary energy currency for cellular processes.

• The ATP-ADP cycle involves the following energy transformations:

  • Releasing energy: $ATP + H_2O \rightarrow ADP + P_i + \text{energy}$

  • Requiring energy: The reverse process where ADP and inorganic phosphate ($P_i$) are recombined into ATP.

• Chemical components of ATP:

  • Adenine (nitrogenous base).

  • Ribose (five-carbon sugar).

  • Three phosphate groups.

• Inorganic phosphate ($P_i$) is specifically represented as $HPO_4^{2-}$.

• The GTP-GDP (Guanosine Triphosphate) cycle operates similarly to the ATP cycle and is particularly relevant for certain cytoskeletal dynamics.

Microfilaments (Actin Filaments)

• Microfilaments, also known as actin fibers, provide a structural framework and serve as a scaffold that determines cell shape and cytoplasmic organization.

• Structural Composition:

  • The fiber is composed of two strands of actin protein monomers wound around each other in a helical structure.

  • The protein monomers are known as G-actin (Globular), while the polymerized fiber is known as F-actin (Filamentous).

  • Individual fibers have a diameter of approximately $7\,nm$ and lengths spanning several micrometers. They are the thinnest of the three main cytoskeletal fiber types.

• Prevalence:

  • Actin is among the most abundant proteins in eukaryotic cells.

  • In non-muscle cells, actin accounts for $1\%-5\%$ of all proteins.

  • In muscle cells, actin accounts for approximately $10\%$ of total protein.

• Microfilaments can organize into parallel bundles or branched networks.

Polymerization and Dynamic Behavior of Actin

• Nucleation involves the initial assembly of 3 actin monomers.

• Polymerization is a reversible process involving the association and dissociation of monomers.

• Actin fibers exhibit polarity with two distinct ends:

  • The (+) end: Characterized by faster polymerization.

  • The (-) end: Characterized by slower growth or shrinkage.

• Dynamic States:

  • At sufficiently high monomer concentrations, the polymer grows from both ends.

  • Treadmilling: This occurs below a specific critical monomer concentration where the (+) end continues to grow while the (-) end shrinks. This results in a segment of the filament that appears to "move" through the cytoplasm even though the total length remains relative.

• Energy Involvement:

  • ATP binding and hydrolysis play a vital role in regulating the assembly and dynamic behavior of actin fibers.

  • The process involves the hydrolysis of ATP and the exchange of ATP for ADP.

Functional Structures of Actin

• Actin filaments form specialized structures in different regions of the cell:

  • Lamellipodia: Flat, sheet-like regions at the front of crawling cells.

  • Stress fibers: Bundles of actin located within the cell volume involved in tension.

  • Actin cortex: A dense network of actin located just beneath the plasma membrane (cell cortex).

• Microvilli:

  • These are protrusions in intestinal epithelial cells supported by microfilaments.

  • They significantly increase the cell’s surface area, thereby improving the absorption of nutrients in the intestine.

• Membrane Anchoring:

  • Microfilaments assist in anchoring proteins to the plasma membrane.

  • Examples of involved proteins in red blood cells include Glycophorin, Band 3 protein, Ankyrin, Spectrin ($\alpha$-chain and $\beta$-chain), and Band 4.1 protein.

Myosin and Actomyosin Interactions

• Myosin proteins are molecular motors that utilize ATP energy to perform mechanical work.

• Myosin fibers are composed of multiple motor heads.

• The interaction between actin and myosin (Actomyosin) generates movement, such as cell motility or muscle contraction.

• Muscle Structure:

  • Muscle fibers contain myofibrils, which are composed of sarcomeres.

  • The sarcomere is the repeating structural unit of the muscle, containing organized actin and myosin filaments.

  • Contraction occurs via the "Sliding Filament" mechanism, where myosin heads pull the actin filaments.

• Regulating proteins in muscle include Tropomyosin and Troponin.

• Stress Fibers and Bundling:

  • Actomyosin groups create stress fibers.

  • Actin bundling proteins, such as $\alpha$-actinin, and cross-linking proteins, like filamin, help organize these structures.

  • The polarity of actin filaments in these bundles is typically not uniform.

Eukaryotic Cell Movement Mechanism

• Cell crawling or amoeboid movement involves a coordinated three-step process:

  1. Pushing: Actin polymerization at the (+) end in the leading edge (lamellipodium) creates a pushing force.

  2. Anchoring: Focal adhesions, which are large multi-protein complexes containing integrins, form mechanical links between the intracellular actin bundles and the extracellular substrate.

  3. Pulling: Actomyosin (actin and myosin) generates contractile forces in the stress fibers that pull the rear of the cell forward.

• Experimental Evidence:

  • In experiments using Amoeba proteus, the drug cytochalasin B—which breaks apart microfilaments—was used.

  • Treated amoebas rounded up and ceased movement, while untreated control amoebas continued to move, proving that microfilaments are essential for amoeboid movement.

• Cytokinesis:

  • During cell division, actin and myosin form a contractile "actomyosin ring" that helps divide the daughter cells.

Intermediate Filaments

• Intermediate filaments are composed of tough proteins that form long, rope-like structures by twisting together.

• Characteristics:

  • They provide high mechanical strength to the cell and stabilize cellular structures.

  • They are highly diverse, encoded by at least 70 different genes.

  • The diameter ranges from $8\,nm - 12\,nm$, with lengths of several micrometers.

  • In the nucleus, they form the nuclear lamina network.

  • In the cytosol, they cross the cell to provide mechanical support and anchor organelles in place.

  • They can interact with both microfilaments and microtubules via proteins like plectin.

• Assembly Hierarchy:

  1. Monomer: Contains an $\alpha$-helical region.

  2. Coiled-coil dimer: Two monomers wound together ($48\,nm$ long).

  3. Staggered tetramer: Two coiled-coil dimers packed together.

  4. Unit-length filament (ULF): Two tetramers packed together.

  5. Full filament: Eight tetramers (ULF) twisted into a rope-like structure ($10\,nm$ diameter).

• Examples:

  • The keratin family, found in hair and nails.

  • Intermediate filaments supporting microvilli in the intestinal epithelium.

Microtubules

• Microtubules are the thickest of the three cytoskeletal fibers, with a diameter of approximately $25\,nm$.

• Structure:

  • Hollow tubes composed of 13 protofilaments.

  • Built from tubulin dimers, each consisting of an $\alpha$-tubulin monomer and a $\beta$-tubulin monomer.

  • They have distinct polarity: The (-) end exposes $\alpha$-tubulin, and the (+) end exposes $\beta$-tubulin.

• Dynamics:

  • Length is modified by adding or removing tubulin dimers.

  • They primarily elongate and shorten at the (+) end; degradation at the (-) end is minimal or non-existent.

  • Dynamic instability: A process where microtubules rapidly switch between phases of growth and shrinkage. This process requires energy derived from GTP.

  • Regulation is provided by proteins such as XMAP 215 and Kinesin-13.

Microtubule Organizing Centers and Transport

• Centrosome:

  • In animal cells, the Centrosome acts as the Microtubule Organizing Center (MTOC).

  • It is located in the center of the cell and contains two centrioles.

  • The (-) ends of microtubules are anchored in the centrosome, and the fibers extend from the center toward the cell periphery.

• Motor Proteins and Intracellular "Tracks":

  • Microtubules serve as tracks for molecular motor proteins, which are enzymes that convert ATP energy into movement. One side binds the microtubule, and the other side binds a cargo (e.g., mitochondria, endosomes).

  • Kinesin: Moves along the microtubule track from the (-) end toward the (+) end (Anterograde transport).

  • Dynein: (specifically cytoplasmic dynein) Moves along the track from the (+) end toward the (-) end (Retrograde transport). It often works with the protein Dynactin.

  • Axonal transport is a prime example of this system, where genes like KIF5A, KIF1A (for Kinesin) and DYNC1H1, BICD2 (for Dynein) are linked to neurological diseases.

• Cell Division:

  • Microtubules are essential for separating chromosomes during the cell division process.

Cilia and Flagella in Eukaryotes

• Microtubules form the stable skeleton for locomotor organelles in eukaryotic cells.

• Types:

  • Cilia: Usually shorter, present in large numbers, found frequently on unicellular protists. In humans, they line the lungs to move mucus containing trapped particles.

  • Flagella: Long (typically $100\,nm - 200\,nm$), usually only one or two per cell (e.g., sperm flagellum). Different from prokaryotic flagella.

  • Movement: Characterized by wave-like bending.

• Internal Structure (Axoneme):

  • Both cilia and flagella have a diameter of $250\,nm$ ($0.25\,\mu m$) and a similar internal structure called an axoneme.

  • "9+2" Arrangement: 9 fused pairs of microtubules on the periphery and 1 unfused pair in the center.

  • Radial spokes connect the central pair to the outer pairs.

  • Nexin: A protein complex that cross-links the outer microtubule pairs to prevent sliding.

  • Basal Bodies: Specialized MTOCs that anchor the cilia and flagella into the cell.

• Movement Mechanism:

  • Dynein motor heads walk toward the (-) end.

  • If the microtubule pairs were not connected, they would simply slide past each other.

  • Because the pairs are cross-linked by nexin, the force generated by dynein is converted into a bending motion in a single plane.

  • Dynein activity must be synchronized across the 9 pairs to create effective motion.

• Non-motile Cilia (Primary Cilia):

  • Some cilia are not involved in movement but serve sensory functions (e.g., in neurons for smell or balance).

  • They play roles in transporting proteins/organelles, supporting structural development, and inter-neuronal communication.