Moving cargo into the cell

Question 1

Why is cell movement required in neurones?

Answer 1

Things made in the nucleus in the cell body need to be transported along the axon to the synapse.

Question 2

What are motor proteins for?

Answer 2

Motile activities are coupled to the interaction of a motor protein with either actin filaments or microtubules.

Question 3

What are motor proteins?

Answer 3

An enzyme that converts chemical energy (ATP) to mechanical energy.
Hydrolysis of ATP is coupled to conformational changes in the protein, this moves the motor protein.

Question 4

What are the types of motor proteins?

Answer 4

Actin based molecular motors - the myosin superfamily.
Microtubule molecular motors - dyenins and kinesins

Question 5

What are the domains of myosin?

Answer 5

Head domain
Neck domain
Tail domain

Question 6

What is the head domain?

Answer 6

Binds to F-actin and ATP.
Uses ATP hydrolysis to generate force.
Found in all myosins.

Question 7

What is the neck domain?

Answer 7

Acts as a linker and works as a lever to transduce the force generated by the head.

Question 8

What is the tail domain?

Answer 8

Mediates interactions with cargo molecules or other myosin tail regions.
Most variable - the motor protein is interacting with something else - a cargo, vesicle, or other protein.

Question 9

What is myosin II?

Answer 9

Muscle myosin.
Made from 6 polypeptides - 2 identical large heavy chains, 2 pairs of light chains.
Heavy chain contains the neck, head and tail domain.

Question 10

What are the light chains of muscle myosin?

Answer 10

Regulatory proteins, not needed for movement, but help interactions with other protein, and help turn motor domains on and off.
Associated with head and neck regions.

Question 11

What is the evidence for the motor activity of the myosin head?

Answer 11

Purified myosin heads attached to glass slide.
ATP and fluorescent actin filaments added and observe.
Actin filaments bind to head regions, causing myosin to migrate and move along.

Question 12

What are the conclusions from the in-vitro motility assays?

Answer 12

Motor (head) and neck domains are sufficient for generating movement.
Motor domains alone can’t move actin, but can still hydrolyse ATP.
A longer neck gives faster actin filament movement.

Question 13

What are the conclusions from in-vitro motility assays - direction?

Answer 13

All myosins except for myosin VI move towards the plus/barbed end of the actin filament.
Actin filaments move with the minus/pointed end leading.

Question 14

What is the actin-myosin crossbridge cycle?

Answer 14

Myosin bound tightly to actin.
ATP binds to myosin head, conformational change in head causes it to unbind from actin filament.
Hydrolysis of ATP. Neck domain undergoes conformational change, head is displaced along the filament, distance determined by length of neck.

Question 15

What is the actin-myosin crossbridge cycle from Pi?

Answer 15

Weak binding of myosin head to actin causes release of Pi and tight binding of myosin to actin.
The release of Pi triggers the power stroke, ADP is released. Head regains original conformation.
At the end of cycle, myosin head is locked onto actin filament at a new position.

Question 16

What are non-processive motors?

Answer 16

Myosin II: The heads of myosin cycle independently of each other.
Net movement of the actin filament results from uncoordinated actin attachment and detachment of many myosin along the filament.

Question 17

What are processive motors?

Answer 17

Myosin V: The heads of myosin work in a coordinated manner - 1 is always bound to filament.
Heads working together ensures that the cargo stays attached to the actin filament long enough to be transferred along it.

Question 18

What is the processive cycle of myosin V?

Answer 18

Leading head has ADP bound.
ATP binding to trailing head promotes dissociation from actin filament.
Hydrolysis causes conformational change, throws trailing head forwards.
New leading head binds to actin and Pi is released, then ADP is released from trailing head.

Question 19

What is the regulation of non muscle myosin II?

Answer 19

Myosin is in an inactive folded state.
Phosphorylation on light chain by myosin light chain kinase causes myosin to take on extended active state, exposing its actin binding sites.

Question 20

What can non muscle myosin II do?

Answer 20

Active myosin II can spontaneously assemble into filaments, can act in cytokinesis and cell migration.

Question 21

What are Kinesins?

Answer 21

Similar structure to myosin, different mechanism:
Head domain and neck domain are the same.
Tail domain mediates interactions with cargo molecules and kinesin regulatory chains.

Question 22

What is the conventional kinesin structure?

Answer 22

Light chains are involved in regulation but also in binding to transported vesicle.
Grouped into cytosolic and spindle kinesins.

Question 23

What are cytosolic kinesins?

Answer 23

Membrane bound vesicles and organelles are transported by their own specific kinesins.
Move long distances along cell along well defined routes.

Question 24

What are spindle kinesins?

Answer 24

Responsible for spindle assembly and chromosome segregation during cell division.
Also called kinesin related proteins (KRP)

Question 25

What are the conclusions from kinesin in vitro motility assays?

Answer 25

Conventional kinesins are plus end motors and always move towards the plus end of the microtubule.
Spindle microtubules are negative.

Question 26

What is the process of conventional kinesin?

Answer 26

Each tubulin dimer contains a single kinesin binding site.
ATP bound to lagging head locks it firmly to microtubule.
The leading ADP bound head is randomly searching for the next binding site.
The leading head weakly docks to its binding site.

Question 27

What is the process of conventional kinesin from Pi?

Answer 27

After the leading head docks to binding site, the lagging head ATP is hydrolysed.
Release of Pi from lagging head weakens its binding. The leading head rebinds ATP and locks down on the microtubule.
This throws the trailing head forward, the trailing head becomes the leading head.

Question 28

What is Kinesin regulation?

Answer 28

Folding at non-coiled coil region hides the cargo binding region.
When folded cannot firmly attach to microtubules.
Cargo binding to tail releases the inhibition, extending the kinesin.
The active cargo bound kinesin can now attach and move along the microtubule.

Question 29

What are internal kinesins?

Answer 29

The motor domain is in the middle of the protein.

Question 30

What is the C-terminal kinesin?

Answer 30

Motor domain is at C terminus.
Moves towards the minus end of the microtubule.
Involved in spindle pole formation.

Question 31

What are bipolar kinesins?

Answer 31

Motor domains at both ends.
The can bind microtubules at either end - either binding them together or sliding them relative to each other.
Important for mitosis.

Question 32

What are dyenins?

Answer 32

Cytosolic - movement of vesicles and chromosomes.
Axonemal - beating of cilia and flagella.

Question 33

What are cytosolic dyenins?

Answer 33

Move towards the negative end of microtubules.
Largest motor protein.
Moves 7x faster than kinesin.

Question 34

What is the structure of cytosolic dyenins?

Answer 34

Contain 2 identical heavy chains which form the head domains, interact with microtubules and contain ATPase.
Attached to heavy chain is a stalk and intermediate chains.
Cargo are attached via accessory proteins.

Question 35

What is the process of cytosolic dyenins?

Answer 35

Processive: ATP hydrolysis causes stalk attachment to microtubule.
Release of ADP and Pi causes conformation power stroke, rotating the head and stalk relative to the tail.
Power stroke causes 8nm step, cycle repeats with other head.