Motor Proteins and Cytoskeletal Movement

Motor Proteins

• These proteins bind to polarized cytoskeletal filaments.
• These proteins use energy from repeated ATP hydrolysis cycles.
• ATP hydrolysis helps them move steadily along the filaments.
• Dozens of motor proteins coexist in eukaryotic cells.
• Motor proteins differ in filament type (actin or microtubules).
• Motor proteins differ in movement direction along the filaments.
• Motor proteins differ in the cargo they carry.
• Cytoskeletal motor proteins use a head region, or motor domain, for filament track association.
• The motor domain binds and hydrolyzes ATP.
• The motor domain determines the track identity and movement direction.
• The tail of the motor protein determines the cargo identity.

Three Groups of Cytoskeletal Motor Proteins

• Myosins bind to actin.
• Myosins move toward the positive end of actin filaments.
• An exception exists, myosin VI moves towards the negative end.
• Kinesins bind to microtubules.
• Kinesins move toward the positive end of microtubules.
• Dyneins bind to microtubules.
• Dyneins move toward the minus end of microtubules.

Myosin Superfamily

• Skeletal muscle myosin, called myosin II, was the first identified motor protein.
• Sequence comparisons indicate at least 37 distinct myosin families across eukaryotes.
• Some myosins are exclusively found in plants or vertebrates.
• Specifically myosins VIII and XI are found in plants, while IX is found in vertebrates.
• Myosin tails diversified for binding to other subunits and cargos.
• Yeast contain 5 myosins, C. elegans have at least 15, humans have 40.
• Myosin II is associated with contraction in both muscle and non-muscle cells.
• Myosin V is involved in vesicle and organelle transport.
• Myosin I is generally involved in intracellular organization.
• Nine human myosins are primarily or exclusively expressed in inner ear hair cells.
• Mutations in five of those nine myosins cause hereditary deafness.

Kinesin Superfamily

• The Kinesin Superfamily was first identified in the giant axon of the squid.
• Kinesins are structurally similar to myosin II.
• They have two heavy chains and two light chains per motor.
• Kinesins share a common motor domain with myosin.
• Yeast have 6 kinesins, C. elegans have 16, and humans have about 45.
• Kinesin-1 has a C-terminal that binds cargo.
• Kinesin-3 functions as a monomer and moves membrane-enclosed organelles along microtubules.
• Kinesin-5 is bipolar and slides microtubules past each other.
• Kinesin-13 loses motor activity but binds to microtubules to increase dynamic instability.
• Kinesin-14 moves towards the minus end, not the plus end, of microtubules.

Dynein Family

• Cytoplasmic dyneins are important for vesicular transport and Golgi localization.
• Axonemal dyneins are highly specialized for rapid sliding of microtubules.
• This powers the beating of cilia and flagella.
• Dyneins consist of 2 or 3 heavy chains with a motor domain and a variable number of intermediate and light chains.
• Dyneins are the largest molecular motors and among the fastest.

Structural Similarity of Myosin and Kinesin

• Motor proteins track along different filaments
• They have different kinetic properties.
• They show no identifiable amino acid sequence similarities.
• Motor domains of both myosin and kinesin are built around nearly identical cores, revealed by 3D structure determination.
• The central force-generating element common to both includes the site of ATP binding.
• The machinery translates ATP hydrolysis into an allosteric conformational change.
• The motor domain of myosin is substantially larger than that of kinesins.
• Myosin's motor domain is about 850 amino acids, compared to 350 for kinesins.

Myosin Mechanochemical Cycle

• Attached: A myosin head lacking a bound nucleotide tightly locks onto an actin filament in a rigor configuration.
• This state is short-lived in actively contracting muscle.
• Released: ATP binds to the myosin head, causing a slight conformational change that reduces affinity for actin.
• This allows the head to move along the filament.
• Cocked: The cleft closes around ATP triggers a shape change displacing the head by about 5 nm.
• ATP hydrolysis occurs, but ADP and inorganic phosphate (Pi) remain tightly bound.
• Force-Generating: Weak binding to a new actin site and inorganic phosphate(Pi) release, tightly binds head to actin.
• The release triggers the power stroke, force-generating change in shape, and head regains original shape.
• The head loses its ADP and returns to the start of a new cycle.

Kinesin Mechanochemical Cycle

• Rear lagging head is tightly bound, leading head is loosely bound.
• ADP exchanges for ATP in front motor causes a small protein that shifts to a forward pointing conformation.
• This shift pulls the rear motor forward, detaching from ATP hydrolysis and Pi release.

Dynein Mechanochemical Cycle

• Dynein contains 6 AAA domains, four can bind ATP, but only one retains major ATPase activity.
• The stalk is detached when ATP is bound.
• ATP hydrolysis causes the stalk to attach.
• The release of ADP and Pi results in the power stroke.
• The power stroke involves rotation of the head and stalk relative to the tail domain.

Motor Protein Kinetics and Cellular Functions

• A single kinesin I dimer moves in a highly progressive manner.
• Kinesin I travels for hundreds of ATPase cycles before dissociating.
• Myosin II cannot move progressively and dissociates within one or two steps.
• Myosin gains in speed what it loses in processivity.
• Linked myosins can move its filament 20 steps during a cycle time.
• Kinesins can only move 2 steps
• Motor protein movement speed varies within each class.
• Myosins range from 0.2 to 60 µm, while kinesins range from 0.02 to 2 µm.
• These differences arise from fine-tuning the mechanochemical cycle.
• Velocity decreases by reducing the ATP hydrolysis rate, or increasing the proportion of time spent bound to the filament track.
• Myosin V spends up to 90% of its cycle bound to the filament track.
• Myosin II only spends 5% of it's cycle bound on the filament track.
• Each step size adjusts according to lever arm length or angle.

Motor Proteins Mediate Organelle Movement

• Dynein and accessory proteins link to membrane-enclosed organelles.
• Dynactin, a large complex, binds weakly to microtubules, Dynein, and the Arp 1 actin-related filament.

Cytoskeleton and RNA Localization

• The cytoskeleton localizes specific RNA molecules.
• This establishes cellular asymmetries.

Regulation of Motor Function

• Cells regulate motor function by organizing proteins and filaments.
• As seen in the assembly of non-muscle myosin II.

Muscle Contraction

• Motor proteins and filaments have specialized organization as seen in skeletal muscle.