Lecture 2 - Protein Trafficking Flashcards
Polarity must be maintained, but how?
Physical barrier
* Axon initial segment
Selective trafficking
* Polarized trafficking in neurons
and also:
Selective retention
* Protein-Protein interactions
Selective degradation
Local Translation
Neurons exhibit and retain morphological and functional asymmetry!
How does the Axon Initial Segment act as a physical barrier to maintain polarity?
Forms after axon establishment
Molecular Sieve (physical “fence”)
* Prevents free diffusion of selected
membrane and cytoplasmic proteins into axons
Regulates initiation of action potentials
What experiment was used to show the role of AIS in polarity maintenance?
Small (10 kD) and large (70 kD) soluble dye was used
The small dye could pass through but not the large dye
AIS acts as a physical barrier that allows motor proteins to selectively enter
How is protein transport in neurons similar to macro-world logistics?
Proteins and organelles (e.g. mitochondria) are in constant motion
* From sites of synthesis to sites of function (synapse formation)
* For degradation (e.g. from distal cellular regions to lysosomes located near center of the cell)
* Where energy is needed (e.g. mitochondria)
Occurs along a system of intracellular highways
* Tracks (cytoskeleton)
* Motor proteins
* Cargoes
How do we know intracellular transport exists?
Axonal transport was observed in large squid axons
Driven by microtubule tracks moving in the axon
Plus end/forward/anterograde transport - ______
Minus end/backward/retrograde transport - ______
Plus end/forward/anterograde transport - kinesin (except kinesin-3)
Minus end/backward/retrograde transport - dynein
What is the structure of a kinesin motor?
Tail
- Cargo binding
Stalk
- Mediates dimerization
Head
- Motor domain, attach to Microtubule
How do we know that kinesin ‘walks’?
In vitro motility assays
Latex beads attached to kinesin were seen to be moving
Fluorescence imaging showed the two dyes moving forward of each other - step-wise alternation
What is the mechanism propelling kinesins forward?
ATP hydrolysis by head region propels kinesins forward
Dimer with two motor heads, consisting of a catalytic core and neck linkers
Heads contain tightly bound ADP
1. Move randomly driven by Brownian motion, binds tightly to microtubule
2. ADP released and ATP enters
3. Triggers neck linker to zipper onto the catalytic core, throwing the second head forward
4. The trailing head hydrolyses ATP and releases phosphate, while the neck linker unzippers from the trailing head
And continue from 2.
How does the tail domain mediate the binding of adapters and cargoes?
Binding is regulated by post-translational modifications at the tail domain
How does the tail domain mediate autoinhibition?
The tail binds to head when there is no cargo/phosphorylation
What is the life cycle of kinesin motors?
It is degraded or recycled as dyenin
How does dynein differ from kinesin?
Dynein is a minus end motor
Dynein is much larger
The structure of the dynein complex?
2 heavy chain (HC)
* Head (motor) domain
–> ATPase activity
* Linker
–> Microtubule binding and generation of movement
* Tail
–> Dimerisation and cargo interaction
2 intermediate chains (IC)
* Scaffold linking HC with LC
* Binds Dynactin
2 light intermediate chains (LIC)
* Attached to HC
* Binds adapter proteins
2 light chains (LC)
* Assembly of dynein complex and cargo interactions
How does dynein motor motion occur?
- ATP binding releases the motor from the MT
- Bending of linker and then ATP hydrolysis
- Diffusing of motor and rebinding to MT
- Linker straightens during a powerstroke to move the whole molecule forward
- ADP release
and continue from 1.
What is dynactin?
What are its functions?
Dynactin is an essential Dynein adapter
* A complex of 23 proteins
Functions
* Attaches cargoes to Dynein
* Activates Dynein motility
* Targets Dynein to plus end of microtubule tracks
* Coordinates transport between Dynein and Kinesin motors (bidirection cargo movement)
–> Front and rear locomotive
What directs motors (and cargoes) to their destinations? (3)
(1) The axon initial segment and motor targeting
(2) Motors exhibit intrinsic directionality
(3) Cargo binding cause differential targeting
- Binding to different combinations adapters and cargo can direct motors to different locations
What are the roles of transport in neuronal function? (5)
During development
* New material to support
–> Axon and dendrite outgrowth
–> Formation of synapses (to form neuronal networks)
In mature neurons
* Addition of new proteins (synaptic plasticity)
* Degradation of faulty proteins (maintenance)
* Transport of signaling molecules from synapse to nucleus
Transport delivers biomaterials for neurite outgrowth. Why is this necessary?
Rapid expansion of size
* Neurite elongation: 0.5 mm/day
* Surface area expansion: ~1μm^2/min
* Cell volume: 0.6%/day
* Surface area: 20%/day
Most components essential for expansion are synthesized in the neuronal cell body
Transport is required from sites of synthesis to destinations of function
Synaptogenesis - formation of new synapses
Why is it essential?
When do synapses form, and what is required?
Essential for polarity and function
(neurotransmission)
Synapses form upon axon-dendrite contact
Delivery of proteins to designated sites along axons and dendrites enables pre- and post-synaptic specialisations to form functional synapses.
Synaptic proteins are transported as vesicular packages. What are the different vesicular packages, and which kinesins are involved in their transport?
Axons (presynaptic terminal)
* Synaptic vesicle precursor transport vesicles (STVs) carry proteins that form synaptic vesicles (KIF1)
* Piccolo-Bassoon transport vesicles (PTVs) carry proteins that form active zones (KIF5)
Dendrites (postsynaptic density)
* PSD95-containing vesicles form post synaptic densities (KIF1)
How does axo-dendritic contact initiates synapse formation?
Neurexin present in axon, forms trans-synaptic complexes with postsynaptic proteins such as neuroligins
Neuroligin present in dendrites, act as ligands for neurexin
How are cargoes delivered to destinations? (2 words)
Membrane fusion
Problems with neuronal transport can cause synapse loss and axon degeneration. How does this occur in Alzheimer’s?
APP aggregates in axons, causing focal blockages (axonal varicosities)
Further swelling (multiple axonal spheroids) leads to degeneration of distal axon (resulting in end bulb)
What are some diseases resulting from axonal transport disruption and neurodegeneration?
Amyotrophic lateral sclerosis
Huntington’s disease
Alzheimer’s disease
Parkinson’s disease
What is synaptic loss correlated with?
Disease severity
What is affected in Alzheimer’s disease?
Disrupted axonal transport
Synapse malfunction and loss
How are tracks and motors affected during neurodegeneration?
Structural (tracks)
- hyperphosphorylation of Tau
- neurofibrillary tangles (accumulation of Tau)
- Disrupt trafficking
Regulatory (motor)
- Impact binding to microtubules
- Reduced levels of motor proteins in AD patients
What molecules are responsible for Alzheimer’s disease? What does it affect?
Aβ oligimers
Disrupt axonal transport of synaptic proteins
How are transport defects an underlying cause for Alzheimer’s disease?
Lead to axonal disconnection
resulting in
Functional deficits
Cell death (which leads to functional deficits and axonal disconnection)
