Lecture 2 - Cytoskeleton II

Question 1

Define the three types of accessory proteins associated with cytoskeletal filaments

Answer 1

1. Motor Proteins - use ATP turnover to move along surface of filament
2. Proteins that influence filament dynamics (e.g., Polymerisers, Depolymerisers)
3. Proteins that influence filament structure (e.g., Branching/Severing)

Question 2

How do Motor Proteins move along the surface of filaments?

Answer 2

Couple ATP turnover to filament binding/unbinding, and to force generating conformational change

Question 3

Define the three main families of motor proteins, and the cytoskeletal filaments they bind to

Answer 3

• Actin Filaments - Myosins
• Microtubules - Kinesins and Dyneins

Question 4

What are Myosins? What are their Functions?

Answer 4

• Large Family of proteins (>40), which are defined by charactistic motor domain that binds to ATP and the actin microfilament
• Couple ATP Hydrolysis to their movement relative to actin, which is used to slide actin (e.g., Muscle Contraction) or to transport cargo

Question 5

(monomers, dimers, polymers?)

How can Myosins function?

(2 Points)

Answer 5

• Monomers - work in teams (e.g., Muscle Contraction)
• Dimers - translocate using hand-over-hand motion (e.g., cargo transport)

Question 6

What are Kinesins? How do they Function?

Answer 6

• Large Family of proteins (>45), which are defined by characteristic motor domain, that is structurally similar to myosin motor domain, and binds to ATP and MTs
• Function as either monomers, dimers, or tetramets

Question 7

What are the two groups of Kinesin proteins?

Answer 7

• Translocating - move directionally along MTs, typically towards +end (Exception - Kinesin 14)
• Regulating - bind to MT ends, and influence filament dynamics

Question 8

How do Dimeric Translocating Kinesins move along MTs?

Answer 8

Hand-over-Hand Motion, with each step moving along MT one tubulin heterodimer (8nm), and consuming 1 ATP

Question 9

What are the Dyneins?

(3 Points)

Answer 9

Family of -end directed motor proteins, consisting of two major branches
1. Cytoplasmic Dyneins - present in all eukaryotic cells, involved in cargo transport
2. Axonemal Dyneins - found in axoneme of cilia/flagella, facilitate motion

Question 10

What is the General Structure of Dynein?

Answer 10

Complex of one or more heavy chains (500kDa) and a variable number of light and intermediate chains

Question 11

What is the Characteristic Unit of Dynein?

Answer 11

Planar Ring consisting of 7 domains:
* 6 AAA ATPase domains - ATP binding (present in one heavy chain)
* 1 Microtubule-binding domain

Question 12

What are Actin Capping Proteins? How do they perform their function?

(3 Points, Give Examples)

Answer 12

Group of proteins which control actin filament length by binding to the ends of the filament and inhibiting dynamics
* Determine filament length by either:
1. Stabilising Actin Filament (e.g., Tropomodulins - caps -ve end, preventing monomer dissociation)
2. Promoting Disassembly (e.g., Capz - binds to +ve, inhibiting polymerisation)

Question 13

What are Formins?

Answer 13

Dimeric actin polymerisers that nucleate and remain associated with growing barbed +ends, protecting the filament against capping proteins and directing addition of actin (5-10 fold faster)

Question 14

Describe the Mechanism of Kinesin-Induced (Kinesin 13 Family) MT Depolymerisation?

Answer 14

• Depolymerising Kinesin undergoes diffuse interaction with MT lattice, then binds to the +/- ends
• Kinesin induces ATP-dependent depolymerisation of MT from both ends

Question 15

(Surfing)

How do MT Polymerizers accelerate MT Growth?

Answer 15

MT Polymeriser proteins (e.g., XMAP215 Family) move processively with growing MT tip (+end), accelerating addition of tubulin dimers

Question 16

What is the ARP2/3 Complex? What is its Function?

Answer 16

• Protein complex which Nucleates actin filament growth, and promotes branching
• ARP2/3 Complex - little intrinsic activity, but when engaged by nucleation promoting factors it functions to initiate formation of daughter filaments

Question 17

How does ARP2/3 Complex Facilitate Actin Branching?

Answer 17

Complex attaches to side of existing mother filament, and templates formation of Y-branch at regular 70º angle

Question 18

State the 3 Known MT-Severing Enzymes

Answer 18

Katanin, Spastin, Fidgitin

Question 19

Summarise the Mechanism of MT-Severing by Katanin

(3 Points)

Answer 19

1. Hexameric ring of Katanin Subunits assemble on MT (recruited by MT itself)
2. ATP-Hydrolysis - results in conformational change in Katanin, which destabilises tubulin-tubulin contacts
3. Tubulin tails may also be captured by MT-severing complex and removed from lattice via partial/total unfolding