Cytoskeleton

Question 1

What is the cytoskeleton and its major roles?

Answer 1

Cytoskeleton = intricate network of filaments present in cytoplasm of all cells

in eukaryotes contains 3 core elements:

1. Microtubules
2. Microfilaments
3. Intermediate filaments

• made from subunits that can disassemble and rapidly diffuse within the cell and reassemble again
• modular nature gives strength and adaptability

→ cytoskeleton is highly dynamic

• important as cells continually test the environment and need to recognise where structure is needed
• if the situation changes, dynamism allows you to rapidly adapt or change

Major Roles:

1. Support cell shape
   - maintaining filopodia, lamelipodia, psuedopodia etc
2. Intracellular traffic
   - guides moving vesicles
3. Cell division
   - mitotic spindle (microtubules)
4. Motion
   - cilia
   - flagella
   - muscle contraction (actin)
5. Wound healing
6. Sperm egg fusion
7. Muscle function

Question 2

Describe the intermediate filaments

Answer 2

Intermediate Filaments are made from elongated fibrous subunits (10nm in diameter)

→ monomer coils to form dimer → dimers associate to form tetramer → associate to form protofilament → form base unit for full filament

→ lateral hydrophobic interactions, not polar, flexible and hard to break

→ least dynamic (but still dynamic, constantly regenerating)

• provide mechanical strength

WHERE YOU NEED MECHANICAL STRENGTH (HOW DO TISSUES RESIST STRETCHING FORCES):
Intermediate filaments pass through cell junctions:
- stretching a sheet of cells with intermediate filaments can resist → cells remain intact and together

TYPES OF INTERMEDIATE FILAMENTS

• made of KERATIN → in epithelial cells - give strength
  
  • found in nails, hair, skin to absorb mechanical stress
• made of LAMIN → give structural support to nuclear envelope
  
  • are signalled to be broken down via phosphorylation events and then reform during cell division

Question 3

Describe the microtubules

Answer 3

• made from polymers of α and β tubulin dimers
• Long stiff hollow tubes of protein (23nm in diameter)
• protofilaments have plus end (β subunit exposed) and minus end (α subunit exposed) which serves as directionality for intracellular transport
  → very dynamic and strong
  → network of tracks that vesicles move along

form centrioles found in centrosome → essential in producing mitotic spindles during cell division to separate chromosomes

• centrosome control no of microtubules formed
• contain nucleating sites from which α and β tubulins grow
• Growth only occurs at POSITIVE END (β subunit)
• radiate in all directions as a device to survey the cell and position the centrosome at its center and thus positioning organelles

form cilia/flagella → control beating of flagella/cilia - essential for propulsion, sweeping of fluids - The motion of the cilia and flagella is created by the microtubules sliding past one another, which requires ATP

Question 4

How are microtubules polymerised and depolymerised?

Answer 4

Tubulin dimers with GTP bind more tightly to each other than tubulin with GDP

therefore microtubules with newly added tubulin tend to keep growing

• Tubulin in GDP bound state puts strain on system and leads to peeling

Growing microtubules occur if addition of GTP tubulin is faster than GTP hydrolysis

Shrinking microtubule occurs if hydrolysis of GTP faster than addition of GTP

→ Dynamic instability - refers to the coexistence of assembly and disassembly at the ends of a microtubule

• switch from growth to shrinking is called a catastrophe
• GTP-bound tubulin can begin adding to the tip of the microtubule again, providing a new cap and protecting the microtubule from shrinking → rescue

Dynamics can be controlled to treat disease:
Colchicine - first known drug affecting tubulin
→ binds tubulin dimers and inhibits polymerisation
- relieved joint pain caused by gout
Taxol - front line drug for treating solid tumours especially in ovarian and breast cancer
→ inserts itself into the the tubulin protein and prevents compaction of α and β subunits so no tension builds up even if microtubules stops growing
- found in bark of pacific yew tree

Question 5

Describe the microfilaments (actin filaments)

Answer 5

• Composed of s.actin subunits which have a plus (fast growing, barbed) end and minus (slow growing, pointed) end
• Thinnest cytoskeleton element (6nm in diameter)
• found in bundles → strong
• Cross linking proteins (actin binding proteins) organise how actin is interconnected
  → highly dynamic
  → very strong
• essential in maintaining shape and movement
• essential for movement involving cell surface
• cytokinesis
• cell movement
• muscle contraction

Dynamics requires energy and polarity:
- actin bound with ADP is weaker → more likely to depolymerise and disassemble
- actin bound with ATP is stronger
Treadmilling = filaments grow faster at positive end (depolymerisation on negative end)

→ profilin recharges ADP-actin to ATP-actin
→ cofilin binds ADP-F-actin and destabilises and unwinds the filament

Question 6

What are motor proteins?

Answer 6

Motor proteins are proteins that move along the cytoskeleton tracks and use energy from ATP hydrolysis to cause conformational changes in protein shape which creates force (convert chemical energy into mechanical work by ATP hydrolysis)

3 classes in eukaryotic cells:

1. Myosins - actin filament motors
2. Kinesins - microtubule motors
3. Dyneins - microtubule motors

Question 7

Describe myosins

Answer 7

Myosins - actin filament motors

movement powered by ATP hydrolysis

Diverse range of myosin types

• myosin II involved in muscle contraction - movement on actin cytoskeleton towards + end
• myosin IV transports vesicular cargo - endocytosis - engulfing things from PM - movement towards - end

Muscle contraction

• skeletal muscles packed with myofibrils
• myofibrils consists of a chain of units called sarcomeres
• sarcomeres are assembles of actin and myosin microfibrils
• myosin binds to and hydrolyses ATP which drives its movement along actin filament

Actin filaments anchored by + end to Z disc

• myosin and actin filaments overlap with the same relative polarity on either side of midline

CONTRACTION - actin and myosin slide past each other without shortening, driven by myosin heads walking towards + end of actin filament

→ each cycle involves myosin binding and hydrolysing 1 ATP molecule

Question 8

Describe kinesins

Answer 8

Kinesins - microtubule motors

Processive Motor

• transports towards minus end or plus end depending on kinesin
• vital to spindle formation in mitotic and meiotic chromosome separation during cell division
• responsible for shuttling mitochondria, Golgi bodies, and vesicles within eukaryotic cells.

Question 9

Describe dyneins

Answer 9

Dyneins - Microtubule motors

Requires accessory proteins called dynactin complex

• transports from golgi to nucleus - towards minus end

Question 10

Explain how filament polarity can be identified

Answer 10

Each unit is polar, giving the filament polarity
Filaments = stacked units bound by non-covalent forces
Filaments have fast (+) and slow (-) growing ends (symbols do NOT represent charge)