L24: Cytoskeletal Motor Proteins

Myosin

Myosin = actin-based motor proteins

• Couple energy from ATP hydrolysis

• Mechanochemical enzymes = convert chemical energy → mechanical energy

• (+) end directed motors = move toward (+) end of actin

  • EXCEPTION: Myosin VI (6) moves toward (-) end of actin

Explain the structure of Myosin complexes

Myosin = 2 heavy chains + 4 light chains

• Myosin heavy chain = Head, neck, tail domain

  • Head domain = actin-binding + ATPase activities

    • Determines direction of movement

  • Neck domain = myosin light chain binding site

    • Structural + regulatory roles

  • Tail domain = different b/w different myosin

    • Determines specific properties

• Myosin light chain = small polypeptide that binds to + stabilizes “neck” domain of myosin heavy chains

What do myosin tail domains define? Explain functions of Myosin I, Myosin II, and Myosin V

Specific properties of myosin determined by tail domains

• Myosin I = bound to membrane ≠ dimerize, single-headed

  • Tails = different

    • Some have 2nd binding site → sliding

    • Some have membrane binding sites → bind to vesicles/organelles

      • Vesicle, membrane association, endocytosis

• Myosin II = assembled into bipolar filaments = contraction (muscle contraction, contractile ring)

• Myosin V = dimerized, organelle transport

  • Oddball = does processive movement (single motor molecule to take multiple steps along filament w/o detaching)

Which myosin have functions that are not as well understood?

Myosin III, IV, and VI-XV

• Conserved head domains

• Variable tail regions

Which myosin is the only known myosin to move towards the (-) end of F-actin?

Myosin VI (6)

Myosin II = conventional myosin

Myosin II = conventional myosin = first myosin discovered, most abundant type in muscle + non-muscle cells

• 2 heavy chains + 4 light chains = 6 proteins total

• Tails = long α helices that mediate further polymerization of Myosin II → bipolar thick filaments

Explain how actin filament sliding assay works

How does ATP affect movement?

What 2 ideas does this sliding assay support?

Actin filament sliding assay = demonstrate motor function of Myosin

• Myosin molecules stuck to glass (tails bound, heads free)

• Fluorescent actin filaments added (labeled w/ phalloidin)

  • Filaments stick to Myosin on glass

• ATP

  • No ATP → myosin binds tightly to actin

  • ATP → myosin moves actin

• Myosin walk to (+) end & are bound to glass

  • (-) end is leading movement

  • MUST BE BOUND TO @ LEAST 2 MYOSIN (directionality)

1. ATP hydrolysis = coupled to Myosin motility

2. Myosin ATPase activity = actin activated

   1. Actin absent = 4 ATP/hour

   2. Actin present = 20 ATP/hour

      1. Ensures myosin = active + using ATP w/ actin presence

Explain the myosin-actin cross bridge cycle + 5 stages

Myosin-actin cross-bridge cycle = fundamental, repeating process of muscle contraction where myosin heads bind to actin filaments, undergo a “power stroke” to pull them inward + detach, driven by ATP

Only 1 head can be undergoing process @ one time

1. Rigor state

   1. ATP binding site = empty

   2. Myosin = tightly bound to actin

      1. Rigor mortis of dead body = all muscles = tense (no ATP) → all myosin tightly bound to actin

2. ATP binding

   1. ATP binding cleft = closes

   2. Actin binding cleft = opens

      1. Interaction w/ actin = weak → myosin lets go of actin

3. ATP hydrolysis

   1. ATP hydrolysis: ATP → ADP

   2. Δ Conformation → head moves forward, head binds to actin

      1. Head moves to new position BEFORE rebinding to filament

4. Pi release

   1. Power stroke = myosin head pulls actin thin filament towards center of sarcomere (powered by release of Pi)

5. ADP release

   1. Myosin = rigor state

   2. ATP exchange releases head from actin

   3. Mechanism = still debated

What is special about myosin V dimers?

Myosin V dimers = unconventional

• Processive movement = 2 myosin V heads coordinated to interact w/ same actin filament

  • @ least 1 head always in contact w/ actin

• Allows myosin V to carry cargo

Myosin II cannot move processively

• 2 heads act independently

What are the 2 models for Myosin V movement?

How can the step size of Myosin V be used to determine the correct model?

Processive movement = motor can walk on filament w/o detaching/falling off

1. Hand-over-hand = rear detaches → moves ahead of other head → alternate positions

2. Inchworm = one head consistently in front

Myosin V step size = 72 nm = 36 nm b/w 2 myosin heads x 2

• Fluorescent tracking experiment = track position of1 myosin head over time

• 72 nm = Hand-over-hand model

What is the major difference b/w conventional and unconventional myosin?

Myosin II = conventional myosin = heads independently move

Myosin V = unconventional myosin = heads coordinate movement

What diseases can be caused by mutations in myosin genes?

MYH9 gene → Myosin IIA → bleeding problems, hearing loss, kidney disease, cataracts

Arg702 → decrease platelets, early-onset renal disease, hearing loss in infancy

Myosin VI ablation = deafness in mice

Myosin VII mutation → deafness + blindness in humans

What was used to isolate two classes of MT motor proteins?

In vitro motility assays

• Kinesins = similar ATP binding/hydrolytic core domain to myosin

• Dynein = unique catalytic core

Kinesins definition + 3 classes

Kinesins = MT-activated mechanochemical ATPases

• Kin-N Kinesin = motor domain @ N-terminus of protein

  • → (+) ends

  • Conventional kinesins

• Kin-C Kinesin = motor domain @ C-terminus of protein

  • → (-) ends (rare)

• Kin-I Kinesin = INTERNAL motor domain

  • Do not move along MTs

  • Bind MT ends → protofilament peeling

  • Kinesin-13

Describe structure of conventional kinesin

Conventional Kinesin = first Kinesin discovered + best characterized

• 2 heavy chains + 2 light chains

• Heavy chains

  • Motor domain head @ N-terminus

  • Neck domain

  • Tail/stalk domain = α-helical coiled-coil

• Light chains

  • @ C-terminus of tail region

  • Opposite to motor heads

  • Bind to cargo (organelles + vesicles)

Explain how the microtubule gliding assay works

MT Gliding Assay = demonstrate Kinesin motor function

• Tails = attached to coverslips

• Head = exposed

  • Fluorescent MTs moved around by head

• Generally = Kin-N Kinesins (walk to (+) end)

  • (-) end MT = leading movement

• Kin-C Kinesin (walk to (-) end)

  • (+) end MT = leading movement

Explain the kinesin cross-bridge cycle

ATP hydrolysis + Pi release → associated w/ Δ conformation of head/neck

1. ATP binds to forward head → Δ conformation in neck linker

2. Rear head swings forward past front head

3. New forward head release ADP

What kind of movement do kinesin motor proteins do?

Processive movement = movement over long distances w/o dissociating

• Important for long-distance transport of vesicles + organelles

Define dyneins + describe its structure + 2 types

Dyneins = mechanochemical enzymes that use energy from ATP hydrolysis to (-) end directed movement along MTs

Structure = 2-3 heavy chains w/ unknown # of intermediate + light chains

• Microtubule binding domain = 2 “feet” walking on MT

• ATPase domain = bind + hydrolyze ATP → Δ conformation

• Dynactin binding domain = bind to dynactin complex

  • Dynactin complex = intermediate b/w dynein + cargo

Cytoplasmic Dynein = most common form, many cellular functions

Ciliary/Flagellar Dynein = molecular motor → MT sliding → bending motions of cilia + flagella (microscopic hair-like appendages on eukaryotic cell surface → facilitate movement)

Describe power stroke of dynein + how it can be observed

Dynein power stroke = driven by ATP hydrolysis

Electron micrographs before + after power stroke

• Δ angle b/w stem + stalk domains

Dynactin complex

Dynactin complex = multiunit protein complex = intermediate b/w Dynein and cargo

• Dynein DOES NOT directly bind to cargo

• Critical for cytoplasmic Dynein function

• Mediate attachment of Dynein to cargo

• ~11 proteins

What part of dynactin complex is used to attach to surface of vesicle cargo?

Actin Related Protein 1 (Arp1) filament of dynactin complex → interact w/ Spectrin/Ankyrin membrane complexes of vesicle cargo membrane

Microtubule motors in disease

• Kinesin deficiencies → Charcot-Marie-Tooth Disease, some kidney diseases

• Dynein deficiencies → chronic respiratory tract infections (cilia ≠ function w/o dynein)

• Dynactin Complex mutations (p150^Glued subunit) → familial + sporadic ALS

• Ciliary + Intraflagellar Transport defects → autosomal recessive polycystic kidney disease (ARPKD), retinal degeneration, other sensory disorders

Explain melanosomes movement

A. Where are the microtubule (+) ends and (-) ends?

B. Are Kinesins or Dyneins important for melanosome localization in high cAMP?

C. Are Kinesins or Dyneins important for melanosome localization in low cAMP?

↑ cAMP → dispersed melanosomes

↓ cAMP → aggregated melanosomes

A. (-) ends = MTOC/centrosome, (+) ends cell periphery

B. ↑ cAMP → dispersed melanosomes → Kinesins

C. ↓ cAMP → aggregated melanosomes → Dyneins