Lecture 2: Cytoskeleton and Motor Proteins

Question 1

What machinery do animal cells use to generate movement?

Answer 1

• cytoskeleton
• motor proteins

Question 2

What is the cytoskeleton?

Answer 2

protein-based intracellular network

Question 3

What are motor proteins?

Answer 3

enzymes that use energy from ATP

Question 4

What are the 4 general types of cellular movement?

Answer 4

• polymerization
• mobile motor
• mobile cytoskeleton
• mobile motor and cytoskeleton

Question 5

4 General Types of Cellular Movement

a) Polymerization

Answer 5

reorganization of the cytoskeletal network (actin polymerization)

• growth of the cytoskeleton in one region of the cell pushes the plasma membrane outward
• know where to move based on signals and receptors
• used in amoeboid movement

Question 6

4 General Types of Cellular Movement

b) Mobile Motor

Answer 6

motor proteins ‘walk’ along relatively fixed elements of the cytoskeleton

• can be used to transport cargo throughout the cell – cargo is bound to the cytoskeleton with a motor protein, which hydrolyzes ATP, allowing it to walk along the cytoskeleton

Question 7

4 General Types of Cellular Movement

c) Mobile Cytoskeleton

Answer 7

motor proteins attached to the cell membrane pull on the cytoskeleton, moving an element of the cytoskeleton

• ratcheting action of motor proteins is important for pulling cytoskeleton in one direction

Question 8

4 General Types of Cellular Movement

d) Mobile Motor and Cytoskeleton

Answer 8

motor proteins and the cytoskeleton are arranged such that they slide over each other, pulling the cell into a different shape

Question 9

What are the 3 components of the cytoskeleton?

Answer 9

• microtubules
• microfilaments (actin filaments)
• intermediate filaments

Question 10

Microtubules

What are microtubules? Where are they found?

Answer 10

• long hollow tubes composed of repeating units of tubulin (dimer of 𝛼-tubulin and 𝛽-tubulin)
• most extend from the nucleus toward the centre of the cell, some found along the cell periphery

Question 11

Microtubules

Do microtubules have polarity?

Answer 11

yes

Question 12

Microtubules

What occurs at the plus and minus ends of microtubules?

Answer 12

• typically grow at (+) end
• typically shrink at (-) end
• microtubules shrink faster than they grow

Question 13

Microtubules

What factors influence the rate of growth and shrinkage of microtubules? (4)

Answer 13

• concentration of tubulin
• whether or not GTP bound by 𝛽-tubulin is hydrolyzed
• microtubule associated proteins (MAPs)
• temperature

Question 14

Microtubules

How does tubulin concentration affect the rate of growth and shrinkage of microtubules?

Answer 14

• critical concentration for the plus end is lower than the critical concentration for the minus end

Question 15

What is taxol and how does it function?

Answer 15

chemotherapy drug

• used to treat solid cancer tumours (ie. ovarian, bladder, lung, etc.)

mechanism of action

• targets microtubules – binds to 𝛽-tubulin and prevents microtubule from disassembling (microtubule stabilizing agent)
• targets mitosis – blocks progression of mitosis (metaphase spindle is not properly formed)

Question 16

Microtubules

What is the microtubule-organizing centre (MTOC)?

Answer 16

• located near the nucleus in most cells
• minus end of microtubules is typically at MTOC
• plus end of microtubules is typically extending out toward the cell membrane

Question 17

Microtubules

How are motor proteins associated with microtubules?

Answer 17

microtubules act as tracks along which motor proteins kinesin and dynein move

• kinesin: moves towards plus end (towards cell membrane)
• dynein: moves towards minus end (towards nucleus)

Question 18

Pigment Movement and Microtubules – Analysis of a Primary Data Paper

Answer 18

see notes

Question 19

Microfilaments (Actin Filaments)

What are microfilaments?

Answer 19

polymers composed of the protein actin

• long strands of the globular protein 𝛽-actin (G-actin)
• found in all eukaryotic cells
• mostly around the cell periphery

Question 20

Microfilaments (Actin Filaments)

How do microfilaments form?

Answer 20

• G-actin polymerizes, forming filamentous actin (F-actin)
• similar to microtubules, growth of F-actin is spontaneous and has polarity (plus/minus ends)

Question 21

Microfilaments (Actin Filaments)

How does microfilament movement arise? (2)

Answer 21

• actin polymerization (amoeboid movement) – important for cell movement, actin filament under the membrane gives cells its shape
• sliding filament model using myosin (more common) – microfilaments act as tracks that myosin moves along by pulling on microfilaments (important for intracellular transport – ie. myosin V)

Question 22

Microfilaments (Actin Filaments)

What is actin treadmilling?

Answer 22

• actin monomers treadmill along F-actin toward the (-) end (similar to microtubules)
• growth on one end and shrinkage on the other end, results in treadmilling

Question 23

Microfilaments (Actin Filaments)

What factors influence the rate of growth and shrinkage of microfilaments?

Answer 23

• concentration of G-actin (more monomers → more supplies for growth)
• capping proteins

Question 24

Microfilaments (Actin Filaments)

How do capping proteins influence the rate of growth and shrinkage of microfilaments?

Answer 24

• increase length by stabilizing minus end
• prevents shrinkage on one end (no treadmilling)

Question 25

Microfilaments (Actin Filaments) What is myosin?

Answer 25

motor protein for actin filaments - is an ATPase – transforms energy associated with ATP hydrolysis into mechanical energy - consists of three distinct regions: head, tail, and neck (associated with light chains)

Question 26

Microfilaments (Actin Filaments) What are 3 main myosin types? What are they each involved with?

Answer 26

- myosin I: involved with cargo transport - myosin V: involved with cargo transport - myosin II: involved with muscle tissue

Question 27

Microfilaments (Actin Filaments) Describe the myosin gene family.

Answer 27

- found in all eukaryotic organisms, therefore likely first appeared around 1 billion years ago - at least 30 classes of myosin expressed in eukaryotes, with multiple isoforms within each class - myosin II has two classes and numerous isoforms - numerous isoforms likely due to genome duplication events

Question 28

Microfilaments (Actin Filaments) Describe the directionality of myosin movement.

Answer 28

- most known types of myosin move towards plus end of actin filament - exception: myosin VI moves towards minus end – functions include intracellular transport and endocytosis

Question 29

Microfilaments (Actin Filaments) – Sliding Filament Model What is the sliding filament model?

Answer 29

basic mechanisms of how actin and myosin interact with each other - shared across myosin isoforms and biological processes from subcellular vesicular transport to muscle contraction

Question 30

Microfilaments (Actin Filaments) – Sliding Filament Model What are two important aspects to remember about proteins?

Answer 30

- proteins change shape when compounds bind to them - changing shape can allow proteins to bind or unbind other compounds

Question 31

Microfilaments (Actin Filaments) – Sliding Filament Model What are the two basic processes that underlie the sliding filament model?

Answer 31

two processes form the cross–bridge cycle (which allows muscle contraction) - cross-bridge: myosin binds to actin (chemical) - power stroke: myosin bends (structural)

Question 32

Microfilaments (Actin Filaments) – Sliding Filament Model Describe the steps of the cross-bridge cycle.

Answer 32

1. ATP binds, causing myosin to detach from actin 2. detachment of myosin causes ATP to be hydrolyzed to ADP and Pi, which remain bound by myosin 3. hydrolysis of ATP causes myosin to extend and attach to (+) end of actin (myosin forms cross-bridge with actin) 4. release of phosphate results in conformational change and promotes power stroke (bending of myosin head) 5. ADP is released

Question 33

Microfilaments (Actin Filaments) – Sliding Filament Model What is unitary displacement?

Answer 33

distance that myosin steps during each cross-bridge cycle - step size depends on length of myosin neck

Question 34

Microfilaments (Actin Filaments) – Sliding Filament Model What is the unitary displacement for myosin monomers?

Answer 34

amount moved is variable

Question 35

Microfilaments (Actin Filaments) – Sliding Filament Model What is the unitary displacement for myosin dimers?

Answer 35

displacement depends on the periodicity of the actin filament - myosin V dimer uses both monomers in tandem, with an average unitary displacement of 36 nm – ‘walks’ along microfilament with ~36 nm steps on average (period of the helical actin filament)

Question 36

Microfilaments (Actin Filaments) – Sliding Filament Model What is the duty cycle?

Answer 36

proportion of time during each cross-bridge cycle that myosin is attached to actin (time spent in cross-bridge aka attached to actin) / (time for full cross-bridge cycle)

Question 37

Microfilaments (Actin Filaments) – Sliding Filament Model What is the duty cycle of non-muscle myosin?

Answer 37

typically 0.5 - means that myosin is tightly bound to actin for only half of each cross-bridge cycle - used for intracellular vesicle transport (ie. myosin V)

Question 38

Microfilaments (Actin Filaments) – Sliding Filament Model What is the duty cycle of muscle myosin?

Answer 38

typically 0.05

Question 39

Microfilaments (Actin Filaments) – Sliding Filament Model What is the unitary displacement and duty cycle of myosin V?

Answer 39

- unitary displacement: 36 nm - duty cycle: 0.5

Question 40

Microfilaments (Actin Filaments) – Sliding Filament Model What is the unitary displacement and duty cycle of myosin in thick filaments?

Answer 40

- unitary displacement: 5-15 nm - duty cycle: 0.05