Lecture Notes on Myosin and Muscle Contraction

Understanding the structural basis of muscle contraction requires knowledge of both the sarcomere and the myosin proteins' structure and function.

Mechanochemical Enzymes
- Myosins are motor proteins that convert chemical energy into mechanical force.
- They belong to three superfamilies of motor proteins: myosins (interacting with actin), and kinesins and dyneins (interacting with microtubules, MTs).
- All motor proteins bind and hydrolyze ATP, utilizing the released energy to translocate along cytoskeletal filaments.
- Myosins specifically move toward the plus ends of actin filaments.
Structural Characteristics
- Myosin proteins have a basic structure comprising:
  - A tail region
  - A neck region
  - A globular head region (also referred to as the motor domain).
- The head region is highly conserved across myosin families and contains the sites for actin binding and ATP hydrolysis.
- The tail region varies among different myosins, allowing for specific cargo binding.
- Currently, 19 families of myosin proteins have been identified, with myosin-II being a focus because it is the myosin found in muscle cells.
- Myosin-II forms dimers with two globular heads and a long α-helical coiled-coil tail, creating a myosin filament consisting of numerous dimers.
- Myosin filaments are structured such that heads on opposite ends face different directions, forming a bipolar filament that constitutes the thick filament in muscle sarcomeres.

The Cross Bridge Cycle
- The cycle involves ATP binding, hydrolysis, and the release of ADP and inorganic phosphate (Pi), coupled with conformational changes in the myosin head, and interaction with actin.
- The cycle can be described in the following stages:
1. Rigor state: Myosin is bound to actin with no nucleotides, producing rigor binding, which is associated with rigor mortis.
2. ATP Binding: When ATP binds to the myosin head, the head releases from actin.
3. ATP Hydrolysis: The ATP hydrolysis to ADP and Pi causes a conformational change (“cocking”) in the myosin head.
4. Tight Binding: The release of Pi enables myosin to bind tightly to actin, leading to the power stroke.
5. Power Stroke: A conformational change occurs during tight binding which generates force and moves actin relative to myosin, followed by ADP release and returning to the rigor state.
Direction of Movement
- Muscle myosin only translocates toward the plus end of actin filaments, requiring an organized actin structure to determine the movement direction of cargo.

Structure of the Sarcomere
- The sarcomere is a fundamental unit of muscle that stacks to form myofibrils, interconnected within skeletal muscle cells.
- Each sarcomere is bounded by structures known as Z discs, which anchor the plus ends of actin filaments and stabilize them.
Interaction of Myosin and Actin
- The myosin thick filaments are located in the sarcomere's center and interact with the actin filaments from both sides.
- As myosin heads engage actin and generate force, the structure allows the pulling of both actin filaments toward the center, causing muscle contraction.
Mechanics of Sarcomere Contraction
- Each sarcomere shares Z discs with adjacent sarcomeres, contributing to a mechanically continuous chain spanning the myofibril.
- During contraction, all myofibrils activate their myosins synchronously, resulting in the overall contraction of the muscle cell, which enables movement through joints.

Tropomyosin and Troponin
- In resting muscle cells, tropomyosin blocks myosin binding sites on actin, preventing continuous contraction.
- Tropomyosin runs along the actin filament and interacts with 7 actin subunits.
- The troponin complex, located at the ends of tropomyosin, contains a calcium-binding protein.
- Calcium concentrations must rise to approximately $5 imes 10^{-5} M$ for myosin to gain access to actin's binding site.
Calcium Signaling
- Calcium spikes occur when muscle cells are stimulated by motor neurons, caused by action potentials that depolarize muscle membranes and are rapidly propagated through T-tubules.
- Voltage-gated calcium channels in the T-tubule membrane release calcium ions into the cytosol, where they bind to troponin, causing tropomyosin to shift, revealing myosin binding sites.
- The sarcoplasmic reticulum (SR), rich in Ca-ATPase complexes, pumps calcium ions back, ending contraction and returning the muscle to a resting state.

Mechanics of Organelle Movement
- Organelles move directionally due to motor proteins attaching to their surfaces and interacting with cytoskeletal filaments, thus employing ATP hydrolysis to translocate along them.
- Mostly, organelle transport occurs on MTs, but some use actin filaments, particularly myosin-V.
Structure of Myosin-V
- Myosin-V possesses a shorter α-helical tail and forms dimers that bind organelles and translocate them toward the plus ends of actin filaments.

Diverse Myosin Functions
- Myosin-II functions in sarcomeres for muscle contraction, while it can also facilitate actin contraction at smaller scales in non-muscle cells, like during cell division.
- Myosin-I is a monomeric form involved in structures such as microvilli, aiding in membrane interactions and creating rigidity due to its pulling on actin filaments.
Understanding Motor Activities
- Any motor protein-based movement should be evaluated for its specific movement goal, filament organization, motor properties, and the tail's anchoring.
- Distinct situations arise where the motor could be anchored to an immovable structure or a movable organelle, impacting the resultant force and direction.
Filament Polymerization as Movement
- Movement can also occur via filament polymerization, evident in cell processes like lamellipodia extension or by pathogens such as Listeria that leverage cytoskeletal dynamics.

Describe myosin motor protein structures and functions and illustrate the mechanochemical cross-bridge cycle of myosin-II.
Depict skeletal muscle cell structures, focusing on the sarcomere and explain actin-myosin regulatory interactions during muscle contraction, emphasizing calcium ion roles.
Illustrate the arrangements involved in organelle movements, inter-filament actions, and the fusions towards the plasma membrane using motor protein principles.