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Cell Theme 2 and 3 > Dominguez - Actin > Flashcards

Dominguez - Actin Flashcards

1
Q

Diameters of actin, intermediate filaments, and microtubules

A

7 nm
9-11 nm
25 nm

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2
Q

Which cytoskeletal filaments are polar?

A

Actin and microtubules are polar, intermediate filaments are not

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3
Q

Basic subunit of actin and microtubules?

A

Actin monomer

Tubulin dimer

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4
Q

Arp2/3

A

7-subunit protein that plays a major role in the regulation of the actin cytoskeleton.
Mutants delay endocytosis
Basic idea is that mutations in critical proteins of the actin cytoskeleton you can disrupt endocytosis

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5
Q

How does salmonella invade?

A

Contact with host causes host cell to send up actin-rich ruffles that engulf the bacteria. They are trapped inside vacuoles and replicate there.

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6
Q

How does listeria invade?

A

Bacteria grow and replicate and rocket around (right??)

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7
Q

How does wound healing happen?

A

Cables of actin run continuously around the wound margin, acting as a purse string to close up the wound.

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8
Q

Where is the dense area of actin in the cell?

A

At the leading edge.

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9
Q

Lamellipodium versus filopodium

A

Lamellipodium - branched

Filopodium - bundle

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10
Q

Microvilli

A

Actin-rich membrane protrusions

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11
Q

Stereocilia

A

Similar to microvilli but inner ear

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12
Q

Does actin make up thin or thick filaments?

A

Thin filaments

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13
Q

Globular/G-actin

A

Monomeric actin

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14
Q

Does actin hydrolyze ATP ?

A

Yes but it can go for weeks without hydrolyzing it, slow, and affinity for ATP only 3-fold higher than for ADP

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15
Q

When does actin polymerize?

A

Monomeric at low ionic strength and polymerizes in presence of salt. Upon polymerization ATP gets hydrolyzed to ADP-Pi and Pi is slowly released.

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16
Q

F-actin

A

Filamentous actin - a double helix of head-to-tail bound actin monomers. Monomers can associate and dissociate only at filament ends.

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17
Q

What is the repeat of the actin double helix?

A

36 nm, which corresponds to 13 actin subunit.

Pitch per turn is 72 nm.

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18
Q

Which end of actin is more likely to grow?

A

The plus end

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19
Q

Barbed end of actin

A

Plus end

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20
Q

Pyrene actin polymerization experiment

A

Helps us to see transition of monomer to filament. Label the monomer with a probe - pyrene-actin. 10% of actin is labeled. Fluorescence increases 7-10 times upon polymerization. Pyrene label has no significant effect on the polymerization rate constants.

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21
Q

Time course of polymerization

A

Nucleation - lag phase
Elongation phase
Termination of elongation

22
Q

Dissociation constant

A

KD = Cc= [Filament][G-Actin]/[Filament+1] = [G-actin]

There is a single dissociation constant, independent of filament concentration or filament length.

KD is the critical concentration.

23
Q

Where is elongation faster?

A

Barbed end

  1. 9 actin subunits/s at barbed end
  2. 3 actin subunits/s at pointed end
24
Q

Nucleators

A

Accelerate nucleation

25
Profilin or thymosin or T4
Inhibits nucleation in cells, monomer-binding protein
26
Elongation factors / formins and Ena, VASP
Elongation can be accelerated by these proteins
27
ATP-actin
Elongation occurs from ATP-actin
28
ADP-actin
Depolymerization occurs from pointed end in form of ADP-actin
29
ADF/Cofilin
Accelerates depolymerization
30
Treadmilling
Actin polymerization can produce force? | Fmax = KT/dln(C/Cc)
31
Actin polymerization inside liposomes?
If you add salt via electroporation into liposomes conaining G actin you will see protrusions growing from liposome at rates from 0.3 - 0.7, similar to cellular protrusions. This is a cool experiment.
32
How fast is actin polymerization in cells?
Much faster than treadmilling - Actin binding proteins are responsible.
33
Tropomodulin, capping protein, gelsolin
Filament capping proteins
34
Gelsolin, Cofilin, Actophorin
Filament severing proteins
35
Villin, Fimbrin, Espin, beta actinin
Filament crosslinking/bundling proteins
36
Arp2/3 complex, formin
Filament nucleation and elongation proteins Arp2/3 complex promotes polymerization and branching at the leading edge of cells by stabilizing a nucleus that grows as a 70 degree branch from the side of a pre-existing filaments. Consists of 7 proteins, including 2 actin related proteins - arp2 and arp3. It binds 2 NPFS - nucleation promoting factors
37
Where is the target binding cleft?
Opposite the nucleotide binding cleft.
38
Profilin
Transitions actin from ADP to ATP. Binds proline rich regions (many cytoskeletal proteins contain proline rich regions). Catalyzes ADP-->ATP exchange, replenishing pool of ATP-actin for polymerization. Present at high concentrations in eukaryotic cells. Inhibits filament nucleation. Cannot add filament to pointed end, but can elongate filament barbed ends at same rate as free actin monomers.
39
Thymosin - beta4
Small protein Binds ATP actin with 50x affinity than ADP-actin. Acts as a buffer/reservoir of actin monomers. 2 helices that bind 2 ends of actin. Actin cannot join at either end. Acts as a buffer keeping actin monomers in cell. You lose this to acquire profilin. Need to keep actin bound to this or you get filaments.
40
ADF/Cofilin
Speeds up rate of pointed end depolymerization (in vitro rate of depolymerization is 20x slower than in vivo). Severs actin filaments, increasing rate of polymerization by generating new barbed ends. Binds weakly to ADP-P-actin, increasing rate of Pi release. Binds tightly to ADP-actin, increasing dissociation from filaments. This can help with locomotion. Enhances turnover of actin filaments by severing and depolymerizing filaments.
41
Gelsolin
Barbed end capping and filament severing protein. Consists of 6 related domains (G1-G6). Regulated by Ca2+
42
Tropomodulin
Pointed end capping protein in muscle and non-muscle cells.
43
Capping protein
Binds to filament barbed-ends with high affinity. Stable heterodimer of 2 related subunits and is essential for actin based motility, as shorter filaments are stiffer and can excerpt force against the membrane.
44
Actin crosslinking/bundling proteins
Contain at least 2 actin-binding domains (ABDs) that bind 2 different filaments, most are symmetric (dimers) - others contain actin binding domains belonging to different structural families
45
Intestinal microvilli
1-2 uM long and 2 uM wide structures, consisting of 20-30 actin filaments crosslinked by the proteins villin and fimbrin
46
Villin and fimbrin
Crosslink actin in intestinal microvilli
47
Stereocilia
Aligned into rows of increasing height with espin and fimbrin
48
Espin and fimbrin
In stereocilia
49
Formins
Nucleate non-branched actin filaments. Bind to filament barbed ends, modulating elongation and protecting filaments from capping proteins. Form caps that walk with the barbed end of the filament as it elongates. They are processive - they stay bound as it elongates Profilin - accelerates formin-mediated filament elongation.
50
N-WASP
Activator of ARP2/3 complex
51
VASP
Filament elongation

Cell Theme 2 and 3 (7 decks)

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