6 Cytoskeletal Component

What are microtubules? Describe their structure and composition.

• Microtubules (MTs) are hollow, tubular structures found in nearly every eukaryotic cell. They are a key component of the cytoskeleton, as well as other structures like the mitotic spindle, centrioles, and the core of cilia and flagella. They have an outer diameter of 25 nm and a wall approximately 4 nm thick, and can extend across the entire cell.

• Microtubule Structure and Composition:

  • The wall of a microtubule is made up of globular proteins arranged in longitudinal rows called protofilaments.

  • These protofilaments run parallel to the long axis of the tubule, and are aligned side by side in a circular pattern, forming the microtubule wall.

  • When viewed in cross-section, a microtubule consists of 13 protofilaments.

  • Each protofilament is composed of αβ-tubulin heterodimers stacked in a head-to-tail arrangement.

  • The αβ-tubulin heterodimer is the basic building block of microtubules.

  • α-tubulin has a bound GTP, which is not hydrolyzed and does not exchange.

  • β-tubulin can be bound to either GTP or GDP, which is important for microtubule dynamics.

Explain the process of microtubule polymerization and depolymerization. What role do GTP and GDP play in this process?

• Microtubule polymerization and depolymerization are the processes of microtubule assembly and disassembly.

• Polymerization:

  • αβ-tubulin dimers bind to an open ring of γ-tubulin molecules within the γ-tubulin ring complex (γ-TuRC). This ring complex is important for microtubule nucleation.

  • The γ-TuRC determines the polarity of the microtubule, with α-tubulin at the minus end and β-tubulin at the plus end.

  • Growth occurs primarily at the plus end by the addition of GTP-bound tubulin dimers.

  • The GTP bound to β-tubulin is hydrolyzed to GDP after the dimer is incorporated into the microtubule.

• Depolymerization:

  • When the rate of GTP hydrolysis exceeds the rate of dimer addition, the microtubule becomes unstable and begins to depolymerize.

  • This depolymerization occurs primarily at the plus end by the loss of GDP-bound tubulin dimers.

• The Role of GTP and GDP:

  • GTP promotes microtubule assembly and stability. GTP-bound tubulin dimers have a higher affinity for each other, leading to polymerization.

  • GDP promotes microtubule disassembly and instability. GDP-bound tubulin dimers have a lower affinity for each other, leading to depolymerization.

What is dynamic instability? How does it contribute to microtubule function?

• Dynamic instability is the ability of microtubules to switch between periods of growth (polymerization) and shrinkage (depolymerization). This behavior is intrinsic to microtubules and is driven by the GTP hydrolysis cycle.

• How Dynamic Instability Contributes to Microtubule Function:

  • It allows microtubules to rapidly explore the cytoplasm and find their targets, such as chromosomes during cell division.

  • It allows for rapid remodeling of the microtubule cytoskeleton in response to cellular needs.

What are microtubule-associated proteins (MAPs)? How do they regulate microtubule dynamics and function?

• Microtubule-associated proteins (MAPs) are proteins that bind to microtubules and regulate their dynamics and function. Examples of MAPs include:

  • Stabilizing MAPs (e.g., tau, MAP2): These MAPs bind along the sides of microtubules and prevent depolymerization, thereby increasing microtubule stability.

  • Destabilizing MAPs (e.g., kinesin-13): These proteins promote microtubule depolymerization, particularly at the plus end. They act as microtubule depolymerases.

  • +TIPs: These proteins specifically associate with the growing plus ends of microtubules and can influence their growth rate or link them to other cellular structures, such as membranes.

• How MAPs Regulate Microtubule Dynamics and Function:

  • Controlling microtubule stability: MAPs can either stabilize or destabilize microtubules, thereby affecting their length and lifetime.

  • Mediating interactions: MAPs can mediate the interaction of microtubules with other cellular components, such as organelles and other cytoskeletal filaments.

Describe the role of the centrosome in microtubule organization. What are the key components of the centrosome?

• The centrosome is the major site of microtubule initiation in animal cells and plays a crucial role in microtubule organization. It is typically located near the nucleus and consists of the following key components:

  • Centrioles: Two barrel-shaped structures arranged perpendicular to each other. They contain nine evenly spaced sets of triplet microtubules and a central hub.

  • Pericentriolar material (PCM): An amorphous, electron-dense material that surrounds the centrioles. It is within the PCM that microtubule nucleation occurs.

• How the Centrosome Organizes Microtubules:

  • Microtubule nucleation: The PCM contains γ-TuRCs, which nucleate microtubules.

  • Microtubule anchoring: The minus ends of many microtubules are anchored at the centrosome.

Explain the mechanism of action of kinesin and dynein motor proteins. How do they use ATP hydrolysis to generate movement along microtubules?

• Kinesin and dynein are microtubule-based motor proteins that use ATP hydrolysis to generate movement along microtubules. They are responsible for a wide range of intracellular transport processes.

• Mechanism of Action:

  • Both kinesin and dynein have a motor domain that binds to microtubules and hydrolyzes ATP. The energy released from ATP hydrolysis is used to induce conformational changes in the motor domain, resulting in movement along the microtubule.

  • The motor domain binds to the microtubule.

  • ATP binds to the motor domain.

  • ATP is hydrolyzed, releasing energy.

  • The released energy causes the motor domain to undergo a conformational change, which results in the motor protein taking a "step" along the microtubule.

  • ADP and inorganic phosphate are released.

  • The cycle repeats, allowing the motor protein to continue moving along the microtubule.

What are the different types of kinesin and dynein motors? How do they differ in their functions and cellular localization?

• Kinesins:

  • Conventional kinesins (kinesin-1): Typically move toward the plus end of microtubules and are involved in outward transport of cargo, such as organelles, towards the cell periphery.

  • Other kinesin families (kinesin-2 to kinesin-14): Exhibit diverse functions, including minus-end directed movement, microtubule depolymerization, and spindle assembly.

  • Kinesin-13: Specifically acts as a microtubule depolymerase.

• Dyneins:

  • Cytoplasmic dynein: Moves towards the minus end of microtubules and is involved in inward transport of cargo, towards the cell center.

  • Axonemal dynein: Specialized for the rapid sliding movements of microtubules that power the beating of cilia and flagella.

How are kinesin and dynein involved in intracellular transport? Give specific examples.

• Kinesin:

  • Transport of organelles: Kinesin-1 transports organelles, such as mitochondria and peroxisomes, from the cell center towards the periphery. For example, the outward movement of mitochondria is disrupted in cells lacking the KIF5B kinesin, indicating the role of this plus-end directed kinesin in the transport of this organelle.

  • Anterograde axonal transport: Kinesin-1 is responsible for the transport of vesicles, organelles, and other cargo along microtubules from the neuron cell body towards the axon terminals.

• Dynein:

  • Retrograde axonal transport: Cytoplasmic dynein transports cargo, including aged or damaged organelles, from the axon terminals back to the cell body for degradation or recycling.

  • Golgi positioning: Dynein is involved in positioning the Golgi apparatus near the centrosome in animal cells by moving Golgi vesicles towards the minus ends of microtubules.

Describe the structure of cilia and flagella. What is the role of the axoneme?

• Cilia and flagella are hairlike structures that project from the surface of eukaryotic cells and function in motility and sensory perception. They are essentially the same structure, differing mainly in their length and beating pattern.

• Structure:

  • Axoneme: The core structure of cilia and flagella, composed of an array of microtubules. It runs longitudinally through the entire organelle and is covered by a membrane continuous with the plasma membrane.

  • 9 + 2 array: The characteristic arrangement of microtubules in motile cilia and flagella, consisting of nine peripheral doublet microtubules surrounding a central pair of single microtubules.

  • Peripheral doublets: Each doublet consists of a complete A microtubule and an incomplete B microtubule.

  • Dynein arms: Motor proteins attached to the A tubules of the doublets. They generate the force for movement.

  • Nexin links: Elastic connections between adjacent doublets that limit sliding and contribute to bending.

  • Radial spokes: Structures that project from the outer doublets towards the central pair, involved in coordinating dynein activity.

How do cilia and flagella generate movement? What is the role of dynein motor proteins in this process?

• Dynein activation: Axonemal dynein arms on one side of the axoneme are activated, while those on the other side are inactive.

• Sliding force: Activated dynein arms attempt to walk along the adjacent doublet microtubule, generating a sliding force.

• Bending motion: Nexin links resist sliding, causing the axoneme to bend instead of fully extending.

• Alternating sliding: Dynein activity alternates between sides of the axoneme, causing the cilium or flagellum to bend back and forth in a whiplike or undulating motion.

• The Role of Dynein Motor Proteins:

  • Force generation: Axonemal dynein arms generate the sliding force between microtubule doublets.

  • Bending motion: Their activity, coordinated by the radial spokes and central pair, causes the bending of the axoneme, resulting in the characteristic beating patterns of cilia and flagella.

What is the primary cilium? How does it differ from motile cilia and flagella?

• The primary cilium is a non-motile, sensory organelle found on almost all vertebrate cells.

• Differences from Motile Cilia and Flagella:

  • Motility: It is non-motile, lacking the central pair of microtubules and the dynein arms necessary for movement.

  • Structure: Typically has a 9 + 0 arrangement of microtubules, meaning it lacks the central pair.

  • Function: Primarily sensory, acting as an antenna for detecting extracellular signals.

What is the role of the primary cilium in sensory perception? Give examples.

• Mechanosensation: In the kidney, primary cilia act as flow sensors, detecting changes in fluid flow.

• Chemosensation: Olfactory cilia in the nose are specialized primary cilia that detect odor molecules.

• Photoreception: The outer segments of rod and cone cells in the retina, responsible for light detection, are highly modified primary cilia.

How is the primary cilium involved in cell signaling?

• Signal reception: Primary cilia contain receptors for various signaling molecules, such as Hedgehog and Wnt proteins, which are important for development and tissue homeostasis.

• Signal transduction: Upon ligand binding, these receptors initiate intracellular signaling pathways that regulate gene expression and cell behavior.

What are the consequences of primary cilia defects?

• Ciliopathies: A group of human genetic disorders caused by defects in cilia structure or function.

  • Polycystic kidney disease: Characterized by the formation of cysts in the kidneys, often due to mutations in genes involved in primary cilia function.

  • Retinal degeneration: Mutations in genes that affect primary cilia in the retina can lead to progressive vision loss.

  • Skeletal abnormalities: Some ciliopathies result in skeletal defects, such as polydactyly (extra fingers or toes) or shortened limbs.

How can studying primary cilia help us understand human diseases?

• Disease modeling: Studying the effects of mutations in cilia genes in model organisms, such as mice and zebrafish, can help us understand the mechanisms underlying human ciliopathies.

• Therapeutic targets: Identifying the molecular pathways disrupted in ciliopathies can lead to the development of targeted therapies for these disorders.

• Early diagnosis: Understanding the role of primary cilia in development and disease can lead to improved diagnostic tools for identifying ciliopathies early on.

Describe the structure and composition of intermediate filaments. How do they differ from microtubules and microfilaments?

• Intermediate filaments (IFs) are strong, flexible, ropelike fibers that provide mechanical strength to cells subjected to physical stress. They have a diameter of about 10 nm. Unlike microtubules and actin filaments (also known as microfilaments), which are found in all eukaryotes, IFs have only been identified in animal cells. However, many other eukaryotes have similar insoluble filaments made from different protein sequences. IFs are composed of about 70 different genes in humans. The subunits of IFs are grouped into five major classes based on the type of cell they are found in as well as biochemical, genetic, and immunologic criteria.

• IFs assemble by forming strong lateral contacts between α-helical coiled-coils. These coiled-coils extend over most of the length of each elongated fibrous subunit. The individual subunits are staggered in the filament, which allows IFs to tolerate stretching and bending better than either actin filaments or microtubules.

• Each monomer of an IF has a pair of globular terminal domains separated by a long α-helical region. Two monomers associate in a parallel orientation to form dimers. Depending on the type of IF, dimers may be composed of identical monomers (homodimers) or nonidentical monomers (heterodimers). Dimers then associate in an antiparallel, staggered fashion to form tetramers. Tetramers are thought to be the basic subunit in the assembly of IFs. Eight tetramers then associate laterally to form a unit length of the IF. The end-to-end association of these unit lengths forms highly elongated IFs. Once they are formed, IFs undergo dynamic remodeling thought to involve the intercalation of unit lengths of filament into the body of an existing filament.

• The nuclear lamina is a meshwork formed from one type of IF. The nuclear lamina is located just beneath the inner nuclear membrane. Other types of IFs extend across the cytoplasm from one cell-cell junction to another, strengthening the entire epithelium in epithelial tissue.

• Intermediate Filament Interaction With Other Cytoskeletal Elements

  • IFs radiate through the cytoplasm of many animal cells and are often interconnected to other cytoskeletal filaments by thin cross-bridges. In many cells, these cross-bridges consist of an elongated dimeric protein called plectin. Plectin can exist in numerous isoforms. Each plectin molecule has a binding site for an IF at one end. Depending on the isoform, it may have a binding site for another IF, an actin filament, or a microtubule at the other end.

What are the major types of intermediate filaments, and where are they found in the cell?

• Keratins are the primary structural proteins of epithelial cells. They are anchored at the outer edge of the cell by connections to the cytoplasmic plaques of desmosomes and hemidesmosomes. Keratin-containing IFs radiate through the cytoplasm. They are tethered to the nuclear envelope in the center of the cell.

• Neurofilaments are found in the cytoplasm of neurons. They are composed of three distinct proteins: NF-L, NF-M, and NF-H. NF-H and NF-M have sidearms that project outward from the neurofilament. These sidearms are thought to maintain the proper spacing between the parallel neurofilaments of the axon. In the early stages of differentiation when the axon is growing toward a target cell, it contains very few neurofilaments but many microtubules. Once the nerve cell has become fully extended, it is filled with neurofilaments that provide support as the axon increases in diameter.

• Vimentin is found in many cells of mesenchymal origin.

• Desmin is found in skeletal, cardiac, and smooth muscle. It forms a scaffold around the Z disc of the sarcomere.

• Glial fibrillary acidic protein is found in glial cells (astrocytes and some Schwann cells). o Peripherin is found in some neurons.

• Type I keratins (acidic) are found in epithelial cells and their derivatives (hair and nails).

• Type II keratins (neutral/basic) are found in epithelial cells and their derivatives (hair and nails).

• Neurofilament proteins (NF-L, NF-M, and NF-H) are found in neurons.

What is the role of intermediate filaments in providing mechanical strength to cells?

The IF network serves as a scaffold for organizing and maintaining cellular architecture and absorbing mechanical stresses applied by the extracellular environment. They accomplish this because of the physical connections between them, microtubules, and actin filaments, which transforms these otherwise separate elements into an integrated cytoskeleton.

How do intermediate filaments interact with other cytoskeletal elements?

Intermediate filaments are connected to the rest of the cytoskeleton by proteins called plakins. Plakins are large proteins that contain multiple domains that connect cytoskeletal filaments to each other and to junctional complexes. One example is plectin, which bundles intermediate filaments and links them to microtubules, actin filament bundles, and myosin II filaments. Plectin also helps attach intermediate filament bundles to adhesive structures at the plasma membrane.

Discuss the role of intermediate filaments in nuclear lamina formation.

The inner surface of the nuclear envelope of animal cells is bound by integral membrane proteins to the nuclear lamina, a thin filamentous meshwork that provides mechanical support to the nuclear envelope. The nuclear lamina is made of lamins, which are polypeptides that are members of the same superfamily of polypeptides that assemble into the 10-nm IFs of the cytoplasm. The nuclear lamina is also a site of attachment for chromatin fibers at the nuclear periphery. It plays a role in DNA replication and transcription that is not fully understood.

Describe the structure of actin filaments. How do actin monomers polymerize to form filaments?

• Actin filaments are flexible structures with a diameter of 8 nm. They are also known as microfilaments. Actin filaments are most highly concentrated in the cortex, which is located just beneath the plasma membrane. They determine the shape of the cell's surface, are necessary for whole-cell locomotion, and drive the pinching of one cell into two.

• Actin filaments are helical polymers of the protein actin. They are organized into a variety of linear bundles, two-dimensional networks, and three-dimensional gels.

• Actin subunits assemble head-to-tail to create flexible, polar filaments. The subunits are positioned with their ATP-binding cleft directed toward the minus end within the filament. This forms a tight, right-handed helix about 8 nm wide called filamentous or F-actin. Because the asymmetrical actin subunits of a filament all point in the same direction, filaments are polar and have structurally different ends: a slower-growing minus end and a faster-growing plus end. The minus end is also called the pointed end, and the plus end is also called the barbed end. These names come from the arrowhead appearance of the complex formed between actin filaments and the motor protein myosin, which is visible in electron micrographs.

• Actin polymerization is controlled by the concentration of actin, pH, and the concentrations of salts and ATP in a test tube.

What are the key factors that influence actin polymerization dynamics?

In cells, actin behavior is also regulated by accessory proteins that bind actin monomers or filaments. These actin-binding proteins dramatically alter actin filament dynamics through spatial and temporal control of filament nucleation, elongation, and depolymerization. They also regulate the association of actin with membranes as well as how filaments are organized. Actin-binding proteins enable actin polymerization to generate forces to support, shape, and move cellular membranes.

Explain the concept of actin filament polarity. How does it relate to the direction of actin polymerization?

Actin filaments are polarized. They have a slower-growing minus end and a faster-growing plus end. The minus end is also called the pointed end, and the plus end is also called the barbed end. This polarity relates to the direction of actin polymerization because subunits are added to the plus end faster than they are added to the minus end. Actin filaments can undergo treadmilling when a filament assembles at the plus end while simultaneously depolymerizing at the minus end.

What are actin-binding proteins, and what are their general functions?

• Actin-binding proteins are accessory proteins that bind to actin monomers or filaments. They regulate actin filament dynamics. Actin-binding proteins can:

  • Promote the formation of branched and parallel filaments

  • Slow or accelerate the kinetics of filament assembly and disassembly

  • Assemble filaments into larger ordered structures

How do actin-binding proteins regulate actin filament assembly and disassembly?

Actin-binding proteins regulate every step of the dynamic behavior and assembly of actin filaments. Approximately half of the actin is kept in a monomeric form by proteins, such as profilin. Nucleation factors, such as the Arp2/3 complex and formins, promote the formation of branched and parallel filaments, respectively. Capping proteins can slow or accelerate the kinetics of filament assembly and disassembly. Proteins that bind or cap actin filaments and proteins that promote filament severing or depolymerization can slow or accelerate the kinetics of filament assembly and disassembly.

What is the role of Arp2/3 complex in actin nucleation and branching?

The Arp2/3 complex is a nucleation factor that promotes the formation of branched filaments. It nucleates actin at the minus end.

How do formins promote actin filament elongation?

Formins are nucleation factors that promote the formation of parallel filaments. They nucleate actin at the plus end.

Explain the function of capping proteins in regulating actin filament length.

Capping proteins bind to filament ends, regulating filament length. For example, CapZ in the Z disc caps the plus ends of actin filaments in the sarcomere, preventing depolymerization.

What is the role of thymosin and profilin in actin monomer sequestration and exchange?

About half of the actin is kept in a monomeric form by proteins such as profilin. Thymosin binds to actin monomers to prevent them from adding to the ends of actin filaments. Profilin binds to actin monomers, promoting the addition of monomers to the plus end of filaments.

How does cofilin regulate actin filament disassembly?

Cofilin is an actin-binding protein that severs actin filaments. It binds ADP-actin filaments and accelerates their disassembly.

Describe the structure of myosin molecules. How do they interact with actin filaments?

• Myosins are a superfamily of motor proteins that move along actin filaments. All myosins share a characteristic motor (head) domain. The head contains a site that binds an actin filament and a site that binds and hydrolyzes ATP to drive the myosin motor. The tail domains of myosins are highly divergent, while the head domains are similar. Myosins also contain light chains.

• Myosin II molecules are elongated proteins formed from two heavy chains and two copies of each of two light chains. Each heavy chain has a globular head domain at its N-terminus that contains the force-generating machinery, followed by a very long α-helical amino acid sequence that forms an extended coiled-coil to mediate heavy-chain dimerization. The two light chains bind close to the N-terminal head domain, and the long coiled-coil tail bundles itself with the tails of other myosin molecules.

• Myosin Interaction With Actin Filaments

  • Myosin heads bind and hydrolyze ATP, using the energy of ATP hydrolysis to walk toward the plus end of an actin filament. The opposing orientation of the heads in the thick filament allows the filament to slide pairs of oppositely oriented actin filaments toward each other, which shortens the muscle.

Explain the mechanism of muscle contraction. What is the role of myosin II in this process?

• Muscle contraction is caused by the sliding of myosin filaments past the actin thin filaments. Neither type of filament changes length.

• The force-generating molecular interaction between myosin thick filaments and actin thin filaments only happens when a signal passes from a nerve to the skeletal muscle, stimulating it. This signal from the nerve triggers an action potential in the muscle cell plasma membrane. This electrical excitation quickly spreads into a series of membranous folds called transverse tubules, or T tubules, which extend inward from the plasma membrane around each myofibril. The signal is then relayed across a small gap to the sarcoplasmic reticulum, an adjacent weblike sheath of modified endoplasmic reticulum surrounding each myofibril. When the action potential activates a Ca2+ channel in the T-tubule membrane, it triggers the opening of a Ca2+-release channel in the closely associated sarcoplasmic reticulum membrane. Ca2+ flooding into the cytosol then initiates the contraction of each myofibril. The increase in Ca2+ concentration is transient because Ca2+ is rapidly pumped back into the sarcoplasmic reticulum by an abundant, ATP-dependent Ca2+-pump in its membrane. The cytoplasmic Ca2+ concentration is usually restored to resting levels within 30 milliseconds, allowing the myofibrils to relax.

• Myosin II is the primary motor for muscle contraction.

What is the structure of the sarcomere, and how does it contribute to muscle contraction?

• The sarcomere is the basic contractile unit of a muscle cell. It is formed from a miniature, precisely ordered array of parallel and partially overlapping thin and thick filaments. The thin filaments are composed of actin and associated proteins and are attached at their plus ends to a Z disc at each end of the sarcomere. The capped minus ends of the actin filaments extend inward toward the middle of the sarcomere, where they overlap with thick filaments, the bipolar assemblies formed from specific muscle isoforms of myosin II. Sarcomere shortening is caused by the myosin filaments sliding past the actin thin filaments, without any change in the length of either type of filament.

• Accessory proteins ensure uniformity in filament organization, length, and spacing in the sarcomere. They also enable it to withstand the constant wear-and-tear of contraction. The actin filament plus ends are anchored in the Z disc, which is built from CapZ and α-actinin. CapZ caps the filaments, preventing depolymerization, while α-actinin holds them together in a regularly spaced bundle. Actin filaments are stabilized along their length by tropomyosin and nebulin. Nebulin stretches from the Z disc toward the minus end of each thin filament, which is capped and stabilized by tropomodulin. Opposing pairs of titin, an even longer protein, position the thick filaments midway between the Z discs. Titin acts as a molecular spring. It has a long series of immunoglobulin-like domains that can unfold one by one as stress is applied to the protein. A springlike unfolding and refolding of these domains keeps the thick filaments poised in the middle of the sarcomere and allows the muscle fiber to recover after being overstretched.

Describe the events that occur at the neuromuscular junction.

The neuromuscular junction is the point of contact of a terminus of an axon with a muscle fiber. It is a site of transmission of the nerve impulse from the axon across a synaptic cleft to the muscle fiber.

How is muscle contraction regulated by calcium ions?

Muscle contraction in vertebrate skeletal muscle is dependent on Ca2+ and therefore on commands transmitted via nerves. This is due to a set of specialized accessory proteins closely associated with the actin thin filaments. One of these accessory proteins is tropomyosin, the elongated protein that binds along the groove of the actin filament helix. The other is troponin, a complex of three polypeptides: troponins T, I, and C. Troponin T binds to tropomyosin, troponin I is an inhibitory subunit, and troponin C binds to Ca2+. Troponin I binds to actin as well as to troponin T. In a resting muscle, the troponin I–T complex pulls the tropomyosin out of its normal binding groove into a position along the actin filament that interferes with the binding of myosin heads, preventing any force-generating interaction. When the level of Ca2+ is raised, troponin C, which binds up to four molecules of Ca2+, causes troponin I to release its hold on actin. This allows tropomyosin to return to its normal position so that the myosin heads can walk along the actin filaments.