Lecture 19- Axon guidance II Flashcards
How can the direction of the growth cone change? (long summary)
-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
What do growth cones grow on?
-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
What substrate do neurons grow on?
- 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
What substrate do neurons prefer: adhesive or non-adhesive?
-prefer adhesive substrate -can attach to the surface due to integrins in the cell membrane -this enables the clutch to engage
How can adhesion be measured?
-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
On which substrate do neurons grow slower? (adhesive/non-adhesive)
-neurons tend to grow more slowly on more adhesive substrates
Do all neurons react to the same substrates in the same way?
different classes of neurons= can attach to different substrates differentially, depending on what proteins they have in their cell membrane
Are very adhesive substrates good for axonal growth?
-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
Is axon navigation dependent on following gradients of adhesion?
-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
Is the axonal navigation done by hierarchical adhesion decisions?
-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
What are the molecules in the extracellular matrix (ECM) that support axon growth?
-laminin -fibronectin -vitronectin -various forms of collagen
Do different neurons differ in their preferences for particular ECM molecules?
- 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
Is the preference for substrate important?
-yes, important for the neurons getting to the right target
What is the example of substrate preference?
-membranes from optic tectum have anterior and posterior (make it into a stripe assay) -axons prefer the anterior part (the temporal part)
What are integrins composed of?
-composed of 2 subunits, alpha and beta -there are 20 different alpha and 10 different beta subunits
Where are the integrins presented?
-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
What is the alpha6 subunit of integrins good for?
-good at binding laminin
What is the alpha5 subunit of integrins good for?
-good at binding fibronectin
Can axons change the expression of integrin subunits?
-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
What are other substrates apart from ECM?
-other neurons -many neurons and axons express cell adhesion molecules (CAMs)
What are the types of molecular cues influencing direction of growth cones?
- Contact adhesion (need cell-cell contact)
- Contact repulsion
- Long-range attraction
- Long-range repulsion
What are the cell adhesion molecules and their role on axon gudiance?
-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
What is often true about the leading and following axon?
- 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
What is the example of the role of CAMs in axon guidance? (FasII in longitudinal axons in Drosophila)
- 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
What is the example of the role of CAMs in axon guidance? (NCAM in chick brachial plexus)
- 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
What is the redundancy in axon pathfinding?
-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
What is the labelled pathway hypothesis?
-different pathways express different CAMs -pioneer axons express particular CAMs that other growth cones follow -axons will follow axons expressing complementary CAMs
Can axons change CAM expression upon changing pathways?
- yes
- axons may have to change pathways
- may have to change CAM on surface!
