L11: Molecular motors I Flashcards
what are motor proteins?
Definition of motor proteins:
Proteins that convert chemical energy into movement or mechanical work.
Cytoskeletal motors: Intracellular transport of proteins, nucleic
acids and membrane bound organelles
Muscle contraction
Cell migration
Mitosis / meiosis
Cell division (cytokinesis)
Chemical energy
ATP → ADP + Pi drives function of molecular motors
Other motors:
DNA and RNA polymerases, helicases,
ribosomes
Bacterial flagellar motor
tracks for the cytoskeletal motor proteins?
Microtubules serve as tracks for kinesin and dynein motor proteins.
Microtubules extend from the nucleus to the cell periphery, forming a crosslinked network that helps in intracellular transport, cell shape, and organization.
Actin filaments serve as tracks for myosin motor proteins.
Actin filaments exist in two main arrangements:
Cortical actin: Just beneath the plasma membrane, providing structural support and helping in cell movement.
Inner actin bundles: Found deeper within the cell, often forming stress fibers for maintaining tension and structure.
These filaments act as scaffolds where motor proteins function.
Structure:
Microtubules are made of tubulin dimers (α-tubulin and β-tubulin). These dimers polymerize to form protofilaments, which then interact laterally to form a hollow cylindrical structure (microtubule).
Actin filaments are made of globular (G-actin) subunits that polymerize into filamentous (F-actin), forming a helical structure.
intrinsic polarity?
Polarity of Microtubules and Actin Filaments
Both microtubules and actin filaments have intrinsic polarity, meaning they have distinct plus (+) and minus (-) ends.
This polarity influences motor protein movement, ensuring that motors travel in a specific direction.
Actin Polymerization & ATP Hydrolysis
Actin monomers (G-actin) bind ATP before polymerizing into F-actin (actin filaments).
Once incorporated into the filament, ATP is hydrolyzed to ADP, providing energy for filament dynamics.
The actin filament forms a helical structure with a periodic repeat of ~36 nm.
Actin monomers assemble head-to-tail, giving the filament its polarity:
Plus (+) end: Faster-growing end.
Minus (-) end: Slower-growing, more depolymerization occurs here.
Motor proteins move directionally:
Most myosins move toward the plus end of actin filaments.
The movement direction for kinesins and dyneins (on microtubules) depends on their structure/ individual properties
common features shared by cytoskeletal families?
a cargo binding region (top), linker region (often alpha helical coiled coil) (middle) and a cytoskeletal binding/motor domain (bottom).
Three Main Classes of Motor Proteins
Kinesins – Move along microtubules, mostly toward the plus (+) end.
Dyneins – Move along microtubules, but toward the minus (-) end.
Myosins – Move along actin filaments, mostly toward the plus (+) end.
General Structure of Motor Proteins
Cargo-binding region → Attaches to vesicles, organelles, or other cellular cargo.
Motor domain → Interacts with the cytoskeletal track (either microtubules or actin).
This is where ATP hydrolysis occurs, converting chemical energy into mechanical movement.
Linker region → Connects the cargo-binding region to the motor domain, allowing force transmission.
Dynein Specifics
Dynein’s motor domain is much larger than those of kinesin and myosin.
It has light chains that help stabilize a longer alpha-helical domain (possibly part of the “lever arm”).
Dynein moves toward the microtubule minus end, playing key roles in organelle positioning and intracellular transport.
kinesin family proteins and their functions?
N-kinesins (motor domain at the N-terminus) – mostly move toward the plus end of microtubules (e.g., Kinesin-1, Kinesin-4).
C-kinesins (motor domain at the C-terminus) – move toward the minus end of microtubules.
some kinesins (motor domain in the middle)
some are involved in microtubule depolymerization rather than transport. (research bipolar kinesins)
Examples of kinesin functions:
Kinesin-1 – specialized for vesicle & mRNA transport along microtubules.
Kinesin-4 – involved in chromosome positioning during mitosis.
Kinesin-13 – depolymerizes microtubules, important in mitotic spindle dynamics.
- check internal kinesins
myosin family
Myosin Superfamily Overview
There are ~39 myosins in the human genome, each adapted for specific functions.
Myosins are actin-based motor proteins that use ATP hydrolysis to generate movement.
Different myosin families have structural and functional specialisations.
Myosin-2 Family (Muscle Contraction & Contractile Forces)
Structure:
Has two motor domains (heads) connected by an α-helical coiled-coil tail.
Forms filaments by polymerisation (multiple molecules work together).
Works in ensembles, meaning does not function as a single motor.
Function:
Generates contractile forces in muscle cells and non-muscle cells.
Moves hand-over-hand along actin filaments, pulling them for muscle contraction.
Found in sarcomeres of skeletal and cardiac muscle.
Myosin-5 (Intracellular Transport & Vesicle Movement)
Structure:
Two-headed myosin with an extended α-helical coiled-coil domain.
Has a cargo-binding tail that interacts with organelles, vesicles, and mRNA.
Function:
Vesicle and organelle transport along actin filaments (e.g., moves melanosomes in pigment cells).
Unlike myosin-2, it functions as a single molecule rather than in filaments.
Uses a hand-over-hand stepping mechanism with large steps (~36 nm).
Myosin-1 (Membrane & Cytoskeletal Linkage)
Structure:
Single-headed myosin with a short tail domain.
Lacks a coiled-coil domain, meaning it does not form filaments.
Function:
Membrane-cytoskeleton interactions, helping with endocytosis, exocytosis, and membrane tension regulation.
Often found in microvilli and cell surface projections
dynein family
Cytoplasmic dynein: The main function is in minus-end directed intracellular transport. It moves cargo toward the minus end of microtubules.
Axonemal dyneins: These are involved in the movement of cilia and flagella. They help in the beating motion of cilia and flagella, which are structures made of microtubules and other proteins.
Dyneins: They fall into two main groups:
Cytoplasmic dyneins: Drive intracellular transport, moving cargo toward the minus end of microtubules.
Axonemal dyneins: Responsible for movement in cilia and flagella
motors and translocation?
Motors can bind to and translocate on cytoskeletal filaments
motor domain interaction:
One motor domain of a motor protein (such as kinesin or myosin) binds to the microtubule or actin filament.
The other motor domain is not directly bound to the track and is free to move (flopping around).
ATP Hydrolysis Cycle:
The ATP binding and hydrolysis cycle drives the movement.
When ADP is replaced by ATP, the motor protein undergoes a conformational change.
This change allows the lagging motor domain (the one that was previously detached) to bind to the track, while the leading motor domain (the one ahead) detaches.
Hand-Over-Hand Movement:
The motor domains move in a hand-over-hand fashion, where one head detaches and moves forward, and then the cycle repeats. This results in the cargo being moved along the filament.
Myosin V on Actin Filaments:
Myosin V moves along actin filaments in a similar hand-over-hand manner.
The position of the myosin molecule changes over time during movement.
This motion can be visualized using techniques like Atomic Force Microscopy (AFM), where changes in the position of the head and the overall molecule can be captured and analyzed.
*check if right
motors pulling cytoskeletal filaments?
motors bind to and pull cytoskeletal filaments
Muscle contraction driven by the relative sliding of the interaction of mysoin with actin.
Myosin II forms filaments overlapping with actin filaments
* Myosin motors attach to actin and pull it in one direction
One motor working at a time.
Uses atp hydrolysis
Big change in conformation of the motor that pulls the actin filament in this reaction.
Gen force and shortening in the muscle
Molecular event responsible for muscle contraction.
methods for studying the structure-function relation of motor protein?
In vitro Motility Assay:
This method involves isolating myosin motors and spreading them onto a glass surface.
Then, actin filaments (labeled with a fluorophore to make them visible under a microscope) are added.
As the myosin motors interact with the actin filaments, the filaments will move across the surface.
Although you can’t see the myosin motors directly, you can observe the filament movement caused by their action (on actin filaments). This assay helps understand the interaction between motor proteins and their tracks.
Optical Traps (Optical Tweezers):
Purpose: This method is used to measure the force generated by motor proteins, such as myosin, as they move along actin filaments.
Force Measurement: It can measure piconewton forces (10^-12 newtons) with high precision.
Mechanism:
A laser beam is focused to trap polystyrene beads at the focal point.
If two laser beams are separated by a distance, they can trap two beads.
These beads are placed between actin filaments, and the setup essentially functions as a force sensor.
Displacement of the beads occurs when the motor proteins (like myosin) apply force, moving the actin filaments and causing the beads to shift.
By analyzing how much the beads are displaced, researchers can measure the force applied by the motor proteins and directionality of the movement.
The stiffness of the system can also be inferred. When myosin attaches to actin, it increases the stiffness of the system, causing the system to become less “noisy” and more rigid. This change in noise can help quantify motor activity.
Trace Length: The length of the trace made by the bead gives insight into how long myosin stays attached and its working stroke, i.e., the amount the motor moves the actin filament in one cycle.
Fluorescence Microscopy:
Purpose: This method is used to track the real-time movement of motor proteins or filaments, such as actin, by tagging them with fluorescent markers.
Process:
Fluorescent molecules are attached to the motor domains or actin filaments.
These molecules emit light when excited, allowing the tracking of the motor proteins’ movement along the filaments.
This technique is especially useful for studying dynamic motor protein behavior in live conditions.
Inhibiting Myosin Function:
Purpose: This method helps to confirm the essential role of myosin in actin filament movement.
Process:
By inhibiting myosin activity (using specific inhibitors), it is observed that all actin filament movement stops, demonstrating the importance of myosin motors in driving this movement.
*check ppt to see what each method looks like
