Axon growth

Question 1

How do ECM molecules promote navigation?

Answer 1

-receptors for ECM are integrin
1. specificity: different subunits results in specificity for different ligands
2. Transmembrane receptors: integrate with actin cytoskeleton intracellulary
3. Signaling: increase FAK125 kinase activity and other signaling elements

Question 2

Are there laminin “roadways” in developing embryos?

Answer 2

Antibody that recognized laminin->there is a pathway btwn the growth cone and the laminin target (only a correlation)

Question 3

Are ECM pathways necessary for navigation?

Answer 3

1. Purify laminin to find a function blocking antibody
2. Examining neural crest cell migrations
   -add antibodies to right side of the embryo
   -results in no migration of neural cells on the right side and normal migration on the left (with no antibody)
   -shows that laminin is necessary for neural crest cell migration

Question 4

are integrins necessary for navigation?

Answer 4

1. infect early chick embryo with retrovirus carrying LacZ (makes cells blue) and beta1 antisense (opposite of beta1 integrin) (wait for 3-9 days)

Results:
1. blue cells (LacZ+) migrated poorly
2. non-blue cells (LacZ-) migrated poorly (have normal beta integrin)
Integrin is necessary for cell migration

Question 5

how do growth cones know which way to go when it found a roadway?

Answer 5

Hypothesis: growth cones that bind to laminin can follow a laminin gradient
Results: laminin gradient doesn’t direct growth cone navigation
-concentrations of adhesive substances allow axon growth but does not INSTRUCT it
-ECM is PERMISSIVE BUT NOT INSTRUCTIVE

Question 6

dorsal neurons are attracted by a netrin gradient

Answer 6

-axons follow a netrin gradient towards the floor plate
-transfection with plasmid of netrin-expressing common cell line->doesn’t confirm that netrin is attractant (could be something else that the COS cells make)
-non-transfected COS cells not expressing netrin=growth cones don’t go towards COS cells->now you can say it is responsible

Question 7

draw netrin and wnt4 gradients and axon growth

Answer 7

wnt4 gradient towards anterior part of the cell
-non-transfected COS cell expressing wnt4 on posterior side of cell->axons grew posteriorly
-fz3 knockout (receptor for wnt4)->axon gets lost after it passes netrin gradient

Question 8

ventral neurons are repelled by a netrin gradient

Answer 8

ventral neurons grow away from floor plate and netrin-expressing COS cell line

Question 9

what is the neuronal cytoskeleton composed of?

Answer 9

-microtubules
-actin microfiliments
-neurofiliments

Question 10

what are microtubules made of

Answer 10

non-covalent polymers
-alpha and beta tubulin subunits
-and MAPs
-MTs are found in parallel bundles in the axon tube
-in growth cones, MTs are unbundled, splayed out and very unstable/dynamic
-polar structures (+/-)

Question 11

actin filiments

Answer 11

-composed of actin polymer
-and actin associated proteins (AAPs)
-located in cortical sheath in axon and growth cone, filipodia
-very dynamic, motile structures
-myosis II is an actin based motor protein

Question 12

location of MT and Actin

Answer 12

draw locations
-actin found in filipodia and lamellipodia
-MT=’palm’ of growth cone and some of the actin rigth periphery

Question 13

where are new microtubules added onto a growing axon?

Answer 13

it adds on to the end
-bleaches an area in the axon->new MTs are added distally

Question 14

where are new actin filaments added?

Answer 14

filipodia and some are left behind in the axon cortex

Question 15

where does new lipid bilayer come from?

Answer 15

adding lipid bilayer along the axon tube

Question 16

how do neuroblasts (sperical) initiate an axon?

Answer 16

1. neuroblasts project multiple filipodia
2. one of the projections becomes dominant, grows long and becomes the axons
   - other projections become dendrites

Question 17

what is the function of Tau on the projections becoming dominant?

Answer 17

-neuronal MT associated protein
-stabilizes MTs by promoting mT growth and suppressing MT shortening
-can mediate actin-MT interactions
-suppression of Tau = no projection ever becomes dominant

Question 18

are MTs necessary to maintain neurite structure?

Answer 18

addition of NGF (neural growth factor) and MT disrupters (vinblastine/colchicine) to neurons in culture
-results in whole neurite collapses
-shows that MTs are necessary to maintain neurite distruptors

Question 19

how do actin microfiliments (MFs) maintain neurite structure?

Answer 19

addition of NGF and chytochalasin (MF disruptor) to neurons in culture on plastic dish
-whole neurite collapses
-MFs are necessary to maintain neurite structure

Question 20

What happens when MFs are added to a polylysine dish

Answer 20

addition of NGF and chytochalasin on polylysine dish
-results in growth cone collapse, but neurite remains intact with slow growth
-suggests that MFs are involved in outgrowth, but not solely responsible

Question 21

what happens to the axon when cytochalasin and nocodazole?

Answer 21

turns axon away
-cytochalasin destabilizes actin on one side
-nocodazole destabilizes MTs

Question 22

what happens to the axon when taxol is used?

Answer 22

axon turns towards
-promotes stability of MTs

Question 23

how do growth cones migrate?

Answer 23

1. growth cone projects ‘explorer’ filipodia
2. receptors on ‘explorers’ bind to extracellular cue; now ‘winner’ filipodia
3. actin filiments in ‘winner’ are stabilized, actin continues to push out filipodia to search for more cues
4. MTs identify and invade winner filipodia and stabilize them
5. loser filipodia retract and new ‘explorers’ are projected
6. MTs and MFs work together to stabilize ‘winner’ filipodia and convert into growing axon tube

Question 24

MF ‘treadmilling’

Answer 24

subunits are added and removed equally
-doesn’t change position

Question 25

draw clutch model

Answer 25

actin not engaged and engaged with ECM
-ECM binds to integrin receptor, which binds to an intermediate, which binds to MF
-complex is stuck in place, whole membrane moves
-integrin=clutch

Question 26

TIP proteins

Answer 26

MT associated proteins (MAPS) that accumulated at the outward facing end of many MTs
-allows pioneer MTs to interact with actin bundles in the periphery
-normally inactive, until they are activated by a signaling event

Question 27

how do MT stabilizers and destabilizers control growth cone steering?

Answer 27

-stabilizers (positive stimulus) bind to one side, causing growth cone to turn in that direction
-destabilizers (negative stimulus) cause repulsion of the affected area

Question 28

how does MT stabilizers result in turning of growth cone?

Answer 28

1. filopodia/lamellipodia engage with positive extracellular cues by receptors
2. receptors induce signal cascades on actin and MT regulating protiens and intracellulary with actin cytoskeleton
3. actin filaments stabilize, pushing ‘winner’ filipodia out, looking for more positive cues
4. MTs extend into ‘winner’ filipodia and stabilize
5. filopodial membrane extends toward more attracting cues