BCHE4040/LSCI5440-Aspects of

Neuroscience

Section 5A: Axoplasmic and Cell

membrane transport

Kim Hei-Man CHOW, PhD

Assistant Professor
Room 604, Mong Man Wai Building
Biochemistry Program
School of Life Sciences
Email: heimanchow@cuhk.edu.hk
Learning objectives (Section 5)
Part A: Axoplasmic transport
1. Anterograde axonal transport
2. Retrograde axonal transport
3. Cytoskeletons
4. Motor proteins—dynein, kinesin, and myosin

Part B: Cell membrane transport

1. Exocytosis
2. Endocytosis
3. Endosome pathway
4. Neurotransmitter recycling pathway
 Part A. Axoplasmic transport

1. Also known as: axonal transport

2. a transport process of chemicals, vesicles and cell organelles along the interior of the axon
3. 2 directions:
• Anterograde axonal transport—movement from cell body towards axonal terminal
• Retrograde axonal transport—movement from axonal terminal towards cell body
 The need for axoplasmic transport
Main reason: Axons could be very long and complex !

1. Some motor neurons have axons longer than 1

meter, and may form thousands of synapses
(inter-neuronal connections) at their terminals
2. In cell body, it is where RNA, proteins, peptides
are synthesized
3. At axons, in addition to enzymes,
neurotransmitters and structural proteins;
there is a high rate of turnover of cell
organelles, all of which need to be replaced
regularly to facilitate firing of action potentials

Imagine: in such a long neuron, how membrane proteins, vesicles, organelle be transported from the cell
body to the end of axon and mediate proper cell function?

Indeed…we have our own intracellular “Railway” system

 Axonal cytoskeleton—the rail tracks
1. The axon has a “railway” and “cargo” systems
2. Unique unipolar orientation of microtubules
3. Actin cytoskeleton (tunnel)—exists as
periodically spaced rings underneath axonal
plasma membrane, organized by spectrin and
adductin-->provides elasticity and supportive
forces along the axon
4. Axon initial segment (NIS)—for initiation of
action potentials and maintaining neuronal
polarization
• contains a very dense meshwork of
cytoskeleton, iron channels (to initiate
firing)
• Contains a scaffold protein Ankyrin-G (the
master organizer of the NIS) links
transmembrane proteins to actin and
microtubule cytoskeleton
 Anterograde and Retrograde Transport
1. Anterograde transport: direct away from
the cell body, mediated by kinesin
complexes (tail-directed trains)

2. Retrograde transport: direct towards the

cell body, mediated by dynein/dynactin
complexes (soma-directed trains)

3. Some cargoes (passengers) can bind to

kinesin and dynein/dynactin complexes,
the same type of “passengers” can be
seen riding in two directions
simultaneously (e.g. endosomes,
lysosomes, mitochondria and mRNA)
 Motor proteins—the “Trains”
1. Three families: myosin, kinesin and dynein
2. They move along cytoskeletons via a force-
dependent mechanism by converting
chemical energy from ATP into motion
3. The motor proteins (trains) propel
themselves along the cytoskeleton (railway)
using a mechanochemical cycle of:
i. Filament binding
ii. Conformational change
iii. Filament release
iv. Conformational reversal
v. Filament rebinding
(just like walking)
Illustration of the mechanochemical cycle
mechanochemical cycle

Filament binding/rebinding

Conformational
reversal Conformational
change

Filament release
 “Peripheral/tail-directed” train—Kinesin
1. Anterograde transport protein
2. A superfamily of molecular motors that
generate force and motility along microtubule
filaments
3. Protein structure contains
1. A conserved motor domain for ATP
hydrolysis and microtubule binding
2. Divergent non-motor domains for family-
specific mechanisms and functions (e.g.
determine which cargo to bind to)
 Examples of cargo transported by kinesin
• All are transported from cell body to axon (also dendrites, but example below are specific
to axon)
• Major function is to establish cell polarity during development, i.e. axon specification from
all neurites
1. PI3K (phosphoinositide 3-kinase)
• A surface protein that is critical for establishing/maintaining neuronal polarity
• Facilitate the accumulation of phosphatidylinositol 3,4,5-trisphosphate (PIP3—its
product) in the growth cone ( the tip) of axon.
2. Shootin-1
• It is found to accumulate in one single neurite during development and this
neurite would subsequently grow and develop into axon (initiate the polarity)
• Regulate accumulation of PI3K and PIP3 at axon
• Actively transported from the cell body to the growth cone of axon
3. Par3/Par6/atypical PKC (aPKC) polarity complex
• Axonal determination requires the localized activity of aPKC
• Par3/Par6/aPKC promotes the production of PIP3
 Kinesin—transportation mechanism
Top:
1. Crystal structure of kinesin I motor heads and neck
linkers docked onto a microtubule.
2. Kinesin motor heads bind to tubulin and walk towards
the plus end of the microtubule (towards cell periphery)

Bottom (mechanism of walking):

1. A motor head (purple) in the empty state binds the
microtubule, with the lagging head (blue)swinged up
behind.
2. The neck linker of the leading head (purple) stiffens
upon ATP binding, which drives the lagging head
forward towards the next binding location (it helps to
twist the linker in such the lagging head (blue) moves
forward).
3. ATP in the now lagging head (originally the leading
head, purple) is hydrolysed to ADP and phosphate Pi,
which results in detachment of this head from the track.
4. The cycle continues with each ATP hydrolysed
corresponding to one step forward
Watch this video: https://youtu.be/YAva4g3Pk6k
“cell body-directed” train—Dynein-dynactin complex
1. Retrograde transport protein
2. Dynein needs to function in complex with dynactin—a
highly conserved co-activator complex for dynein and
act as a scaffold to facilitate dynein binding to the cargo
3. Each functional unit consists of 2 dynein and 2 dynactin
4. Dynein (orange/blue/yellow) is bound to dynactin
complex (pink/white/blue) through interactions +
between dynein light chains and dynamatin (pink)
subunits of dynactin
5. The Glued subunits (of dynactin, blue balls) and the
stalk (of dynein) bind to microtubules.
6. Powered by ATP hydrolysis (by Dynein ATPase),
transforming chemical energy to mechanical energy
Examples of cargo transported by dynein-dynactin
complex
• All are transported from the distant end of dendrites (also axon but examples below
illustrates cargos from dendrites ) towards cell body
• Major function is to sustain cell survival, dendrites growth

1. Nerve growth factor (NGF)/Tropomyosin receptor kinase A (TrkA) signalling

endosomes
• NGF binds and enhances activities of TrkA
• Activated NGF/TrkA complex forms signalling endosomes, which is endocytosed
into the cell and activates downstream signalling cascade

2. Brain-derived neurotrophic factor (BDNF)/TrkB signalling endosomes

• BDNF binds to TrkB, triggers internalization of the BDNF/TrkB signalling endosomes
• BDNF regulates dendritic arborisation and outgrowth
“short ranged” train—myosin
1. Either retrograde or anterograde
direction short-ranged transport
2. Binds actin but not microtubules
3. N-terminal motor (head) binds actin
filaments, hydrolyses ATP and generates
force
4. The head is followed by a neck region,
which act as a lever arm
5. The tail comprises a proximal region
contains a dimerizing coiled-coil
sequence and a distal region, which can
be globular or non-helical.
 Actin and myosin at the CNS synapse
1. Neuronal synapses are highly dynamic and
undergo molecular and structural changes
linked to the regulation of synaptic
plasticity.

2. Myosins make essential contributions to

these processes by mediating actin
cytoskeleton rearrangement and cargo
transport

Examples:
1. Myosin Va transports the endoplasmic reticulum into the spines of Purkinje neurons, thereby allowing cerebellar
long-term depression
2. Myosin VI associates with membranes, walks towards the actin filament's minus end and functions in AMPA receptor
and type A GABA receptor trafficking

Synaptic myosins might be linked to psychiatric disease (non-muscle myosin IIa and myosin Vb) and neurodegenerative
disease (myosin VI). Mutations in myosin Va cause severe neurological defects including ataxia (cerebellum) and seizures.
Non-muscle myosin IIb is required for normal nervous system development
Important concepts
1. Neurons grow via transporting newly synthesized membrane from the
Golgi to the plasma membrane at cell body, axonal growth cone and
dendrites
2. Anterograde transport: away from the cell body, mediated by kinesin
complexes on microtubules
3. Retrograde transport: towards the cell body, mediated by dynein-dynactin
complexes on microtubules
4. The key steps in mechano-chemical cycle.
5. Kinesin and Dynein-dynactin complex: interacts with tubulin, involved in
long-range transport
6. Myosins: interacts with actin, involved in short-ranged transport
7. The roles of kinesins in axonal growth; Dynein-dynactin complex in cell
survival and dendritic growth and myosins in synaptic plasticity
Concept check (T/F)
1.Microtubules are unipolar in nature
2.Axon initial segment lacks microtubules and cytoskeletons
3.Anterograde transport is mediated by dynein complex
4.Motor proteins function by utilizing ATP as a source of energy for
electrical work
5.Kinesin transport facilitate neuronal polarization during
development
6.Dynein complex moves along actin cytoskeleton
7.Myosin moves along the microtubule cytoskeleton
Reference and further reading: