Moving cargo into the cell Flashcards
Why is cell movement required in neurones?
Things made in the nucleus in the cell body need to be transported along the axon to the synapse.
What are motor proteins for?
Motile activities are coupled to the interaction of a motor protein with either actin filaments or microtubules.
What are motor proteins?
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.
What are the types of motor proteins?
Actin based molecular motors - the myosin superfamily.
Microtubule molecular motors - dyenins and kinesins
What are the domains of myosin?
Head domain
Neck domain
Tail domain
What is the head domain?
Binds to F-actin and ATP.
Uses ATP hydrolysis to generate force.
Found in all myosins.
What is the neck domain?
Acts as a linker and works as a lever to transduce the force generated by the head.
What is the tail domain?
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.
What is myosin II?
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.
What are the light chains of muscle myosin?
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.
What is the evidence for the motor activity of the myosin head?
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.
What are the conclusions from the in-vitro motility assays?
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.
What are the conclusions from in-vitro motility assays - direction?
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.
What is the actin-myosin crossbridge cycle?
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.
What is the actin-myosin crossbridge cycle from Pi?
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.
What are non-processive motors?
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.
What are processive motors?
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.
What is the processive cycle of myosin V?
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.
What is the regulation of non muscle myosin II?
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.
What can non muscle myosin II do?
Active myosin II can spontaneously assemble into filaments, can act in cytokinesis and cell migration.
What are Kinesins?
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.
What is the conventional kinesin structure?
Light chains are involved in regulation but also in binding to transported vesicle.
Grouped into cytosolic and spindle kinesins.
What are cytosolic kinesins?
Membrane bound vesicles and organelles are transported by their own specific kinesins.
Move long distances along cell along well defined routes.
What are spindle kinesins?
Responsible for spindle assembly and chromosome segregation during cell division.
Also called kinesin related proteins (KRP)
What are the conclusions from kinesin in vitro motility assays?
Conventional kinesins are plus end motors and always move towards the plus end of the microtubule.
Spindle microtubules are negative.
What is the process of conventional kinesin?
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.
What is the process of conventional kinesin from Pi?
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.
What is Kinesin regulation?
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.
What are internal kinesins?
The motor domain is in the middle of the protein.
What is the C-terminal kinesin?
Motor domain is at C terminus.
Moves towards the minus end of the microtubule.
Involved in spindle pole formation.
What are bipolar kinesins?
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.
What are dyenins?
Cytosolic - movement of vesicles and chromosomes.
Axonemal - beating of cilia and flagella.
What are cytosolic dyenins?
Move towards the negative end of microtubules.
Largest motor protein.
Moves 7x faster than kinesin.
What is the structure of cytosolic dyenins?
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.
What is the process of cytosolic dyenins?
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.
