Lecture 19: Muscle Cells and Myosin

What is the structure of muscles?

Muscles → bundle of muscle fibers → multinucleated muscle cell (individual muscle cells) → myofibril → sarcomere

How are sarcomeres attached?

end to end

What is the basic unit of a muscle?

a sarcomere

What are sarcomeres delineated by?

Z-disks

What attaches to Z-disks?

the plus ends of actin filaments

Where do the minus ends attach to?

the center of the sarcomere

What is in the middle of sarcomere?

bundles of myosin proteins (motor proteins)

True or False: Motor heads of myosin can pull the filaments—moving the filaments toward the center.

true

True or False: Z-disks are actually a cylinder (3-D structure).

true

What happens when myosin is not nailed down?

myosins will walk along the actin filaments with cargo

What happens when actin filaments are not nailed down and myosins are?

myosin motors are not able to move, but actin filaments able to move inwards (muscle contraction)

What type of motors are myosin?

plus-end directors

True or False: Myosin motors only move in 1 direction.

true

What did Kuhne discover in 1859?

myosin

What did Huxley discover in 1880?

A bands were lost after treatments which removed myosin; A bands were composed mainly of myosin

What did Szent-Gyorgui discover?

vitamin C

What was the glycerinated muscle fiber prep (Szent-Gyorgi)?

• Dissect out small bundles of mussel; place 50% glycerol at 0 C then transfer to -20 C (store for months)

• Plasma membrane is disrupted; soluble components leak out, but insoluble components of contractile apparatus remain intact

  • Glycerol prevents formation of ice crystals which disrupt structure

• Conclusion: Showed that purified actin/myosin filaments could contract after addition of ATP

What are the different types of light microscopy?

• Bright field

• Differential interference contrast

• Phase contrast

What was the contracting myofibril study with Huxley and Hanson?

• Blended glycerol-extracted psoas muscle to generate fine myofibrils (2 um)

• Imaged contraction with phase-contrast microscopy

• A bands remain constant length during contraction

• Extract myosin, A bands disappear

Who is credited for the sliding filament model?

Huxley and Hanson

What is the sliding filament model?

• Thick and thin filaments maintain constant length during contraction; slide past each other during contraction

• Experimental evidence from phase contrast microscopy; differential interference contrast microscopy; electron microscopy; X-ray diffraction patterns

What happens to actin in rigor mortis?

• When ATP is depleted, myosin heads bind tightly to actin, creating cross-bridges; muscle becomes rigid

• ATP can be added back and muscle will lose its stiffness

Where is myosin II found?

muscles

What are the 3 regions of myosin?

• Head region

  • Binds to actin filaments

  • Binds ATP and hydrolyzes ATP

• Neck region

  • Hinge at base of neck 

  • Head and neck can rotate–important for generating movement

• Tail region

  • Connects to the cargo

  • Binds to other myosins

What is the process for myosin movement?

1. Myosin binds ATP and head releases from actin

2. Hydrolysis of ATP to ADP + P causes myosin head to rotate into “cocked” position

3. Myosin head binds actin filament

4. Release of phosphate, elastic energy straightens myosin and moves actin filament left (the power move)

5. ADP is released and ATP bound, and the head releases from actin

True or False: Each step in the myosin movement process corresponds to a conformational change and functional properties of the myosin motor protein.

true

If sarcomeres only contract, how can we flex or extend our limbs?

• Opposing motions:

  • Flexion (bending of limb at joint)

  • Extension (straightening of limb)

• Different muscles are responsible for these opposing motions

  • Flexor

  • Extensor

  • Adductor

  • Abductor

What are the sarcomere caps?

CapZ and tropomodulin

Where is CapZ located?

near the Z-disk

What is Titin?

the “spring” that extends from one Z-disk to another and runs through the middle of the myosin thick filament

True or False: Titin is the largest know protein in the human genome, and winds during hyyperextension.

true

What is Nebulin?

the “ruler” to form filaments of fixed size; binds to and wraps around the actin filaments

What does Nebulin bind to?

one end binds to tropomodulin and the other end binds to CapZ

What keeps thick filaments centered during contraction?

the connection between the Z-disk and Titin

What domains of Titin provide elasticity, and what do these domains do under mechanical stress?

PEVK domains and unfold from mechanical stress

What component of the sarcomere is involved with limb-girdle muscular dystrophy?

telethonin

True or False: Muscles are maintained in a relax state, contraction occurs when brain or neurons send signal to muscle/

true

What is the relaxed state of muscles maintained by?

proteins that bind to actin and block the binding of myosin

What happens when there’s a signal for a muscle to contract?

the “blocking” protein will expose the myosin binding site—allowing myosin to bind actin and move the actin filaments

True or False: An electrical signal in the neuron is converted into a chemical signal within the cell.

true

Which type of protein blocks myosin from getting to the actin filament?

tropomyosin

What is important to know about tropomyosin?

• Winds around the actin filament

• Contains a Troponin complex

  • Set of 3 proteins

  • Binds to one end of tropomyosin

  • Can bind calcium

Where is there a low concentration of calcium?

inside of muscle cells

When is calcium let into the cells?

when there’s a chemical signal from the brain

What does calcium bind to?

troponin

What happens when calcium binds troponin?

there’s a conformational change in troponin that moves tropomyosin off of the myosin binding sites

What is the sarcoplasmic reticulum?

the network of tubes that surround the sarcomeres; a specialized ER

What does the sarcoplasmic reticulum store?

calcium

What is the transverse tubule?

invaginations of the plasma membrane

A neuron contacts the surface of the muscle cell and will travel down to the what?

the T-tubule

What happens when calcium is pumped out of the cell?

the muscle cell goes into a relaxed state

What is the process for releasing stored calcium?

1. Action potential

2. Channel opens and calcium rushes out

3. Calcium interacts with troponin

4. Pump uses ATP hydrolysis to put calcium into the ER

5. Channel in the T-tubulin is opened to let calcium in from outside of the cell

6. Pump will then push calcium of the cell again

How is calcium removed from the cytoplasm, and what happens to other proteins?

• A calcium “pump” in the sarcoplasmic membrane pumps the released calcium back into the sarcoplasmic reticulum to be stored

• Troponin and tropomyosin revert back to the conformation in which they block myosin binding

Most energy from cell movement comes from where?

contraction of stress fibers

True or False: The leading edge of stress fibers are nailed down more than the trailing edge, so the trailing edge is pulled forward/back of the cell.

true

What is important to know about stress fibers?

• Sarcomere-like structures

• Contains contractile bundles

• Bundles contain integrins (attaches to the ECM)

  • Integrins are similar to Z disks

• Focal adhesions = attachments from the integrins to the ECM

Did contractile bundles in stress fibers or sarcomeres evolve first?

Contractile bundles in stress fibers because individual cells could move before multicellular organisms/muscle cells could move

What are the basic steps in locomotion?

Extension, adhesion (contraction of contractile bundles), translocation, and de-adhesion and endocytic recycling

What does the contractile ring present during cellular division contain?

myosin proteins

What is the motor domain of the myosin?

the head region

True or False: The head and neck domain undergo conformational changes to result in movement.

true

Why does the neck/lever arm change conformation?

due to ATP hydrolysis

What pieces result with chymotrypsin cleaves myosin protein?

HMM (S1 and S2) and LMM

What is important to know about the in vitro sliding filament assay (Spudich)?

• Motor proteins are attached to coverslip on microscope

• Actin and ATP is added—resulting in actin movement

• Conclusion: motor proteins are plus-end directors

• Contributions: this assay allows other motor proteins to be purified

What are some characteristics of the myosin superfamily?

• Heads are similar

• Tails are different and have different functions

• Myosin drags cargo by attaching cargo to the tail

• Variate in monomer-dimer, lever arm length, and tail domain

What does myosin I do?

attaches to membranes and will therefore attach the membrane and the cytoskeleton together

What does myosin V do?

uses actin filaments as a roadway to move vesicles around

What can optical trap measure?

• Can measure how far a single myosin step is

• Can measure how much force is generated

• Can measure how fast myosin moves

What is important to know about an optical trap?

• Involves refraction (light changes direction when it goes through one medium to another)

• Laser hits clear plastic bead and changes direction (refraction)

• Laser hits the bead from both directions—resulting in no net force on the bead

• Bead gets caught on the laser and will move when the laser does

• Hold the laser in a fixed position and the bead is trapped in that position

• Laser tweezers move the laser beam and the bead follows along (it remains trapped in the moving beam)

What is the step size of a myosin protein?

36 nm per step or one half turn of the actin helix

True or False: Myosin only steps on the monomers pointing upwards.

true

What determines the step size and rate of motion of a myosin?

the lever arm

What model explains myosin V movement?

the “hand over hand” model

What is the hand over hand model?

the trailing head moves 72 nm forward

What is the distance between bound heads?

one half rotation of the actin helix

What is the distance moved by step of a single head?

one full rotation of the helix

What is meant by myosin moving processively?

myosin walks down the filament without failing off because one of the heads must be bound to the actin at all times

What is the myosin V movement process?

• 1. Leading head has ejected Pi and wants to undergo powerstroke but can’t because trailing head is bound

• 2. Trailing head releases ADP, ATP binds and trailing head lets go of actin

• 3. Release of trailing head allows leading head to undergo powerstroke

• 4. Powerstroke in leading head throws trailing head forward

• 5. Leading head hydrolyzes ATP to ATP-Pi

• 6. Leading head contacts actin -> binds and ejects Pi

• 7. Tension between monomers = conformation in which ADP released from the trailing head only

What throws the trailing head forward?

a conformational change of the leading head

What is the duty ratio?

the fraction of the ATP-ase cycle in which the myosin head is bound to actin

True or False: For a myosin to walk processively, the duty ratio must be above 50%. If it was below 50%, then there is a point where both heads are not bound to actin. Heads must be in different phases of the ATP-ase cycle.

true

What does yeast do with myosin V?

yeasts build roadways of actin for myosin to transport vesicles to the bud

What does cytoplasmic streaming occur due to?

the dragging of the ER along the cortical actin bundles—circulating much of the cytoplasm

Which parameters of the myosin superfamily have changed during evolution and resulted in the specialization for different tasks?

• Changed:

  • Duty ratio

• Constant:

  • Step size

  • Tail interactions

What is the duty ratio of myosin II and myosin V?

low and high, respectively