Cytoskleton

Question 1

What is the cytoskeleton?

Answer 1

• a network of proteinaceous filaments found in cells

- important in intracellular transport, cells division, cell motility, cell organisation and polarity

Question 2

What are the different cytoskeletal components?

Answer 2

microtubules (tubulin), intermediate filaments (IF) and microfilaments (actin filaments)

Question 3

What is the actin protein?

Answer 3

• one of the most abundant proteins
• binds ATP/ADP
• makes up microfilaments

Question 4

How does actin generate force?

Answer 4

Through polymerisation and protein/membrane interactions

- needs ATP/ADP and Mg

Question 5

What are the 3 isoforms of actin and where can they be found?

Answer 5

α - striated muscle
β - found in all cells
γ - smooth muscle

Question 6

What are the 2 structures actin can form?

Answer 6

G actin (globular) and F actin (filamentous)

Question 7

Which actin structure has directionality/ polarity?

Answer 7

Actin Filaments

Question 8

How do actin filaments grow?

Answer 8

• actin growth occurs on the barbed end - the plus end
• ATP hydrolysis means that the negative end shrinks, actin falls off
• Recycling purpose
• actin tread milling
• Stays the same length but moves to the right direction
• actin filament length remains the same even though there is a net flux of monomers through the filament

Question 9

Why do actin filaments grow faster at the plus end than the minus end?

Answer 9

One end will have more ATP’s as ATP likes binding to the positive end

• ATP does not like to bind to the negative end
• degradation of ATP and ATP hydrolysis occurs in the negative end
• ATP hydrolysis results in a conformational change that reduces affinity for neighbouring monomers
• affinity lowers in the negative end

Question 10

What regulates the polymerisation, length and organisation of microfilaments?

Answer 10

Actin-binding proteins

Question 11

Name actin monomer binding proteins that regulate filament formations and what they do.

Answer 11

Thymosin:
- ensures a pool of free G-actin
- prevents binding so there is no plus-end growth
Profilin:
- increases affinity of free actin for the filament
- promotes growth and assembly an nucleotide exchange (ADP->ATP)

Question 12

Name some nucleating proteins that promote growth of the actin filament.

Answer 12

ARP2/3:
- nucleating proteins
- needs promoting factor nWASP
- creates branches
Formin:
- produces "fingers" that branch out and bind to G-actin (grabs it) and then binds it to the growing filament

Question 13

How does actin interact with membranes and organelles?

Answer 13

Through anchoring protein Spectrin

• forms a lattice under the plasma membrane and can be stretched and compressed
• links to actin and membrane proteins
• allows cells to be flexible (useful in rbc)

Question 14

Which actin binding proteins prevent growth and stabilisation of the filament?

Answer 14

CapZ:
- capping protein
- binds to plus end so addition of subunits is prevented
- slow growth will have to happen in minus end
Tropomodulin
- also capping protein
- binds to minus end preventing loss of subunits
- important in muscle cells

Question 15

Which actin binding protein helps with depolymerisation and recycling of actin?

Answer 15

Cofilin

• binds to F-actin, promoting ATP to ADP hydrolysis
• binds to G-actin, prevents ADP to ATP exchange
• promotes de-assembling of filaments by chopping them up

Question 16

What is Cofilin dependent on?

Answer 16

Concentration dependence

• low conc: severs F-actin, promotes depolymersation of minus end
• high conc: increases polymerisation by nucleating new filaments

Question 17

What are motor proteins?

Answer 17

class of molecular motors that can move along the cytoplasm of animal cells

Question 18

Name the 3 major classes of motor proteins?

Answer 18

Myosin, dynein, and kinesin

Question 19

Which motor protein is responsible for vesicle transport?

Answer 19

Kinesin: vesicle transport walking from + to - and from - to +
Dyneins: vesicle transport walking from + to - only
- 7 times faster than Kinesins

Question 20

What do microtubules do?

Answer 20

• maintenance of cell shape
• help separate chromatins during mitosis
• tracks for intracellular transport
• organelle contacts and organisation
• controls polarised cell migration

Question 21

What are microtubules made of?

Answer 21

α and β tubulin dimers

• form together to make protofilaments
• laterally associate to form tubulins
• made up of 13 protofilaments

Question 22

How do microtubules grow?

Answer 22

• similar to actin growth
• treadmilling towards positive side
• dimers reversible attach to both ends of microtubules but 2X faster at positive end

Question 23

What causes microtubules filaments to curve?

Answer 23

• GTP hydrolysis changes subunit conformation and weakens bond in the polymer
• creating a curved protofilament

Question 24

How does microtubule shrinking occur?

Answer 24

• if filaments are growing faster than the rate of GTP hydrolysis, GTP cap stops it from growing and is stable
• GTP hydrolysis catches up with the GTP cap
• curving occurs and it peels back away from the microtubule
• shrinking

Question 25

Where does microtubule shrinking usually occur?

Answer 25

the positive end

Question 26

What does catastrophe and rescue mean in terms of dynamic instability of microtubules?

Answer 26

``` catastrophe: - filament is falling apart as there is no addition of monomers - not stable - shrinking rescue: - when the filaments are being rebuild - growth ```

Question 27

What is "search and capture" in terms of dynamic instability of microtubules?

Answer 27

- what occurs in mitosis - allows tubules to explore cell to find interacting partners - constant growth and movement - catastrophe and rescue happening

Question 28

Which type of tubulin nucleates filaments at the minus end?

Answer 28

γ tubulin

Question 29

What are microtubule organising centres (MTOC)?

Answer 29

structure found in eukaryotic cells from which microtubules emerge - pair of centrioles inside - spherical centrosome matrix - γ tubulin ring complexes are nucleating sites that microtubules grow from, anchoring from the minus end

Question 30

What is the microtubule organising centre called for cilia and flagella?

Answer 30

basal bodies

Question 31

Which proteins can promote rescue and catastrophe for microtubules?

Answer 31

TIPs - plus end tracking proteins EB1: - promotes rescue - Binds to and interacts to the plus end - Helps to promote the binding of new monomers - Interacts surrounding proteins - Can change the microtubules behaviour in many ways MCAK: - promotes catastrophe - Uses ATP hydrolysis to speed up the depolymerisation of the microtubules - Linked to kinesin complex - Actual disassembly of the molecule helps it to pull

Question 32

What doe microtubule associated proteins (MAPs) do?

Answer 32

- can stabilise microtubules - Provide the "highway" for the movement of products - If there is a defect it causes neurodegenerative diseases - Provide overall structure - e.g. Tau stabilised filaments in neurons

Question 33

Name a severing protein and explain what they do.

Answer 33

Katanin - cuts at specific locations - uses ATP hydrolysis - forms rings which stick to the side and destabilises structures between components - promote tubule longevity

Question 34

What is hyperglutamylation?

Answer 34

Adding too much glutamate to tubulin components

Question 35

Why is hyperglutamylation dangerous?

Answer 35

- can contribute to neurodegenrative diseases in absense of deglutamylases - neuron loss and astrogliosis - axonal swellings - reduced mitochondria traffic - impairs movement - triggers initiation of spastin causing falling apart of cell

Question 36

How do microtubules contribute to cell migration?

Answer 36

- mictrotubule-driven intracellular transport towards the front of the migrating cell - regulation of actin cytoskeleton assembly at the leading edge of a migrating cell - delivery of factors which stabilise the rear of the cell to maintain directional migration - targeting of focal adhesions for disassembly to allow rear retraction

Question 37

What do integrins do?

Answer 37

allow the ability to grab onto components - pick up the back and moved to the front so they can be engaged with other components - can be endocytosed - can move motor proteins to the location needed to speed up processes - directionality

Question 38

What specialised structures can microtubules form?

Answer 38

Cilia and flagella - can form dimers - interactions between dynein and MT often seen such as in airway moving mucus

Question 39

What is the function of intermediate filaments?

Answer 39

``` 6 different classes provide mechanical support for the plasma membrane - forms a stress network - forms specialised structures such as keratin - hair and ocular lens (class VI) ```

Question 40

How are intermediate filaments formed?

Answer 40

- helical bundles that coil themselves into dimers - laterally polymerise to form unit length filaments - unit length is approx 60nm long - average diameter (10 nm) - non-polar

Question 41

How are keratin proteins formed?

Answer 41

class 1 and class 2 intermediate filaments form hetreodimers with each other

Question 42

What is keratin responsible for forming?

Answer 42

Specialised structures such as hair and nails - associated to larger structures - bundles are packaged and excreted from cells "simple" keratins on the epidermis layer can resist force and absorb energy by forming a stress network - interact with desmosomes and provide mechanical strength

Question 43

What happens if there is a mutation in Keratin 14?

Answer 43

Keratin 14: mutation causes cells not being able to hold on to the interactions in the layers and the top layer of skin starts to come off