Midterm 2 Flashcards by Y. David Zhu

What is the difference between filopodia and lamellipodia? In a neuron, where can these structures be found?

Filipodia are small extensions while lamellipodia are broad extensions. In the growth cone where they facilitate motility.

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(T/F) Actin is a globular protein that hydrolyzes ATP to ADP.

True

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What are the plus and minus ends also known as, respectively?

Barbed end (+) and pointed end (-). The pointed end is so because it looks like the end of an arrow.

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What is G-actin? F-actin?

G - globular, F - filamentous

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What is the role of Pyrene?

What is it attached to?

It is a fluorescent tag used to study actin.

Pyrene is conjugated to Cys374.

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How many times greater is the intensity of Pyrene from polymerized actin versus monomeric form?
How is polymerization initiated?

20 times.

Addition of 100mM KCl (physiological salt concentration).

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How many molecules of actin are required for nucleation?

Three are needed to stabilize the group.

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What do actin-binding proteins do?

Bypass the nucleation step to rapidly deploy actin filaments.

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What type of relationship is the assembly of actin in terms of monomer concentration?

Linear. y=mx+b

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What is the critical concentration? Is the critical concentration for the plus and minus ends of actin the same?

Critical concentration is the concentration below which the actin filament will begin to depolymerize. No, the minus end has a higher critical concentration than the plus end.

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What is treadmilling?

At a concentration when the plus and minus ends of actin are the same rate so the filament appears to be the same length.

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(T/F) If the condition is correct, the minus end can polymerize and depolymerize faster than the plus end.

False

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What is one way you can keep track of how old an actin filament is?

See how many of its ATP-actin has been hydrolyzed to ADP-Pi-actin.

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In regard to the orientation of intracellular actin filaments, which end faces the cell body?
Which faces the leading edge?

Minus end face the cell body.

Plus end face the membrane.

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What is the significance of the integrin molecular clutch that engages the actin filaments?

Engagement of the clutch allow the actin filament to propel forward. Otherwise, there will be a retrograde flow.

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What have we learned about actin polymerization through the Listeria model?

Actin binding proteins speeds up actin polymerization tremendously.

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Which Listeria surface protein is responsible for actin polymerization?

ActA

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With regard to Listeria movements, which end, plus or minus, faces the bacterium?

Plus end

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What is the mechanism of Arp2/3? (2)

Which organism is Arp2/3 found in?

(1) Nucleates actin filament by binding to the side of actin filaments and (2) initiates polymerization by capping minus end.
Listeria

(T/F) Arp2/3 requires ActA.

True

What is the mechanism of ADF/cofilin?

How does this increase the chance of dissociation?

Binds to ADP-actin (“rare” conformation) which destabilizes the conformation and severs the filament.
By generating two uncapped ends.

What is thymosin?

A monomer binding protein that increases the critical concentration of actin in the cytosol. (i.e. inhibits polymerization)

What is profilin and what does it do?

Profilin, like thymosin, is a monomer binding protein that can (1) promote monomer addition to barbed-end to decrease critical concentration, (2) prevent monomer addition to point-end, (3) inhibit spontaneous nucleation, and (4) catalyze nucleotide exchange from ADP to ATP. (i.e. promotes polymerization)

What property of the cell do actin cross-linking proteins contribute to?

Viscoelasticity

Which end does formin bind to?

The barbed, or plus, end.

What is the significance of formin-profilin complex

Formin, bound to the plus end of the actin filament, has unstructured domains or "whiskers" for profilin-actin monomer binding that may, under certain conditions, enhance filament polymerization.

Where is ActA found? | What does it do?

ActA protein is found on the surface of the bacterium cell. | It activates the Arp2/3 complex to nucleate new filament assembly along the sides of existing filaments.

What is the Classic Brownian Ratchet model?

Through Browninan Motion, space is created between the bacterium and actin filament. Elongation of the filament results in the propulsion of the bacterium in a forward direction. However, this model did not match experimental data.

What is the Elastic Brownian Ratchet model? | How is the different from the Classic Browninan Ratchet model?

The filament is wiggling because of its elastic properties that creates space between itself and the bacteria. This results in monomer entery and actin filament elongation to propel the bacterium. In this model, there is an angle between the actin filament and the bacterium.

What is N-WASP?

An endemic protein closely related to WASP that activates the Arp2/3 complex and, thus, expedite actin polymerization.

What is significant about the VCA region of WASP?

It is the activation site of Arp2/3 complex. This region is protected during the inhibited phase of the protein (e.g. "closed" configuration) during which it can bind to Arp2/3 but cannot initiate actin polymerization.

What is PIP2 and CDC42? Where are they located?

These two proteins are located on the plasma membrane and recruit/activate the WASP protein, which leads to actin polymerization.

(T/F) CDC42 is a GTPase.

True

(T/F) ATP hydrolysis on either Arp2 or Arp3 is required for nucleation of actin filaments.

False

What is the difference of Arp2 Q137A-GFP cells compared to WT? (i.e. these cells have their Arp2 gene suppressed by double-stranded RNA)

Mutant produced dendritic actin network disassembly defects resulting in longer lifetimes and greater distance traveled of Arp2/3 speckles as compared to WT.

What is a kymograph?

An intensity or line scan across the outer membrane of the cell that has distance on the x-axis and the number of frames on the y-axis. It is used to show the lifetime and temporal qualities of the actin speckle.

What are the role of GTPase in actin polymerization?

GTPase activates various localized pathways to affect different types of actin structures.

What is processivity in terms of myosin binding? Are skeletal muscles processive?

Processivity is the number of steps that a single motor can take along a filament before it falls off. Skeletal muscle myosins are non-processive in that they move one cycle and detach. In these cases, a large complex must be formed in order to generate continuous movement.

What is duty ratio? Does processive motion have high or low duty ratio?

The fraction of time that each motor domain spends attached to its filament. High

Describe the structure of myosin II.

6 subunits: 2 identical heavy chains, 2 pairs of light chains.

What does ATP binding do to myosin head?

It releases the myosin head from the actin filament.

Which step of the myosin-actin cycle is associated with the power stroke?

Phosphate release triggers tight binging and power stroke. ADP is then released and the myosin returns to its original conformation.

What is saponin?

Saponin is a mild detergent that interacts with membrane cholesterol, selectively removing it and leaving holes in the membrane. In this course, it is used to break holes in the membrane to introduce actin monomers and phalloidin.

Is the plus or minus end attached to the z-disc in a sarcomere arrangement?

Plus end

What are the three main functions of actin?

(1) Determine cell's surface shape (2) Necessary for whole-cell locomotion (3) Drive pinching of one cell into two