Lecture 7: Cytoskeleton

Question 1

List the 3 components of cytoskeleton (and their approximate sizes)

Answer 1

• Microfilaments (actin) (7 nm thick)
• Intermediate filaments (8-10 nm thick)
• Microtubules (25 nm in diameter)

Question 2

Describe the characteristics of actin microfilaments

Answer 2

• Highly conserved among eukaryotes
• Are 7 nm thick, or at least, filaments may be up to 7 μm in length.
• Filaments are organized into bundles and 3-D networks. (Such as sarcomeric actin bundles)
• Bind to specific transmembrane proteins either directly or indirectly
• Exist as monomers (G-actin) and long chains (F-actin)
• Constitute three major varieties:
• α-actin(found in muscle tissue), β-actin(non-muscle actin), and γ-actin(non-muscle actin)

Question 3

How are actin microfilaments polymerized/nucleated?

Answer 3

• Each actin monomer (G actin) has a binding site for ATP, which binds tightly to G-actin.
• Each actin monomer can bind tightly with two other actin monomers to form filamentous actin (F-actin).
• Because all actin monomers are oriented in the same direction, actin filaments display polarity.
• The first step in actin polymerization is nucleation:
• (A trimer is formed.
• Additional actin monomers can then be added to either end. )
• Actin polymerization is reversible.
• ATP-actin associates with the growing (plus or barbed) ends, and the ATP is hydrolyzed to ADP following polymerization.

Question 4

Describe the rate of polymerization and treadmilling.

Answer 4

• The rate at which monomers are added to the growing filament is proportional to the cytosolic concentration of actin monomers.
• ADP-actin dissociates more readily from filaments than ATP-actin.
• The rate of addition of new G-actin to actin filaments occurs more rapidly and at a lower concentration at the plus (barbed or polymerization) end.
• The barbed (+) end grows 5-10 times faster than the pointed end.
• The minus end (pointed or depolymerization end) is the slower growing end.
• Very low concentrations of G-actin favor the disassembly of actin filaments.
• Intermediate concentrations favor a dynamic equilibrium between the minus end and the plus end = treadmilling.

Question 5

What exactly is occurring during treadmilling?

Also, review slide 9 for polymerization picture

Answer 5

• Treadmilling results in equlibrium and zero net growth.
• Higher concentrations of G-actin favor net addition at both ends and, therefore, growth of the actin filament.
• Actin microfilaments consist of a double helical chain of G-actin subunits.

Question 6

Describe 2 drugs that affect Actin polymerization

Answer 6

Cytochalasins:
- Bind to barbed ends
- Block elongation
- Can inhibit movements (e.g., cell division)
Phalloidin:
- Binds to actin filaments and prevents dissociation
- Can be labeled with fluorescent dyes to allow visualization of actin filaments

Question 7

Actin is so versatile because it itself doesn’t dictate it’s function. That’s the job of it’s actin binding proteins it’s associated with.
Name and describe the first 6 actin binding molecule/proteins?

Answer 7

• Spectrin: Found in RBCs, Binds cortical cytoskeleton to the plasma membrane
• Dystrophin: Binds cortical cytoskeleton to the plasma membrane
• Villin and Fimbrin: Cross-links in microvilli
• Calmodulin and Myosin I: Cross-links actin to plasma membrane in microvilli
• α-Actinin: Cross-links stress fibers and connects actin to protein-plasma membrane complex complexes
• Filamin: Cross-links actin at wide angles to form screen-like gels.

Question 8

List the “first 3” actin binding proteins that control treadmilling

Answer 8

• Thymosin: Captures actin monomers; prevents monomers from being polymerized.
• Profilin: Binds to actin monomers and prevents monomers from being polymerized. Facilitates exchange of bound ADP for ATP—which favors polymerization. Note that only ATP-actin monomers can be assembled into F-actin.
• Gelsolin: Destabilizes F-actin and caps actin filaments, preventing loss and addition of G-actin. In presence of Calcium ion, fragments actin filament and remains bound to plus end.

Question 9

List the other 4 actin binding molecules that control treadmilling

Also, review diagram on Slide 14 - Lecture 7

Answer 9

• Cofilin: Triggers depolymerization of ADP-bound actin at the minus end
• Arp2/3: Initiates growth of F-actin from sides of existing filament—causes branching
• Phalloidin: Prevents depolymerization by binding to actin filaments.
• Latrunculins: Binds to G-actin and induces F-actin depolymerization

Question 10

Give the characteristics of intermediate filaments

Answer 10

• Are 8-10 nm thick
• Are abundant in cells subject to mechanical stress
• Provide tensile strength in cells such as neurons and muscle.
• Strengthen epithelial cells as desmosomes and hemidesmosomes.
• All have a common monomer consisting of a central α-helical rod flanked by head and tail domains.
• Head and tail domains determine specific functions.
• Intermediate filament functions: Form a cytoplasmic network in most cells, and associate with other cytoskeletal elements to form a scaffolding that organizes the internal structure of the cell.

Question 11

Describe intermediate filament assembly

Review slide 18 on Lecture 7

Answer 11

• Central rod of two polypeptides form a coiled dimer.
• Rods are aligned tail-to-tail and head-to-head.
• Dimers associate in a staggered antiparallel fashion to form tetramers.
• Because of the antiparallel association of the dimers, polymerized filaments do not have distinct ends.
• Therefore, they are more stable than actin and do not demonstrate dynamic behaviors such as treadmilling.
• Tetramers assemble end to end to form protofilaments.
• Eight protofilaments are wound together to form filaments.

Question 12

Loosely describe the intermediate filament types

Answer 12

• Type I and Type II are the keratins.
• Type III is a bunch of different crap.
• Type IV are straight neurofilaments
• Type V is lamins (Not to be confused with laminins. Those are different.)
• Type VI: Nestin - more on this much later.

Question 13

Describe the characteristics of microtubules

Review picture on slide 20

Answer 13

• 25 μm in diameter
• Composed of tubulin dimers: Alpha unit + beta unit
• Protofilaments are longitudinal rows of tubulin dimers.
• Microtubules consist of 13 protofilaments arranged parallel to form a cylinder with a hollow core.
• Protofilaments have a fast growing plus end and a slow growing minus end. Like actin microfilaments.

Question 14

Describe treadmilling and dynamic instability in microtubules

Answer 14

• Tubulin dimers with GTP bound to the β-tubulin associate with the growing end.
• Plus end: Grows more rapidly than minus end in presence of low calcium ion concentration.
• After polymerization, GTP is hydrolyzed to GDP and the tubulin is less stable.
• Dimers at the minus end dissociate.
• At high concentrations of tubulin-GTP, the dimers are added more rapidly than GTP is hydrolyzed, and the microtubule grows.
• If concentration of tubulin-GTP drops, GTP at the plus end is hydrolyzed and dimers are lost. So a lot like actin. Except uses calcium ions (not sure why) and GTP

Question 15

What are the functions of the cytoskeleton?

Answer 15

• Cell movement
• Support and strength for the cell
• Phagocytosis
• Mitotic spindle formation
• Cytokinesis
• Cell-to-cell and cell-to-extracellular matrix adherence
• Changes in cell shape

Question 16

What is the role of microtubules in mitosis?

See slide 27 in Lecture 7

Answer 16

This question’s a little confusing but I think…
Within the mitotic center, there’s the microtubule organizing center. Which is held in place by Radiating microtubules.
There’s two mitotic centers on either side of the cell, and the mitotic spindle(s) connect the two together.
The spindle is made of two types of microtubules: Kinetochore Microtubules are anchored to the centromere of metaphase chromosomes. Polar microtubules run along the sides of the cell membrane and overlap with each other at the center of the cell. These aren’t attached to any chromosomes.

Question 17

Describe the role of intraciliary transport

See Slide 29 - Lecture 7

Answer 17

1. Anterograde transport of cargos along a microtubule is mediated by Kinesin
   - At the tubulin assembly site is where raft proteins, ciliary axonemal proteins or any other material is returned to the body
   - A raft protein may provide a mechanism for the transport of multiple cargos.
2. Retrograde transport of cargos along a tubule is mediated by cytoplasmic dynein
3. At the basal body, disassembly of the raft protein complex-cargo-molecular-motor-machinery.

Question 18

Describe the role of microtubules and motor proteins in axonal transport

Answer 18

1. From the minus end to the positive end, anterograde transport along a microtubule is mediated by kinesin. (Towards the synapse)
2. From the plus end to the minus end (away from the synapse, towards the neuron), retrograde transport of a vesicle along a microtubule is mediated by cytoplasmic dynein

Question 19

Experiment:
Myosin I: (# of heads, what the tail binds to, what the head binds to, direction of the head moves towards the.._)
Same for Myosin II
Kinesin
Cytoplasmic Dynein

Answer 19

• Myosin I: (One head, tail goes to the cell membrane, head binds to actin, Head moves towards the Barbed (+) end
• Myosin II: (Two Heads, Tail binds to myosin II, Head Binds to Actin, Head Moves towards the Barbed (+) end)
• Kinesin: (Two Heads, Tail binds to vesicle, head binds to microtubule, head moves towards the Plus End)
• Cytoplasmic Dynein: (Two Heads, Tail binds to vesicle, head binds to microtubule, head moves towards the Minus End)

Question 20

Describe myosin II phosphorylation

See Slide 32, Lecture 7 for Review

Answer 20

• In skeletal muscle, the regulation of actin-myosin interaction is mediated by Calcium ions binding to troponin
• In smooth mucle and non-muscle, contraction is regulated by phosphorylation of one of the myosin light chains.
• Inactive myosins have their tail regions folded back and closely apposed (whatever that means) to the myosin head.
• After the myosin light chain is phosphorylated, inactive myosin is activated so that their tails are stretched out.