Lecture 19- Axon guidance II

Question 1

How can the direction of the growth cone change? (long summary)

Answer 1

-in culture, growth cones move straight ahead (similar numbers of filopodia on each side) -depolymerize actin filaments on one side, growth cone steers to other side -in culture= growth cone is symmetrical, grows in straight line when stable culture -if you locally locally depolymerize actin then growth cone with steer to the other side= the filopodia on the depolarised side are smaller -axon grows in the direction of the most filopodia, where the majority form -local de-stabilization of microtubules on one side (turns away from that side) -local stabilization of microtubules on one side (turns towards that side) -can also affect the direction of axon via destabilization of microtubules -if stabilized on one side (like when SCG10 is degraded) it will turn to the direction of stabilized microtubules

Question 2

What do growth cones grow on?

Answer 2

-growth cones have preferences for particular molecular and physical substrates -axons really like the cracks -like junctions between things -injured axons will migrate on some artificial mechanical pathways across a wound

Question 3

What substrate do neurons grow on?

Answer 3

• neurons grown in culture on plain glass or plastic often attach but rarely extend neurites with growth cones
• but they stick well to polycationic substrates such as polylysine (because of -negatively charged cell membrane) and extend axons
• in order for growth cone to advance must attach to substrate (clutch engage hypothesis) so don’t extend axon when on glass and plastic
• if on adhesive substrate( likes it)
• if less adhesive= more rounded (doesn’t like the substrate) minimizes area in contact with substrate

Question 4

What substrate do neurons prefer: adhesive or non-adhesive?

Answer 4

-prefer adhesive substrate -can attach to the surface due to integrins in the cell membrane -this enables the clutch to engage

Question 5

How can adhesion be measured?

Answer 5

-measure how long a growth cone maintains its adhesion while being squirted with a consistent force of tissue culture medium -the longer it takes to dettach/dislodge the more adhesive the substrate

Question 6

On which substrate do neurons grow slower? (adhesive/non-adhesive)

Answer 6

-neurons tend to grow more slowly on more adhesive substrates

Question 7

Do all neurons react to the same substrates in the same way?

Answer 7

different classes of neurons= can attach to different substrates differentially, depending on what proteins they have in their cell membrane

Question 8

Are very adhesive substrates good for axonal growth?

Answer 8

-more is not better -the more adhesive substrates do not support axon outgrowth -the ability to detach is just as important as the ability to attach -the optimal amount of adhesion for axon growth is not too little not too much

Question 9

Is axon navigation dependent on following gradients of adhesion?

Answer 9

-is likely to be true for some neuron populations -eg. sensory neurons in moth grow towards the CNS along a basal lamina of increasing complexity (higher adhesion there) -axons avoid distal transplants moved proximally (if change the gradient they like it) -remember this is true only of some neuronal populations

Question 10

Is the axonal navigation done by hierarchical adhesion decisions?

Answer 10

-ocellar axons of Drosophila -grow on basal lamina of epithelial cells to get to the brain -in mutants for a subunit of laminin, axons of ocellar neurons follow the axons of mechanosensory neurons and do not make the correct connections in the brain -remember this was only shown in this particular neuronal population

Question 11

What are the molecules in the extracellular matrix (ECM) that support axon growth?

Answer 11

-laminin -fibronectin -vitronectin -various forms of collagen

Question 12

Do different neurons differ in their preferences for particular ECM molecules?

Answer 12

• yes -can perform stripe assays to determine preferences -retinal neurons prefer laminin to fibronectin when grown in a strip assay, but will grow on fibronectin if there is no choice

Question 13

Is the preference for substrate important?

Answer 13

-yes, important for the neurons getting to the right target

Question 14

What is the example of substrate preference?

Answer 14

-membranes from optic tectum have anterior and posterior (make it into a stripe assay) -axons prefer the anterior part (the temporal part)

Question 15

What are integrins composed of?

Answer 15

-composed of 2 subunits, alpha and beta -there are 20 different alpha and 10 different beta subunits

Question 16

Where are the integrins presented?

Answer 16

-present at the growth cone determine which ECM molecules a growth cone will respond to (key molecules in engaging the clutch) -are receptors for many different ECM proteins

Question 17

What is the alpha6 subunit of integrins good for?

Answer 17

-good at binding laminin

Question 18

What is the alpha5 subunit of integrins good for?

Answer 18

-good at binding fibronectin

Question 19

Can axons change the expression of integrin subunits?

Answer 19

-yes, can change expression of integrin subunits with developmental age -e.g young chick retinal ganglion cells express alpha6 and grow well on laminin, but no longer respond laminin when they reach the tectum -the preference for substrate depends on the integrin subunit, but that can be changed, expression of the integrin subunits during development

Question 20

What are other substrates apart from ECM?

Answer 20

-other neurons -many neurons and axons express cell adhesion molecules (CAMs)

Question 21

What are the types of molecular cues influencing direction of growth cones?

Answer 21

1. Contact adhesion (need cell-cell contact)
2. Contact repulsion
3. Long-range attraction
4. Long-range repulsion

Question 22

What are the cell adhesion molecules and their role on axon gudiance?

Answer 22

-include IgG superfamily, which have an extracellular domain similar to antibodies -cadherins -can bind homophilically= proteins of the same type bind to each other -axons expressing the same CAMs often fasciculate with each other

Question 23

What is often true about the leading and following axon?

Answer 23

• very frequently the following axon will be expressing the same cell adhesion molecule (CAM)
• fasciculate= if multiple is in a group, bundle formed -defaciculation= bundle gets spread

Question 24

What is the example of the role of CAMs in axon guidance? (FasII in longitudinal axons in Drosophila)

Answer 24

• a subset of longitudinal axons in the CNS express fasciclinII (FasII) a Drosophila CAM
• loss of function mutants exhibit defasciculation of the tracts
• defasciculation rescued by expression of FasII specifically in the pathways
• FasII gain of function prevents normal defasciculation

Question 25

What is the example of the role of CAMs in axon guidance? (NCAM in chick brachial plexus)

Answer 25

• neural cell adhesion molecule (NCAM) is expressed in almost all vertebrate neurons and glia
• extracellular domain can be modified by the addition of carbohydrates, particularly sialic acid residues; sialyation decreases adhesion
• axons of motor neurons express highly sialyted form of NCAM while in brachial plexus -non sialyted forms are more adhesive than sialyted forms
• don’t want them too tight together otherwise won’t go to the right spot

Question 26

What is the redundancy in axon pathfinding?

Answer 26

-phenotype of mutant animals lacking a single molecule involved in axon guidance often subtle -multiple molecules involved in guiding each class of axon to their targets -double mutant embryos with deletions in 2 CAMs often have a severe phenotype whereas each single mutant is relatively normal -Drosophila fasI mutants= no defects; fas;abl double mutants have significant defects (two distinct molecular pathways, either of which may suffice for axon guidance -even when in vitro effect seems huge, knockout in vivo will not have much effect

Question 27

What is the labelled pathway hypothesis?

Answer 27

-different pathways express different CAMs -pioneer axons express particular CAMs that other growth cones follow -axons will follow axons expressing complementary CAMs

Question 28

Can axons change CAM expression upon changing pathways?

Answer 28

• yes
• axons may have to change pathways
• may have to change CAM on surface!