neuronal cytoskeleton Flashcards
homeostasis in axons
after neurogeneis in development
the same neurons remain throughout life
so maintaining homeostasis in them is v important
cytoskeleton plays role in this
Neuromuscular junction
v large
see distinct structure in microscopy
terminus of motor neuron contains cluster of synaptic vesicles
lots of fusion of membranes - so many mitochondria present to provide ATP
lots of cytoskeleton organises synaptic structure
myelin secreted by glial cell to protect axon
synapse types
terminal:
-synapse formed at end of axon
-common for neuromuscular junction
en passant:
-littered along axon’s length
-branch out slightly (not to extent of dendrite)
-many synapses in brain are en passant
Axonal cytoskeleton components
acin
MT (^conserved)
neurofilaments (neuron specficic)
importance of axonal transport
neurons are non dividing
cannot regenerate
machinery used in them needs repleneshing
axonal transport does this
many neurodegenerative diseases (eg motor neuron disease) result from transport machinery malfunction
axonal transport cargo types
lots of cargo are cyroskeletal proteins (tubulin, G-actin, neurofilament subunits)
lots are synaptic vesicle precursors or components of synapse
-NT receptors
-mitochondria
-SV precursors
-lysosomes, autophagosomes - degradation machinery
-endosomes
lots of transport synapse -> cell body too
fast v slow axonal transport
fast - 50-400mm/day
>vesicles
>membrane bound organelles
slow - 0.2-10mm/day
>cytoskeletal components
>harder to observe - less well understood
identification of slow and fast transport
radiolabel AA
inject into dorsal ganglion of Spinal cord
labelled AAs incorporate into axons
follow pulse over time period
segment nerve along its length
look at presence of labelled proteins in that section
bands corresponding to some proteins were detected in later segments earlier than other proteins
FASTER transport
could identify proteins present in segments by using IP for known proteins (from the mWt on the autoradiograph)
and using mass spec for unknown ones
axonal transport machinery
same essential machinery used for fast and slow
act in diff way
MTs - the track
gives directional polarity for transport
minus ends towards body
plus ends facing axon tip - more dynamic closer to synaptic structure
motors:
kinesin (anterograde soma-> synapse) towards plus end
cytoplasmic dyenin - retrograde - towards minus end
this grants directionality to the cargo
axonal cargo machinery and disease
mutatiosn in kinesins in vertebrate axons
give neurodevelopmental or neurodegeneration disease phenotypes
dyenin mutations linked too
motor types in axonal transport
only one cytoplasmic dyenin
many kinesins
some primerily dendritic (excluded from axons)
some kinesins are shared
some exclusively axonal
kinesin superfamily
can be + or - end directed (most + directed)
bind diff cargoes
primarily are transporters
kinesin-1 present in axons
but is used generally in many transport processes in many cell types
Kinesin-3 (KIF1A)
exclusive to axons
transports SV structures
Kinesin-3 (KIF1A) discovery screen
first discovered in C. elegans genetic screen
look for mutants with specific uncoordinated movement phenotype
curl up instead of move in sinusoidal wave
Unc mutants (uncoordinated)
Unc-104 protein - conserved across organisms
known as Kinesin-3
transports mature AND precursor SVs
mutant was uncoordinated because cargo wasnt reaching synapses properly
Visualising axonal transport w Kinesin-3
GFP-Rab-3
Rab-3 part of SV
can see GFP fluorescence
use Kymograph to plot fluorescence distance along axon (x-axis) against time (y-axis)
diagonal lines indicate movement along axons (steeper=faster?)
do this with Kinesin-3 mutant known to cause disease phenotype
see synaptic vesicles not moveing
kymograph shows straight vertical lines
shows that kinesin-3 is transporting SVs
KIF1A (kinesin-3) associated neurological disorder KAND
many diseases associated with KIF1A mutations
dont know what the mutations are doing specifically
can test them using model systems
Testing KIF1A human disease mutations in model systems
PH - membrane binding domain
human and C elegans KIF1A relatively conserved
mutated residues in C. elegans that are associated w human KIF1A-associated disorders
replicate them in model
when we do this - generates loss of function uncoordinated phenotype seen in unc-104 mutations
molecular level:
look at c. elegans tail motor neuron
en passant synapses
make R254Q mutation in unc-104 (KIF1A)
get uncoordinated phenotype
synapses seen to be formed in wrong place - in the dendrite - flips the conformation
either because Unc-104 id tranasporting vesicles to the wrong place and not at all
get no snyaptic transmission as they are in dendrite and not at NM junction
can mimic mutations in human disease
and see effects on model systems to get more info than could in a vertebrate system
Dyenin basic
megadalton enzyme
only one kind of them in cells
instead uses accessory subunits to confer specificity - uses ADAPTERs
is main minus oriented motor (apart from v specific minus directed kinesins)
transports all minus end directed cargo
dyenin adapter proteins role
confer cargo specificity to dyenin
not just interactor between motor and cargo
also are activators of motility
need right adapter on right protein to activate motility
in vitro molecular motor assay
hard to do with recombinant dyenin as it just wouldnt move
instead can use TIRF microscopy to watch individual fluorescently labelled motors move in cells
Dyenin on own - see binding and unbinding of MTs
but no movement
add in adapter proteins - allowed dyenin movement to begin
adapters need to be present to activate movement
prevents dyenin from wasting energy with no cargo bound
dyenin dysfunction in axon trafficking
mutations in dyenin v rare
as is important in many cellular processes (as is present in all cells - only one dyenin)
DYNC1H1 cytoplasmic dyenin 1 heavy chain 1
mutant leads to neurodevelopmental/degeneration symptoms
hypothesised from this that inhibition of dyenin mediated axonal transport is sufficient to cause motor neuron degeneration
targeted inhibition of dyenin in neurons
engineer cell specific KO of dyenin in neuron subset of cells after brain has developed in transgenic mice
KO dyenin
shows neurodegenerative symptoms
these mice displayed Gait abnormalities
dyenin absence = decrease in movement parameters
dyenin mediated transport important to keep homeostasis of neuron and prevent neurodegeneration
Dyenin and huntington’s disease
mutation in HTT protein
HTT interacts w Dyenin activating adapter HaP1
adapter for lysosomes
so cant do retrograde lysosome transport
no clearing of degraded products
this could be causing neurodegeneration
Niemann-Pick disease and dyenin
mutations in NCP1/2
change in lipid composition of lysosome membranes
excess lipids esp. cholesterol accumulating in lysosome membrane
effect not clear
suggestes that this change in composition causes kinesins and dyenins to no longer bind them properly
affecting where lysosome ends up in cell
-increased dyenin clustering
-anterograde transport reduced
-causes childhood neurodegeneration
growth cone cues
1 can move
2 can respond to external cues - repulsive and attractive
attractive cues trigger growth cone to move there
repulsive cause contraction of cone
growth cone cytoskeleton
v enriched for actin and MTs
Actin throughout lamellipodium like structure
MTs v dense in the shaft and get sparse towards the growth cone structure
MTs invade the spiky Filopodia regions of the growth cone
actin dynamics and growth cone protrusion
actin is v polar polymer
pointed and barbed end (more growth from barbed??)
filopodia = linear F-actin bundled together by actin bundling proteins
lamellipodia = branched actin network
actun depolymerisation collapses growth cones
drugs eg Cytochalasin D/Latrunculin
see no F-actin staining, see no growth cone movement
growth cone motility similar to cell migration
MT instruction of F-actin remodelling in G cone
aren’t necessary for movement
important for telling the actin structures where to go -give direction to actin growth
also stabilise actin structures
also allow trafficking of machinery required for growth cone propulsion
so 2 diff jobs here
microtubule drugs effect on growth cone direction
Taxol - stabilises MT filaments
>control: G cone moves towards cue
>Taxol+: growth cone movement strays from path of cue
- so MT turnover dynamics are essential for axon growth/guidance
perturb dynamics w MT stabiliser
=cones dont move in right direction
so axonal tips dont go right place
Growth cone MT dynamics modulation by MAPs
MTs undergo dynamic instability
grow and shrink
tightly regulated by many diff proteins that help to stabilise or depolymerise
can control the growing and shrinking
MTs are a lot less dynamic in the axon shaft than in the growth cone as the MTs need to remain there as tracks
can create more MT turnover by either
-changing plus end dynamics
-or generating more MT ends by cutting MTs
MT severing enzymes
Spastin
Katanin
Fidgetin
hexamers that can slice MTs
can create dynamic ends where tehre werent before (ie by moving the tubulin-GTP cap)
in vitro these proteins fragment the MTs up
doesnt happen like this in growth cones
these proteins are locally regulated in growth cone tips to generate new structures
MT severing enzymes and disease
most mutated gene in hereditary spastic Paraplegia
katanin involved in autism related disorders
Spastin depletion
RNAi KO of spastin translation
reduces axonal branches
dendrite architecture remains normal
no. of primary axon branches is greatly diminished
primary branches are a result of the growth cone being stabilised
and this growth cone stabilisation isnt occurring in absence of spastin
the MT dynamics involved in g cone stabilisation is being impaired in spastin KO
-need constant MT turnover to stabilise growth cone
-perturb protein that affects MT turnover
-so g cone less stable
-so reduced primary branches (rely on stable growth cone)
correlated evidence saying that this is possible mechanism for how dynamics affect growth cone guidance
KIF21A and disease
ocular synkinesis linked to mutations in kinesin KIF21A
Congenital fibrosis of extraocular muscles type 1 - CFEOM1
patients with this mutation have issues w eye blinking movement pattern
many mutations associated w this are in KIF21A
most of these in either motor domain or coiled coil region
KIF21A mutant study in mice
recapitulate the KIF21A motor domain and coiled coil domain mutations in mouse model
ocular nerves found to have an axon guidance defect
leaves no coordinated movement between ocular muscles and neuron
reduce number of neurons involved in eye movement
because if neurons dont reach right target they can degenerate
in KIF21A mutant knock in mice
saw this reduced no.
KIF21A deletion, and MT dynamics
if delete this kinesin completely
there is not this reduce neuron phenotype
so this mutation is a dominant effect on patient phenotype
KIF21A regulates MT dynamicls
labelled KIF21A seen by the PM
links the MT to the cell cortex allowing local regulation of cell dynamics
KIF21A mutations in CFEOM1 generate OVERACTIVE Kif21A
WT Kif21A dont move very well
these mutants move a lot more (gain of function)
effect of overactive Kif21A
in WT - motors in inhibited state
has to bind particular structure or be in particular location to be activated
this inhibition is not occuring in the GOF CFEOM1 mutants
knock in animals have larger growth cone
and more filopodia
so overactive growth cone
gives lots of unregulated movement
makes it hard for ocular nerves to reach target
so get uncoordinated ocular muscles
axon regeneration
brain/CNS not well
PNS/sCord - better
factors:
- neuronal survival
-axon re-extension
-synapse reformation
-myelination
-experience dependent refinement of newly formed circuits
axons stay in same place for long time
when sever axon
need to reactivate the extension machinery to re-extend
Aplysia model system for axon regen
has good regen capacity
cut neuron
growth cone like structure appears
propels axon growth
regeneration needs to bring back growth cone dynamics
effect of severing axon
massive restructureing of MT and actin cytoskeleton after axon severing
lots of filament polymerisation/growth
lots of vesicles delivering new membrane to growth cone
non regenerating neuron phenotype
retraction bulb appears
axon dies :(
need to sever MTs w Severing proteins to get growth cone reactivation and avoid this i think
identifying genes involved in axon regen
invertebrate models more suited to regeneration (vertebrates regen less) and are also more genetically tractable
C. elegans genetic screen
-has axonal branches across body’s width
-sever one of these
-grows back to other side after developing g cone like structure
- mutagenesis screen to identify mutations that cant generate this g cone-like structure
-mutants have retraction bulb-like structure instead
DLK-1 pathway
conserved pathway across species
DLK-1 MAPK activated after axon is cut
activates signalling pathway
activation of TFs
a lot of MT associated proteins - MAPs
control MT turnover and dynamics at axonal tips
allows extension of axonal growth cone
investigating DLK-1 effect on MT dynamics during axon regeneration
track MT plus ends with GFP-EBP2
sever axon
3hrs-not many EBP2 tracks
6hrs-lot more EBP2 tracks
in absence of DLK-1
3hrs-not much change from control
6hrs-fewer GFP-EBP2 tracks at axonal tip
DLK-1 mutant has reduced no. of plus ends and MT length at later time point
so DLK-1 important for burst of axonal growth after Axotomy
can push axons to regenerate by affecting MT dynamics
DLK-1 specific target to give axon regen
DLK-1 KO
cut axon
no regrowth
DLK-1, KLP-7(MT depolymerising kinesin) KO
rescues axonal growth in teh DLK-1 deletion strain
DLK-1 is promoting MT polymerisation by directly/indirectly inhibiting Klp-7
OR otherwise affecting MT dynamics by promotion of some other factor
this is needed for MT growth forwards
DLK-1 mechanism use in regenerative medecine
create sCord injury in rat
use MT stabilising epoB drug (like taxol but it can pass blood-brain barrier)
gave greater sCord axon regeneration
kind of stabilising the MTs in the axons after injury - cna regain ability to regen part of axons
controlling MT dynamics is one way to promote regen mechanisms
these drugs dont just target one particular MT type so dont know MT specificity
but can say that affecting dynamics is sufficient to increase sCord regen
