Moving cargo in the cell

Question 1

What is the benefit to intracellular trafficking?

Answer 1

Allows us to direct the movement of organelles within cells

Question 2

What are motor proteins?

Answer 2

Enzymes that convert chemical energy to mechanical

Question 3

How do motor proteins work?

Answer 3

Work by the hydrolysis of ATP coupled to conformational changes

Question 4

What are the 2 types of motor protein families?

Answer 4

Actin based - Myosin family

Microtubule based - Dyneins and kinesins

Question 5

What is the general structure of a myosin motor protein?

Answer 5

3 Domains:

Head domain - Binds to F actin and ATP and uses ATP hydrolysis to generate force

Neck domain - Acts as a linker and works as a lever to transduce the force generated by the head

Tail domain - Mediates interactions with cargo molecules or other myosin tail regions

Question 6

What is the conventional myosin?

Answer 6

Myosin II (muscle myosin)

Question 7

What is myosin II made up of?

Answer 7

6 polypeptide chains

2 large identical heavy chains
4 lighter chains

Question 8

What evidence was used to show the motor activity of the myosin head?

Answer 8

Myosin heads attached to the glass slide

Add fluorescently labelled actin filaments and view their movement

Question 9

What were the conclusions from the myosin motility assay?

Answer 9

Head and neck domains required for movement

All myosins more towards the plus/barbed end of the molecule (except myosin VI)

Question 10

What is determined by the size of the myosin neck domain?

Answer 10

Speed of movement!

Longer necks = faster movement

Question 11

Explain the Actin-Myosin cross bridge cycle

Answer 11

1. Myosin is tightly bound to actin filaments
2. ATP binds to the myosin head and causes a conformational change to unbind it from the actin filament.
3. Hydrolysis of the ATP molecule causes a conformational change to the myosin head into its high energy unstable state.
4. The myosin head can lightly bind to actin molecules
5. upon release of the Pi group, the power stroke is initiated and ADP is released during the process
6. The power stroke pulls the actin filaments along and the cycle repeats

Question 12

What are processive and non-processive motors?

Answer 12

Non processive - Heads move independently and have uncoordinated attachment

Processive - Heads move in a coordinated manner ensuring cargo stays attached to actin filaments to be transported

Question 13

Explain the processive cycle of myosin

Answer 13

1. Myosin heads are both attached to the actin filament
2. The leading head has ADP bound to it
3. ATP binds to the trailing head and promotes dissociation of the trailing head from the actin filament
4. ATP hydrolysis causes conformational change throwing the trailing head forward
5. New leading head binds to actin filaments as the Pi group is released
6. ADP is released from the trailing head and the cycle begins again.

Question 14

How is non-muscle myosin II regulated?

Answer 14

Action of Myosin Light Chain Kinases (MLCKs)

1. non muscle Myosin II exists in an inactive folded state
2. Phosphorylation occurs on the regulatory LC by MLCK
3. This phosphorylation causes myosin II to take on an extended active state exposing its actin binding sites 4. Active myosin II can spontaneously assemble into filaments
4. Active myosin II filaments can act in cytokinesis, cell migration, etc

Question 15

What are the types of microtubule motor proteins?

Answer 15

Kinesins

Dyneins

Question 16

What is the structure of the kinesin motor protein?

Answer 16

Similar to myosins

Head domain - binds to MTs and ATP and use ATP to generate force

Neck domain - linker domain to transduce the force from head

Tail domain - binds to cargo and kinesin regulatory chains

Question 17

What are the types of kinesin?

Answer 17

Cytosolic - Membrane bound vesicles and organelles are transported by their own specific kinesin

Spindle - Spindle assembly and chromosome segregation during cell division

Question 18

Explain the processive motor cycle of a kinesi

Answer 18

1. ATP bound to lagging head to lock it to the MT
2. Leading head has ADP bound and is searching for the next binding site
3. Leading head binds to MT and the lagging head hydrolyses its ATP
4. Release of Pi from lagging head weakens the binding and from the MT
5. The leading head rebinds ATP and is locked into the MT
6. Trailing head begins searching for the next binding site and the process repeats

Question 19

How is the kinesin transport regulated?

Answer 19

Folding of kinesin tail acts as a regulator

Folding hides the cargo binding region and the folded from cannot bind to the MT firmly

Question 20

How is the kinesin family organised?

Answer 20

N terminus motor domain - Moves towards + end

Middle motor domain - Little known

C terminus motor domain - Move towards - end

Both terminus motor domain - Binds to MT at both ends

Question 21

What are the two types of dyneins?

Answer 21

Cytosolic - Movement of vesicles and chromosomes

Axonemal - Beating of cilia and flagella

Question 22

What are some properties of cytosolic dyneins?

Answer 22

All move towards - end of MT

Largest and fastest motor protein

2 heavy chains with a stalk of several intermediate chains attached

cargo attached by complex of accessory proteins

Question 23

How does Dynein movement occur?

Answer 23

1. In ATP bound state, stalk not attached to MT
2. ATP hydrolysis causes stalk attachment to MT
3. Release of ADP and Pi causes power stroke rotating the head
4. Power stroke causes a step on MT and cycle repeats

Question 24

What type of motors are dyneins?

Answer 24

Processive motors