The Cytoskeleton

Question 1

Cytoskeleton

Answer 1

A network of filaments extending throughout the eukaryotic cells. They are highly dynamic ‘bones and muscles’ of the cell.

Question 2

What is the cytoskeleton responsible for?

Answer 2

Cell movement, shape and muscle contraction
Organelle movement and disruption
Vesicle transport, secretion and uptake
Chromosome separation at mitosis
Separation of daughter cells at mitosis
Resistance of cells and tissues e.g. to mechanical stress

Question 3

What is the actin cytoskeleton required for?

Answer 3

7-9mm
Cell movement
Cell division
Vesicle transport, phagocytosis an movement of organelles
Provides the cells ‘muscles’
Most filaments are dynamic but some can be stable e.g. in microvilli
Signalling pathways regulate acton organisation and dynamics
Actin binding proteins help to organise the structures

Question 4

How abundant is actin and what are the building blocks?

Answer 4

10% protein weight in muscle. Building block = monomeric globular G-actin

Question 5

How do G-actin polypeptides fold?

Answer 5

Into 4 subdomains that generate 2 lobes separated by a deep cleft. At the base an ATPase fold - structure binds ATP complexed with Mg2+ ion
G-actin polymerises to F-actin

Question 6

Assembly of F-actin

Answer 6

Many cellular processes depend. Can take place in vitro - depending on conc of G-actin and cations.

Question 7

Actin filament

Answer 7

2 strands of monomers are arranged helically, winding around each other. Each strand complete turn - 72nm = 14 subunits
F-actin has polarity - opposite ends are distinguishable. This is because the ATP-binding cleft in each monomer is always oriented towards the same direction within the filament - to the -ve end of the filament (exposed cleft)

Question 8

How can the polarity of actin filaments be demonstrated?

Answer 8

Electron microscopy using an actin-binding domain of muscle myosin decorate actin filaments. This generated an arrowhead pattern - so one head = barbed / +ve end which is where addition of new subunits take place and actin grows. Opposite end = pointed / -ve. The loss of a subunit takes place preferentially at this end. not charge!

Question 9

What does the addition and loss of subunits depend on?

Answer 9

Concentration of available G-actin. If one starts with only G-acton, without any previously available filaments, the initial start of filament formation is slow, followed by a rapid elongation phase until a steady state is reached.

Question 10

Critical concentration, Cc and actin tread milling

Answer 10

Conc free acton at which growth at one end is balances by the loss at the same end. Cc + lower than -, so in a steady situation subunits added + and lost - => actin treadmilling

Question 11

Rate of ATP-G actin addition and ATP

Answer 11

Much faster at + end, dissociation similar. After ATP containing G actin is added to + end of F-actin, ATP slowly hydrolysed to ADP. - end will contain ADP-actin. Steady state = ATP-actin monomers are added to + and ADP-actin subunits disassemble at -

Question 12

What is actin tread milling powered by?

Answer 12

Hydrolysis of ATP. After ATP-bound G acton added to + end, ATP hydrolysed to ADP+Pi. Pi slowly released so that towards -, actin subunits contain ADP. Hydrolysis and release of Pi result in conformational change of actin, which explains different association and disassociation rates.
Basically controlled by actin-binding proteins

Question 13

Profilin

Answer 13

Captures ADP-G-actin and catalysis exchange of ADP to ATP.

Question 14

Cofilin

Answer 14

Severs ADP-containing F-actn to fragments, generating more - ends and accelerates release of ADP-G-acton.

Question 15

Thymosin-beta

Answer 15

Sequesters ATP-G-actin and provides a reservoir of actin subunits for polymerisation

Question 16

What structures can actin filaments adopt for its various functions?

Answer 16

Contractile stress fibres, thin needle-like spikes at cell surfaces, branched meshworks, bundles in microvilli and adherent belt of actin bundles in epithelia.

Question 17

Myosin head domains

Answer 17

Convert ATP hydrolysis to mechanical work

Question 18

Myosin II

Answer 18

4 separate polypeptides, 2 heavy and 2 light chains. Talk can bundle to help form thick myosin filaments in muscle.

Question 19

Myosis are motor proteins

Answer 19

They walk along actin towards + end. Different myosin classes have variable neck domain lengths that determine step size.

Question 20

A muscle sarcomere

Answer 20

Myosin forms thick, bipolar filaments and actin thin filaments. Actin + ends tethered to Z discs so that 2 actins have opposite orientation.
Sliding actin and myosin during muscle contraction is accomplished by myosin head domain moving along actin towards the Z-disk. Requires ATP and Ca2+ ions. Myosin thick filament is bipolar so draws the actin filaments towards the centre of the sarcomere and shortens it.

Question 21

Accessory proteins

Answer 21

Preserve sarcomere integrity so sarcomere actin is not tread milling.

Question 22

CapZ

Answer 22

Caps actin thin filaments at + end

Question 23

Tropomodulin

Answer 23

Caps actin thin filaments at - end

Question 24

Nebula proteins

Answer 24

Extend along actin from Z-disk to tropomodulin and help regulate all thin filaments to same lengths.

Question 25

Titin

Answer 25

A giant, spring-like protein that extends from Z-disk to the middle of the sarcomere through the thick filament. A pic of titans extends through the whole sarcomere

Question 26

The sliding filament theory

Answer 26

At the beginning of the myosin head power stroke, myosin head lacks bound nucleotide and is locked to actin. When ATP binds to the head domain, the conformation of the head changes which reduces the affinity of myosin to actin and allows myosin head to move along the actin.
ATP is hydrolysed and the released energy rotates the myosin head that now binds to F-actin. This is the cocked position of the myosin head.
In the power stroke, release of Pi tightens the grip of myosin to actin and triggers the force-generating change of the shape of myosin back to the original conformation moving the actin filament relative to the position of the myosin head. In the course of the power stroke, the head loses its bound ADP, they returning to the start of the cycle.

Question 27

Microtubules

Answer 27

Hollow cylinders of tubular, 25mm outer diameter. One end is usually attached to a microtubule organising centre (MTOC), e.g. centrosome. 13 protofilaments are assembled together to make a MT.
Form tracks along which vesicles and organelles can move and the mitotic spindle during mitosis

Question 28

How can microtubulins grow and shrink?

Answer 28

Rapidly by tubulin addition/loss. Grow out the centrosome towards the cell periphery.

Question 29

Protofilament

Answer 29

A row of tubular dimers.

Question 30

Tubulin dimer

Answer 30

A heterodimer of 2 globular, closely related proteins: alpha and beta tubilin

Question 31

Alpha-tubulin

Answer 31

Always bound to nucleotide GTP

On the - end of MT => preferentially shrink at this end, but in cells, MT - end is anchored to a MTOC

Question 32

Beta-tubulin

Answer 32

Nucleotide = GTP or GDP form and is exchangeable

On the + end of MT => preferentially grow at this end

Question 33

MTOC named as…

Answer 33

A centrosome, spindle pores or basal body, depending on the MT function.
MTOCs have 9 short triplet Mts embedded in pericentriolar material that contains gamma-tubulin ring complexes that nucleate growth of new MTs.

Question 34

Inherent dynamic stability

Answer 34

Sudden shortening of a MT, a catastrophe, followed by rescue and rewowth. Displayed by Mts growing out from MTOC. Can be also demonstrated in living cells expressing fluorecently labelled tubules.

Question 35

What is the in-built stability due to?

Answer 35

GTP hydrolysis by beta-tubulin. GTP bound beta tubular caps the growing end. If the addition is faster than hydrolysis of GTP to GDP, tubule grows. When cap is lost (GTP hydrolysed and GDP-bound beta tubular exposed at + end), tubule shrinks rapidly. GTP hydrolysis changes the subunit conformations, leading to weaker bonds in the polymer. Protofilaments curve with loss of lateral associations and tubular dimers de-polymerise.

Question 36

Post-translational modifications of tubulin

Answer 36

Affect MT stability and function. In a-tubulin subunit, a specific lysine reside can be activated which results in more stable MT found e.g. in centrioles and primary cilia. . C-terminal tyrosine residue of a-tubulin can be removed by a specialised carboxypeptidase enzyme also resulting in a more stable MT

Question 37

MT-binding proteins

Answer 37

Affect MT bundling or stability

Question 38

MTs important for directional transport within cells

Answer 38

A polarised microtubule system allows cargo movement along microtubule tracks. Motor proteins mediate e.g. the intracellular transport of membrane-enclose organelles.

Question 39

Kinesins

Answer 39

Motor protein - drives movement + end

Some preferentially bind stabilised MT, some can enhance depolymerisation at + end

Question 40

Dyenin

Answer 40

Motor protein - drives movement to - end

Question 41

Kinesin I

Answer 41

Comprises of 2 heavy chains and associated 2 lighter chains
Globular head domains of heavy chains connected with a flexible linker and a long stalk to tail domains that bind cargo. The 2 kinesin heads use ATP hydrolysis for co-ordinated walking on microtubules. ATP binding to motor head domain causes conformational change that swings the neck and the previously trailing head becomes the leading head

Question 42

Dyenin power stroke

Answer 42

ATP binding and hydrolysis rotates head domain. Dyenin-mediated transport requires additional protein complex, dynactin that links dyenin to its cargo

Question 43

Distinct MT in the mitotic spindle

Answer 43

All grow from spindle pole MTOCs
Kinetochore Mts attach to chromosomes, polar Mts overlap in the middle of the spindle and astral Mts that point outwards and extend to cell cortex
Kinetochore Mts shorten both at spindle pole and at kinetochore. Kinesin-13 helps depolymerisation.
Spindle poles are pushed further apart. A bipolar kinesin causes sliding of polar Mts and dyenins at cell context pull apart astral MTs

Question 44

Intermediate filaments

Answer 44

10-12nm thick
Lamins = subfamily found in nucleus
Monomeric IF and rod like polypeptides with a central alpha helix help to form a coiled-coil fire, which constitute the boiling blocks of the filaments
Key function is to provide structural integrity to cells and tissues. AN example is skin fragility and bliserting caused by mutations in epidermal keratins.

Question 45

Filament assembly

Answer 45

2 dimers form an antiparallel tetramer. Symmetric structure that aggregates to unit length filaments that further assemble into filaments

Question 46

Indistinguishable ends

Answer 46

Due to symmetry - non-polar and cannot provide directional info to motor proteins.

Question 47

Tissue-specific expression patterns

Answer 47

e.g. keratins form the cytoplasmic intermediate filaments in epithelial cells and another IF called desmin is found in muscle. Based on similarity and details of assembly mechanism.