Module 7

Question 1

Cytoskeleton [three fibre types]

Answer 1

1) Actin
2) Microtubules
3) Intermediate Filaments

Question 2

Actin Filament [labelling]

Answer 2

a) labelled with a fluorescently tagged phalloidin molecule or through protein fusion such as actin:GFP
b) phalloidin is a toxin derived from a mushroom called the death cap
c) phalloidin stabilizes the filament when bound with high affinity and high specificity

Question 3

Microtubules [labelling]

Answer 3

a) labelled using antibodies specific to the tubulin subunit or through protein fusion such as tubulin:GFP

Question 4

Intermediate Filaments [labelling]

Answer 4

a) labelled using an antibody specific to the filament or through GFP-fusion

Question 5

What are the monomers of each cytoskeletal fibre?

Answer 5

[actin filament] actin subunits (THINNEST)
[microtubule] dimeric subunits of alpha and beta-tubulin (THICKEST)
[intermediate filaments] depends

Question 6

T or F: In the epithelial cell, an example of an intermediate filament that provides structural support is the lamin protein, which forms the nuclear lamina, providing shape and structure to the nucleus

Answer 6

True

Question 7

Which two cytoskeletal filaments have filament-specific motor proteins? Name the motor proteins

Answer 7

1) Actin Filaments (myosin proteins)
2) Microtubules (kinesin, dynein)

Question 8

The highest density of actin is in the…

Answer 8

Cell periphery

Question 9

What structures are supported by actin filaments?

Answer 9

a) Microvilli
b) Contractile bundles (form sarcomeres that power muscle cell contraction)
c) Lamellipodia and filopodia (cell migration)
d) Contractile Ring (directs cell division)

Question 10

T or F: Each actin filament (F-actin) contains two strands of helical polymers that are both built from single actin monomers (G-actin)

Answer 10

True

Question 11

How did researchers discover that actin filaments are polar (plus end and minus end)?

Answer 11

a) They used myosin which binds in just one orientation on the actin filament
b) The plus-end grows more quickly through addition of more actin subunits (barbed appearance)
c) The minus-end grows more slowly and may shrink (pointed appearance)

Question 12

G-Actin [def]

Answer 12

[def] a single actin monomer

a) divided into four structural domains
b) large ATP-binding cleft between domains 2 and 4
c) each actin monomer is polar
d) ATP binding pockets face the minus-end so they aren’t exposed, except for the terminal monomer at the minus-end

Question 13

F-actin

Answer 13

a) created through the polymerization of G-actin monomers
b) an actin filament is dynamic, constantly engaged in polymerization and depolymerization
c)

Question 14

Which end does polymerization and depolymerization operate at?

Answer 14

They both occur at the plus and minus-ends but there tends to be more growth at the plus-end and more shrinkage at the minus-end

Question 15

How does ATP regulate the growth and disassembly of the actin filaments?

Answer 15

a) ATP-bound actin monomers in the cytosol join at the plus-end
b) Actin hydrolyzes ATP to ADP, releasing inorganic phosphate
c) Most of the actin filament is made up of actin-ADP
d) ADP is not released since the nucleotide-binding site is covered
e) Rate of polymerization is greater than depolymerization at the plus-end, but at the minus-end, it’s the opposite, so Actin-ADP readily dissociates, exchanging ADP for ATP in the cytosol, ready for a new cycle

Question 16

Critical Concentration [def]

Answer 16

[def] the concentration of actin at a single filament end where the rate of actin monomer addition is equal to the rate of removal (no net growth)

a) Filament end will grow if concentration of actin monomers is greater than the critical concentration, and vice versa
b) These concentrations and dynamics are different at both ends of the actin filament

Question 17

T or F: Profilin can bind to actin-ATP and promote ATP binding, activating the monomer. However, in opposition, thymosin can bind to the monomers and inhibit polymerization.

Answer 17

True

Question 18

Treadmilling [def]

Answer 18

[def] there’s no net increase in the length of filament because the rate of polymerization at the plus-end is equal to the rate of depolymerization at the minus-end

a) However, the relative position of the filament is changing and effectively, the filament is moving forward
b) Important for cell movement or migration

Question 19

How do actin filaments power the movement of cells through reorganization of filaments? [steps]

Answer 19

1) Formation of leading edge of the cell
2) Formation of fan-like expansions of the cell membrane, called lamellipodia
3) Formation of finger-like filopodia extensions of the cell membrane
4) Finally, forward movement

Question 20

Myosin Motor Proteins

Answer 20

a) They power intracellular cargo trafficking
b) Eight families - Myosin I, II, and V are present in nearly all eukaryotic cells
c) Each has a N-terminal head domain which binds actin filaments and binds and hydrolyzes ATP
d) Heads are similar but tail domains are very different to carry different cargo at different rates
e) Most myosin motor proteins move toward the plus-end of actin filaments

Question 21

Myosin II

Answer 21

a) two heavy chains that form a coiled-coil motif
b) four light chains (two types)

Question 22

MLCK & Myosin II Thick Filament [def]

Answer 22

[def] myosin light chain kinase

a) In the inactive state, light chains are unphosphorylated and the myosin tail is bent
b) MLCK initiates the extension of the myosin tails and activates the binding domains on motor heads
c) Causes the formation of the Myosin II Thick Filament (15-20 myosin II proteins)
d) Myosin II Thick Filament is a bipolar filament with motor heads to the left and right of a bare zone

Question 23

Sarcomeres

Answer 23

a) Within muscle fibres, myosin II thick filaments are associated with thin actin filaments to form sarcomeres
b) Plus-ends of actin filaments are fixed to Z-discs
c) CapZ protein caps the plus-end, tropomodulin caps the minus-end
d) Nebulin binds together parallel actin filaments
e) Myosin thick filaments are also attached to Z-discs but by a giant spring called the titin protein

Question 24

T or F: During muscle contraction, myosin thick filaments interact with actin filaments, and the result is Z-lines moving further apart

Answer 24

False, the Z-line moves closer together

Question 25

Microtubule Fibres [def]

Answer 25

[def] tubes comprised of 13 protofilaments, arrayed in a circular pattern a) basic subunit of a protofilament is a dimer of alpha and beta tubulin

Question 26

How is muscle contraction a calcium-dependent process?

Answer 26

a) Calcium leads to exposure of myosin binding sites along the actin filaments b) Following contraction, calcium dissociates from the actin filaments and myosin heads release the actin c) This allows thick and thin filaments to slide past one another, allowing muscle relaxation

Question 27

Mechanical Cycle of Myosin [steps]

Answer 27

The steps of the mechanical cycle are coupled to the chemical cycle 1) Myosin is attached to actin 2) ATP binding to myosin releases actin 3) ATP is hydrolyzed to ADP and inorganic phosphate, relaxing myosin 4) The release of inorganic phosphate increases the affinity of the myosin head for actin and allows binding 5) The release of ADP changes myosin conformation, since myosin is attached to actin, the actin filament is pulled to the left (power stroke) [now back in first step orientation]

Question 28

Myosin V [melanosomes]

Answer 28

a) Powers intracellular trafficking of cargo along actin filaments b) EX: Melanosomes are membrane-enclosed organelles containing melanin, found in melanocyte cells c) Myosin V (along with microtubules) distributes the melanosomes to the cell membrane along actin filaments to protect against UV radiation

Question 29

How does the rate of myosin motor protein movement vary?

Answer 29

a) varies with different myosin proteins, ranging from 0.2 to 60 μm/sec b) rate varies with [1] rate of ATP hydrolysis by ATPase in each myosin head and [2] proportion of time myosin is bound to the actin filament c) EX: Myosin V spends 90% of the cycle bound to actin compared to 5% for Myosin II, so Myosin V moves more slowly along actin filaments

Question 30

T or F: In addition to proportion of time myosin is bound to actin, different myosin proteins can differ by their lever arm length, the lever of myosin V (36nm steps) is 3x longer than myosin II (7nm steps). Myosin V moves in a hand-over-hand fashion and moves towards the plus end of the actin filament

Answer 30

True

Question 32

α and β tubulin [GTP]

Answer 32

a) α-tubulin monomer is tightly bound to GTP (never hydrolyzes) b) β-tubulin monomer is less tightly bound to GTP (cyclically hydrolyzed to GDP) c) both α and β subunits are added and removed as dimers, they serve as the microtubule subunit

Question 33

In microtubules, which end is fast-growing and which end is slow-growing?

Answer 33

The plus-end is fast-growing and the minus-end is slow-growing

Question 34

In microtubules, how are the tubulin dimers oriented?

Answer 34

a) β-subunit is closer to the plus-end b) α-subunit is closer to the minus-end

Question 35

Rescue Phase & Catastrophe [def]

Answer 35

[rescue phase] tubulin dimers containing α-β-GTP are added to the plus-end of a growing filament [catastrophe] tubulin dimers containing α-β-GDP are released from a shrinking filament

Question 36

T or F: Microtubules consist mostly of tubulin dimers that contain α-β-GTP

Answer 36

False

Question 37

What's the importance of a GTP cap on microtubules?

Answer 37

The GTP cap at the plus-end favours growth rather than shrinkage, preventing catastrophe

Question 38

In microtubules, do GTP dimers or GDP dimers have a slower dissociation rate?

Answer 38

GTP dimers have 4x slower dissociation rate compared to GDP dimers due to a higher affinity of the microtubule for GTP

Question 39

EB1 [def]

Answer 39

[def] a plus-end binding protein that prevents premature catastrophes and acts as a positive regulator of microtubule growth a) Used in mouse embryonic fibroblasts and labelled using GFP

Question 40

What kind of behaviour does the microtubule exhibit? [def]

Answer 40

[dynamic instability] oscillating behaviour between growth (polymerization, rescue phase) and shortening (depolymerization, catastrophe)

Question 41

MAP [def]

Answer 41

[def] microtubule-associated proteins - control assembly and disassembly of microtubules a) many interconnect microtubules to help form bundles (cross-bridges) b) two groups - those that stabilize or destabilize the filament

Question 42

What are examples of MAPs that stabilize and destabilize microtubules?

Answer 42

[stabilize] Tau, EB1 [destabilize] catastrophin

Question 43

γ-tubulin [def]

Answer 43

a) involved in nucleation of microtubules b) present in much smaller amounts than α‐ and β‐tubulin c) forms the γ-tubulin ring complex (γ-TuRC), which nucleates at the minus-end of a new microtubule by forming a template for the growing plus-end d) γ-TuRC acts as a cap of the minus-end

Question 44

MTOC [def]

Answer 44

[def] microtubule organizing centre - where microtubule nucleation occurs a) In animals, the MTOC is called a centrosome b) [centrosome] two cylindrical structures called centrioles and a cloud of pericentriolar material (PCM) that contains multiple γ-TuRC complexes

Question 45

T or F: During mitosis, microtubules of the mitotic spindle attach to chromosomes, centrosomes are duplicated in mitosis to create two MTOC's but as they separate, microtubules are nucleated at the γ-TuRC complexes and the plus-ends grow outwards.

Answer 45

True

Question 46

Colchicine [def]

Answer 46

[def] a microtubule toxin derived from meadow saffron or autumn crocus that inhibits polymerization a) binds and stabilizes free αβ‐tubulin dimers b) these dimers can still be added to the growing microtubule but will prevent the addition or loss of other tubulin dimer subunits

Question 47

If colchicine was added to a cell in mitosis, what would happen?

Answer 47

The cell would arrest in metaphase WITHOUT chromatid separation

Question 48

Taxol [def]

Answer 48

[def] a microtubule toxin derived from the Pacific yew tree that inhibits mitosis, AKA paclitaxel a) binds to β-tubulin b) increases affinity of the dimer for the plus-end, preventing depolymerization and stabilizing the microtubules c) this prevents the assembly of the mitotic spindle and inhibits mitosis

Question 49

What two chemicals cause rapid depolymerization of microtubules?

Answer 49

Vinblastine and Nocodazole

Question 50

Kinesin [def]

Answer 50

[def] a tetrameric complex that's made up of two identical heavy chain and two identical light chains a) [motor domains] the globular heads at the N-termini of the heavy chains (they bind to MTs and generate movement through ATP hydrolysis) b) kinesins move cargo (vesicles, organelles) toward the plus-end of MTs - periphery of the cell c) across family members of the kinesin protein, the tail is highly variable because it determines the specificity of cargo binding

Question 51

Mechanochemical Cycle of Kinesin

Answer 51

a) hand-over-hand motion b) kinesin has two motor domains - one is always attached to the MT c) lagging head is bound to ATP, leading head is bound to ADP d) ATP-bound kinesin has a higher affinity for the MT than ADP-bound kinesin e) in the lagging head, ATP is hydrolyzed into ADP and inorganic phosphate, reducing affinity of lagging head for MT f) in the leading head, ADP is exchanged for ATP, increasing the affinity of the leading head for MT g) these changes induce a conformational change in the neck region, causing the lagging head to swing forward

Question 52

Explain the two in vitro assays used for kinesin movement

Answer 52

1) Bead Assay - Nomarski Microscope 2) Gliding Mobility Assay - Fluorescence Microscope

Question 53

Dynein [def]

Answer 53

[def] a microtubule-binding motor that travels towards the minus-end of MT (MTOC) a) [cytoplasmic dynein] direct movement of organelles and vesicles in the cytoplasm b) [axonemal dynein] found in structures that power the movement of whole cells like cilia and flagella c) dynein contains two identical heavy chains and a variety of intermediate and light chains

Question 54

Dynein Power Stroke [steps]

Answer 54

1) ATP binding releases the motor head group from the microtubule 2) ATP is hydrolyzed into ADP and inorganic phosphate, allowing it to attach to the MT 3) The release of the inorganic phosphate powers the power stroke, pulling cargo 8nm closer to the minus-end

Question 55

If kinesin and dynein are motors that move cargo in opposite directions, how does cargo end up moving?

Answer 55

A model suggests that proteins are engaged in a molecular tug of war, the final direction of movement is decided by the winner of the battle

Question 56

T or F: An example of the switch between plus-end and minus-end movement is illustrated by the transport of melanosomes in the skin cells of fish, where signalling is controlled by signals that use cAMP as a secondary messenger

Answer 56

True