Gradients

Gradients

1. Gradients can act over long distances

   • In mammals, the majority of C axons turn rostrally after Xing the floor plate. What determines this?
     • One possibility is an A-P gradient of either an attractant or a repellant, which could be a long range diffusible cue
       • or a short range, non-diffusible.
       • How can we distinguish between these possibilities?
         • Could distinguish by culturing large vs small explants:
       • Evidence for a long range attractant
         • Prediction is that a relatively small explant will lose its gradient if the chemotropic agent is diffusible (whereas a larger explant will not), which should not be true if the guiding factor is non-diffusible
           • Also, in larger explants, expect anterior-most axons to turn aberrantly if attractant, but posterior-most axons if repellant (think about it!)
       • If a repellant…

2. Detecting gradients

   • Two ways in which to measure a gradient
     • Change of concentration over time (temporal detection) - cell must compare amount of ligand at two different time points
     • Change of concentration across the cell (or growth cone; spatial detection) - cell must compare the amount of ligand at two different points on it surface.
       • For slow-moving eukaryotic cells (ie not bacteria) spatial detection likely to be most important.
     • Most of what we know has come from studies of the chemotaxis of white blood cells (neutrophils and leukocytes) and the slime mould Dictyostelium
   • Accuracy of orientation
     • Accuracy of orientation towards a source depends on:
       • Absolute concentration of the chemoattractant - most accurate closest to the KD (dissociation constant) of the receptor*.
         • * At the KD concentration, half of the receptors will be occupied giving greatest capacity for detecting increases or decreases in receptor occupancy
       • Steepness of the gradient
     • Gradient is expressed as % change per micron (µm)
       • Leukocytes only orient with accuracy when gradient is ≥0.2%, e.g. 2% change over a 10µm diameter
         • Note that larger diameter cells orient more accurately than small cells in shallow gradients
           • Recent studies indicate that neurons have similar characteristics
   • Mechanisms to enhance range over which gradients can be detected
     • Even relatively steep gradients (2% over 10µm) require very small differences in receptor occupancy to be detected across the growth cone.
       • In mid-gradient, a growth cone with, say, 1000 receptors would require the cell to detect a difference of just 5 occupied receptors across its width for such a gradient (250 x 1.02):

3. Amplification and adaptation

   • Two methods to enhance sensitivity of GCs to gradients
   • Amplification
     • Amplification requires local enhancement of signal together with inhibition of signal reception in other parts of the cell
       • One suggested mechanism involves clustering of receptors and/or signalling components in regions where receptors activated by transporting components from other parts of the cell:
         • Evidence for clustering of downstream components can be found in chemotaxing Dictyostelium…
           • Chemoattractants in leukocytes and Dictyostelium activate PI3Kinase via a G-protein coupled receptor (GPCR)
             • Chemoattractant detected by a GPCR (7TM protein)
             • This activates PI3kinase, which then phosphorylates PI(4,5)P2 (PIP2) to give PIP3 (PI(3,4,5)P3)
             • This provides a docking site for proteins containing Pleckstrin Homology (PH) domains, (such as Akt), creating a localised signalling domain on the membrane
             • Note that PI3K activity is antagonised by PTEN phosphatase
               • This is critical to ensure that signals are highly localised and labile, dependent on continued external activation….
           • Recruitment of PH domain proteins to leading edge of chemotaxing cell
             • See ppt
     • Amplification: primes machinery to ‘test’ cue found?
   • Adaptation
     • If growth cones operate most discriminatively when the chemoattractant concentration is closest to the receptor KD, then clearly receptors will saturate as concentration increases.
       • Simple calculations show that even the shallowest gradients required to turn cells (0.2% per µm) will result in 90% receptor occupancy just 250µm from the optimal ligand concentration.
         • In other circumstances - e.g. synaptic transmission - signal saturation leads to desensitization and resetting of thresholds (or resensitization if signals fall).
           • Is there evidence for such ‘adaptation’ in responses to gradients?
             • Frog axon turning assay
             • Adaptation in response to netrin?
               • Pulsed netrin forms an exponential gradient which is revealed by including a fluorescent marker in the pipette
                 • Growth cones responding to such a gradient initially exhibit a ‘zig-zagging’ trajectory
                   • The alternating attractive and repulsive turning suggests cycles of desentization and sensitization of the growth cone to netrin.
             • Growth cones adapt to chemoattractants
               • To determine whether growth cones adapt to increasing attractant concentrations, Ming et al pretreated axons with differing concentrations of netrin:
                 • They discovered that immediately (30min) after treatment, growth cones no longer responded (b)
                   • However, after a longer period (90mins), growth cones resensitized (c)
                     • This resensitization was dependent upon MAPKinase activation and local protein synthesis in the growth cone.
                       • Responses to Sema3A are subject to similar adaptation (see Piper et al 2005)
               • See ppt

4. Stretching gradients to the limits

   • Over what distance could growth cones be guided by gradients?
     • Depends upon the the shape of the gradient, which depends on how it is set up:
       • Diffusible molecules e.g.
         • Wnts: expression gradient along axis
         • Netrins: point source (floor plate)
       • Non-diffusible molecules e.g.
         • Ephrins (tectum): surface bound molecules expressed in gradients
       • These two different types of gradient have inherently different ranges of action (discussed in Goodhill (1998). Trends Neurosci 21: 226-31)….
   • Two different types of gradient
     • Point source
       • A gradient set up by diffusion from a point source (such as that used by Ming et al) - typically an exponential gradient - has a theoretical maximum range of 1mm, which corresponds well to supposed point source ranges in vivo:
       • Point sources depend on:
         • a limited diffusion rate.
         • some mechanism for removing the guiding molecule - a ‘sink’ - otherwise gradient will flatten with time (depending on diffusion rates)
     • Linear substrate bound gradients
       • can be longer
       • By contrast, a substrate bound gradient, typically more linear in shape, has a theoretical maximum range of close to 1cm.
         • Again, this corresponds well with what is observed in vivo.

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