Aplysia - Sea slug
- Simple NS
- Large neurons
- Easy to examine in culture
What cellular machinery would you target if you want to regulate neurite growth &
guidance? - Actin and microtubule cytoskeleton
Cytochalasin b - prevents polymerization of actin
Rho family GTPases - Function upstream of cascade that regulates the cytoskeletal
proteins. Molecular switches
,Rho, Rac, Cdc42
- Protein has a lipid anchor
GTPase Activating Proteins (GAPs) - activates the GTPase activity of Rho, helping
convert GTP to GDP (inactivating the G protein)
Guanosine Nucleotide Exchange Factor (GEF) - Activate Rho family GTPase by
facilitating the exchange of GDP for GTP
Activated Rho GTPases - - Bind to many effectors: kinases, actin-binding proteins
- directly or indirectly affect the assembly or dissasembly of Filamentous (F)-actin
lamellipodia - a cytoskeletal protein actin projection on the leading edge of the cell
filopodia - Cytoplasmic extensions
focal adhesions - docking sites where cells adhere to their substratum and send signals
to the cell interior
Stress fibres - bundles of actin filaments that keep cells elongated
Focal complexes - Transient, small provide early cellular attachment
Rac - Rho family GTPase
- Affects the focal complexes (FX) and lamellapodia
Cdc42 - Rho family GTPase
- affects the focal complexes (FX) and filopodia
What are the extrinsic factors that regulate filopodia and axon guidance? - Rho family
GTPases
What can you use to discover
cell non-autonomous factors
that regulate axon guidance - cDNA library
Rita Levi-Montalcini limb bud experiment: Remove the limb bud - peripheral neurons die
Rita Levi-Montalcini limb bud experiment: Remove limb bud and replace with tumor -
Peripheral neurons aggressively innervate the tumor
, Rita Levi-Montalcini limb bud experiment: Simplest conclusion? - The tumor is
producing some type of survival & growth factor
What was Stanley Cohen & Rita Levi-Montalcini's assay in finding nerve growth factor -
Sensory & sympathetic ganglia from chick embryos + fragments or extract of tumor
Readout: observation of neuron growth
Stanley Cohen fractionation column - Run sample of fractionation column to get
different pools. Determine which pool contains factor/activity. Fraction that fraction even
more until you get a single protein
Nerve Growth Factor (NGF) - - Related to neurotrophins (neurotrophin 3, BDNF, GDNF,
CNTF)
- Mediate migration in other cell types (angiogenesis, neural crest)
- Uncontrolled expression of these in cancer allows migration
- Regulate Rho family GTPases
End result: formation or disassembly of actin filaments
Ephrins & Netrins lead to _____ polymerization and disassembly. Also ______
contraction.
Upstream: activate ___ and ___ - F-actin, actinomyosin
RhoA, Rac1
Slit & Semaphorins inhibit to ____ disassembly and assembly and ____ contraction.
Inhibit ___, ___, ____ - F-actin, actinomyosin
RhoA, Rac1, Cdc42
NGF responsive neurons - 1. Afferent to low-threshold mechanoreceptors
2. Temperature and pain receptors
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