neuronal cytoskeleton Flashcards

Question

growth cone cytoskeleton

Answer 1

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

Answer 2

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

Answer 3

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

Answer 4

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

Answer 5

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

Answer 6

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

Answer 7

most mutated gene in hereditary spastic Paraplegia katanin involved in autism related disorders

Answer 8

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

Answer 9

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

Answer 10

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.

Answer 11

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)

Answer 12

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

Answer 13

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

Answer 14

has good regen capacity cut neuron growth cone like structure appears propels axon growth regeneration needs to bring back growth cone dynamics

Answer 15

massive restructureing of MT and actin cytoskeleton after axon severing lots of filament polymerisation/growth lots of vesicles delivering new membrane to growth cone

Answer 16

retraction bulb appears axon dies :( need to sever MTs w Severing proteins to get growth cone reactivation and avoid this i think

Answer 17

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

Answer 18

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

Answer 19

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

Answer 20

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

Answer 21

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