protein transport and insertion

Question 1

The three key cytoskeletal filaments that maintain neuronal “shape” are

Answer 1

microfilaments
actin microfilaments
intermediate filaments

Question 2

Microtubules and actin

Answer 2

• critical for transport of proteins to dendrites and the axon
• These proteins are arranged in very specific orientations to provide a region unique transport ability: dendrites (distal versus spine) and axons (distal versus axon initial segment) are arranged very differently

Question 3

Microtubules role and location

Answer 3

• critical for intracellular transport and contribute to the morphology of the cell.
• Microtubules are located in dendrites and axons.

Question 4

Microtubules Polarity

Answer 4

• The polarity differences (+ and -) guide transport of cargo via the motor proteins
• Axons tend to have their + end distal to the cell body and the – end proximal, whereas dendrites can have the +end either distal or proximal

Question 5

Microfilaments location and role

Answer 5

A. Microfilaments are present throughout a neuron, though concentrated in the presynaptic terminal, dendritic spines and growth cones.
B. Microfilaments are involved with intracellular transport and cell movement
- Additionally, microfilaments are key for positioning receptors and ion channels at specific locations on neuronal surfaces.
C. There are numerous proteins associated with microfilaments

Question 6

Intermediate filaments

Answer 6

• Role is to stabilize and maintain neuronal morphology
• Unlike with microtubules and microfilaments, intermediate filaments are not in oligodendrocytes.
• Some subtypes of intermediate filaments, such as α-internexin and nestin are present in early development but disappear in matured neurons

Question 7

Neurofilaments

Answer 7

1) High level of phosphorylation at their tail end
2) Glutamate rich region also at the tail end—importance is that silver staining, a critical stain used by histologists since Cajal’s time

Question 8

Molecular Motors

Answer 8

Myosins - microfilaments
Dyneins - microtubles
Kinesins - microtubules
transport cargo on the cytoskeleton in the nervous system

Question 9

Molecular motors 3 points

Answer 9

1) In axons, where there is little/no protein synthesis machinery, protein complexes or vesicles containing proteins are transported down the axon.
2) Because the needs of axons are so high, and because axons can be very long, vesicles are often recycled in axon terminals to have a local availability of proteins.
3) In dendrites, where there is some protein synthesis machinery, mRNA can also be transported directly to the dendrites and synthesized locally

Question 10

How do molecular motors work?

Answer 10

• In general, the way these molecular motors work is by utilizing energy (via ATP) to move in an anterograde (i.e. towards the axon terminal) or retrograde (towards to the cell body) fashion.
• One end of the molecular motor will recognize the cytoskeleton while the other will recognize proteins on the cargo.
• Movement occurs when the hydrolysis of the ATP causes a conformational change in the cargo-carrying protein, causing it to move on the microfilament or microtubule.

Question 11

Kinesin (microtubule)

Answer 11

1) Kinesin is comprised of two heavy chains (i.e. greater molecular weight) and two light chains.
2) The heavy chains form the “head” of the molecule and contain the ATP-and microtubule-binding regions.
3) The light chains are specialized to interact with membrane bound organelles (i.e. vesicles)
4) Kinesins are typically associated with fast transport

Question 12

Dynein (microtubule)

Answer 12

• Dynein is comprised of two heavy chains and multiple intermediate light chains.
• Dyneins are associated with both retrograde fast transport and anterograde slow transport

Question 13

Axonal Transport

Answer 13

• Two types of axonal transport: fast transport of membranous organelles (i.e neurotransmitter) and slow transport of cytosolic proteins and cytoskeletal proteins
• The neuron is not uniform, so the cytoskeletal layout of dendrites differ from axons, which is what creates speed differences
• Remember: dendrites and axons have different functional needs

Question 14

Dendrites/dendrites spines

Answer 14

• Dendrites are special in that neuronal plasticity, such as LTP/LTD, depend on changing dendrite architecture
• The soma/dendrite boundary is characterized by having “mixed” microtubules polarity
• The microtubules also have different post-translational modifications compared to axons, and these modifications make the dendritic cytoskeleton less stable (more “dynamic”)

Question 15

dendrite spines continued

Answer 15

• Dendritic spines are major sites for excitatory synapses, and their role is to limit changes of plasticity to that synapse
• Spines have more actin compared to the rest of the dendrite
• With activity through NMDA receptors, microtubules come in to help enlarge the spine. This is what helps facilitate long term potentiation (LTP)

Question 16

Axonal Initial Segment AIS

Answer 16

• This is the region that contains the axon hillock, and demarcates the fairly similar soma/dendrites architecture from the very distinct axonal one
• The density of cytoskeletal elements and proteins creates a difficult to permeate region, which keep soma organelles/proteins out of the axon (and vice versa

Question 17

AIS proteins and transport

Answer 17

• The critical protein for the AIS, considered the “master organizer” of the AIS, is called ankyrin G (AnkG).
• AnkG is located throughout the neuron and cells in general, but certain isoforms are located preferentially in the AIS
• AnkG is found first in the AIS, and recruitment of all other AIS proteins depends on AnkG
• AnkG helps anchor the microtubules with the plasma membrane, and possibly helps bundle the microtubules together in the process
• AnkG is also thought to be the “barrier” between soma and axon

Question 18

Axon

Answer 18

1) The most distal point of an axon “can be about 20,000-fold greater than the diameter of the soma.” Efficiency and continuous support are critical here
2) At the proximal axon (along the axonal shaft), the microtubules are tightly wrapped together
3) At the distal axon (i.e. axon terminal), the microtubules are sparser
4) Cargo is not “released” from the transport and allowed to diffuse to the membrane, and instead though various phosphorylation dependent reactions, guided to the appropriate axon membrane portion

Question 19

axon transport

Answer 19

• Axons are unique in that retrograde transport back to the cell body occurs regularly. Signaling endosomes, which are internalized portions of the axon terminal membrane, get transported back to the soma
• These endosomes contain neurotrophic signaling receptors and can modify genetic expression and survival of the neuron
• The sparsity of axon terminal microtubules can make this transport difficult to initiate, and to overcome this, the axon terminal has enriched dynein and actin.
• This enrichment might allow for the cargo to be better attached to the spare microtubules

Question 20

Slow Axonal transport

Answer 20

• (cytosolic proteins and cytoskeletal proteins)
• Slow axonal transport is unidirectional, meaning anterograde to axon terminal or distal dendrites
• Dynein plays a major role in slow axonal transport
• There are two components to slow axonal transport: Slow component A (SCa) and slow component B (SCb)

Question 21

SCa

Answer 21

• the movement of cytoskeletal components, mostly neurofilaments and microtubules.
• The rate of transport is about 0.1-1mm/day, meaning for a meter long axon (like from your spinal cord to your toes) it can take up to 1000 days (~3 years!) to reach.

Question 22

SCb

Answer 22

• involves movement of soluble enzymes to polypeptides for cytoskeletons and moves at a rate of 2-4mm/day.
• For nerve growth or regeneration, this is the limiting step

Question 23

Fast Axonal Transport

Answer 23

• (membranous organelles with proteins)
• Fast transport is bidirectional, meaning there is both anterograde and retrograde transport of materials. This is due to the polarity of the microtubules and the molecular motor present on the axon.
• Kinesin and dynein both play a role in fast axonal transport

Question 24

Axonal transport disorders

Answer 24

• neurodegenerative disorders in particular cause (or are caused by) cytoskeletal pathologies
  A. Massive neuronal loss likely involves many different core “housekeeping” processes failing, and maintaining the cytoskeleton and transportation are critical life functions
• Alzheimer’s disease:
  A. Neurofibrillary tangles are created when a microtubule associated protein (MAP), tau, is hyper-phosphorylated and clumps together
  B. Tau normally helps form microtubule bundles and stabilizes them, and the hyper- phosphorylated tau prevent this and disrupt intracellular transport in the process

Question 25

How are proteins transported to their appropriate destinations?

Answer 25

adaptor proteins (APs) present on the vesicle surface.

Question 26

3 targets of adapter protein transportation

Answer 26

1) Directly to plasma membrane for integral proteins – Distinct proteins on the vesicles target them for the membrane. Once there, the SNARE/SNAP complex can help the vesicle fuse with the membrane.
A. Endocytosis of plasma membrane proteins for either recycling or degradation occurs primarily due to two special proteins: Caveolae and Clatherin
2) To lysosomes for degradation – sugar side chains still left on proteins after going through Golgi mark the proteins for lysosomes, which are responsible for degrading proteins.
3) Plasma membrane for on-demand secretion

Question 27

Lipid rafts

Answer 27

• The rafts are rich in certain types of lipids and cholesterol, compared to adjacent areas
• The microdomain structures help compartmentalize the relevant ion channels/GPCRs, along with associated proteins needed for signaling
• all synapses (post-synaptic) will be set up with many lipids rafts to optimize signaling
• Neurotransmitter can bind to channels within the rafts, or in those not associated with the rafts

Question 28

Benefits of lipid rafts?

Answer 28

1) Upregulation/downregulation of receptors is a common response to neurotransmitter binding; rafts can help keep the required adapter proteins and signaling moleculs (G protein like Gq, Gs, Gi) together during these movements
2) Localization of signaling components – more efficient/better regulated?
- Interestingly, the G-proteins that associate with their receptor also have a high affinity for the rafts. The rafts help stabilize the G-proteins to the receptor
- Secondary signaling components of GPCRs, such as adenylyl cyclase and PLC, and their effectors (ion channels) are present in lipid rafts

Question 29

How do cytoskeletal components interact with the lipid rafts?

Answer 29

1) The lipid bilayer is “fluid” meaning that if a component can insert itself into the membrane, in theory that component could “swim” around the length of the bilayer.
2) What stops this from happening? Proteins, like your cytoskeletal proteins, help anchor rafts so the raft is stabilized in one place.
a. Microtubules and actin specifically have associated with lipid raft stabilization
3) Thus, your lipid rafts are critically important for determining what a “synapse” actually looks and functions like

Question 30

What happens when lipid rafts are disrupted?

Answer 30

1) In rat glial cell experiments, certain SSRIs (depression) over time accumulated in lipid rafts and caused a decrease in G-proteins in the lipid rafts. These G-proteins instead co-localized with their signaling components outside the rafts.
a. Helps depression by: The G-proteins can now increase signaling “tone” without needed continual stimulation
2) In Alzheimer’s disease (AD), the enzyme BACE1, which is normally not in the same lipid raft as amyloid protein, starts aggregating with amyloid protein
a. This aggregation produces excess of the amyloid beta protein subtype that is associated with AD pathology
b. BACE1 inhibitors have failed clinical trials, unfortunately.