MCB 2210 L23: Actin Binding Proteins + Microtubule Associated Proteins

Define the 3 actin drugs and 3 microtubule drugs used to study polymerization

Actin

1. Phalloidin → stabilizes actin

2. Cytochalasin B → depolymerizes (capping)

3. Latrunculin → depolymerizes (sequestering)

Microtubules

1. Taxol → stabilizes MTs

2. Colchicine → depolymerizes MTs (capping)

3. Nocodazole → depolymerizes MTs (binding)

What are 4 factors that actin binding proteins (ABPs) control?

1. Actin assembly

2. Actin disassembly

3. Actin organization

4. Actin movement

What are 2 types of actin binding proteins that bind to both monomers & filaments and nucleate actin?

What are 2 types of actin binding proteins that bind to monomers and control actin polymerization?

1. Formins = nucleate unbranched actin

2. Arp2/3 complex = nucleates branched actin w/ WASP protein

1. Profilin = actin polymerization (Exchange factor, ADP-actin → ATP-actin)

2. Thymosin β4 = sequesters protein → decrease actin polymerization → depolymerization

What are 2 types of actin severing proteins?

What 2 types of actin capping protein?

What is a common actin stabilizing protein in muscle cells?

1. Cofilin = sever (-) ends of actin

2. Gelsolin = sever actin filaments → cap (+) ends actin

   1. Increase [Ca²⁺] activates gelsolin

1. CapZ = caps (+) ends of actin

2. Tropomodulin = caps (-) ends of actin

1. Tropomyosin = stabilizes actin filament in muscle cells (actin = contractile muscle)

   1. Binds to sides → less likely G-actin monomer depolymerizes

What are 3 types of actin cross-linking proteins?

1. Fimbrin = rigid, parallel, tightly bundled actin (protein = short)

2. α-actinin = rigid, parallel, not as tightly bundled actin (protein = longer)

3. Filamin = flexible, gel-like actin networks

What are the 2 types of actin membrane-linker proteins?

1. Spectrin = link actin to membrane

2. ERM Family proteins = link actin to membrane

Describe how ABPs control assembly, disassembly, & organization of actin filaments

Help push membrane outwards

1. Arp2/3 Complex + WASP protein → branched actin

2. Filamin = stabilizes actin branchesx

3. CapZ = actin capping protein = caps (+) end of actin

4. Cofilin = actin severing protein → ADP-G-actin

5. Profilin = actin binding protein → ADP-G-actin → ATP-G-actin

What are cross-linking proteins + 2 types of longer, more flexible spacers, and 3 types of shorter, more rigid spacers?

Cross-linking proteins = 2 actin-binding domains separated by spacer domain

• Longer, more flexible spacers → gel-like networks

  • Filamin

  • Spectrin

• Shorter, more rigid spacers → tight bundles

  • Fimbrin

  • Villin

  • Fascin

• α-actinin = crosslink filaments → parallel/antiparallel bundles → contraction

What are membrane-actin linker proteins and what are 3 types?

Membrane-actin linker proteins = attach cytoskeleton → plasma membrane

• ERM proteins → link b/w F-actin + integral membrane proteins

• Spectrin → connect actin + membrane proteins

• Dystrophin → connect actin + muscle cell membrane

Describe arrays of actin bundles that α-actinin can form

Form antiparallel arrays in contractile structures (stress fibers or sarcomeres)

Define cell cortex and primary actin binding protein associated with it.

What actin binding protein links actin to membrane of microvilli?

Cell cortex = actin-rich layer beneath plasma membrane

Spectrin = closely associated w/ cell cortex

Ezrin = links actin to microvilli membrane

What genetic mutation can lead to easily ruptured RBCs or hereditary spherocytic anemias?

What genetic mutations can lead to muscular dystrophy?

Spectrin-based skeleton of cell cortex = critical for RBC shape

• Mutations in spectrin → easily ruptured RBCs

Dystrophin = actin cross-linking protein = link actin in muscle → membrane proteins

• Mutations in dystrophin → muscle cell membrane weakens → ruptures

Define functions of 3 microtubule associated proteins. γ-TURC, +TIPS, Stathmin

γ-TURC = nucleating protein @ centrosome, associated w/ (-) end

+TIPS = bind to GTP cap of MTs @ (+) end (Ex. EB1)

• Can link to membrane

• Fluorescently tagged to demonstrate MT (+) end dynamics

Stathmin = fragments of MT dimers

• Bind subunits → prevent MT assembly

What are 2 types of microtubule associated proteins that depolymerize microtubules?

1. Kinesin-13 = MT motor protein that depolymerizes @ MT end

   1. Force tubulin dimers → Δ conformation → catastrophe

2. Katanin = severing protein → depolymerizes @ MT middle

   1. CHOPS → catastrophe @ segment ends

What are 3 types of microtubule associated proteins that stabilize MTs?

1. MAPs = stabilize MTs by binding along sides

2. Tau = stabilize MTs by linking MTs → tightly bundled MTs

   1. Taiwan = country on a MAP

3. MAP-2 = stabilize MTs by linking MTs → wider bundled MTs

What microtubule associated protein links microtubules to intermediate filaments?

Plectin

• Pleakley = alien = IF MT (martians)

Describe mechanism of Kinesin-13 and Stathmin

Kinesin-13 = depolymerization @ MT end “13 = unlucky”

1. Strain protofilaments → curls

2. Peels away

3. DOES NOT INVOLVE GTP CAP HYDROLYSIS

Stathmin = disassembly from (+) end “Stalling”

1. GTP cap = GONE (rate of polymerization < rate of hydrolysis)

2. Bind when protofilaments = exposed

3. Take tubulin heterodimers out of circulation

Define polymer-binding MAPs + structure + 2 examples

Polymer binding MAPs = stabilize MTs by binding to sides, enhance assembly (stabilize nuclei), organize MTs → bundles, mediate MT interactions w/ other proteins (Ex. Intermediate filaments + actin)

Polymer-binding MAPs = 2 major domains

• MT binding domain = binds tubulin dimers together → stabilize MT

• Projection domain = interact w/ MTs + other structures (Ex. intermediate filaments)

Tau + MAP2 = organize MTs in neuronal axons + dendrites

Spacing b/w MTs = greater for MAP2 than Tau

• MAP2 (longer projection domain)

Note MAP2 + Tau = expressed in cells that do not normally form axons → long axon like structures are still induced

What do MAPs help do to microtubules?

Stabilize microtubules + enhance growth by suppressing frequency of catastrophes → enhance growth rate

Which MAP is associated with Alzheimer’s disease? What are the is the two-step process that can explain this?

Tau = abundant in neurons + stabilizes MT

• Tau hyperphosphorylation → reduces binding of Tau to MTs

  • Tau becomes “sticky” → accumulates + clumps

• Sequestration of hyper-phosphorylated Tau into Neurofibrillary Tangles (NFTs) = reduces amount of Tau available to bind to MTs

• Reduced Tau binding to MT → MT instability + reduced axonal support

  • Could contribute to Alzheimer’s disease