C. elegans L1 cell adhesion molecule functions in axon...
Transcript of C. elegans L1 cell adhesion molecule functions in axon...
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C. elegans L1 cell adhesion molecule functions in axon guidance
Lihsia ChenDept. of Genetics, Cell Biology & Development
Developmental Biology Center
Biorad
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Goldstein lab, UNC-Chapel Hill
C. elegans embryogenesis
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CELL ADHESION
cellsorting
tissue boundaries
epithelial adherens junctions
axon guidancesynapse targeting & adherens
cell migration
Adapted from Molecular Biology of the Cell, Alberts et al.; Gumbiner et al., 2005, Nature review Mol Cell Bio.
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IMPAIRED CELL ADHESION
cellsorting
tissue boundaries
epithelial adherens junctions
axon guidancesynapse targeting & adherens
Adapted from Molecular Biology of the Cell, Alberts et al.; Gumbiner et al., 2005, Nature review Mol Cell Bio.
cell migration
DEVELOPMENTAL DISORDERS
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IMPAIRED CELL ADHESION
Adapted from Molecular Biology of the Cell, Alberts et al.
Loss of cell adhesionCell delamination
cell migration
inappropriate
METASTASIS
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Cell Adhesion ReceptorsCADHERIN INTEGRIN IgCAM
PLASMA MEMBRANE PLASMA MEMBRANE
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Cell Adhesion ReceptorsCADHERIN INTEGRIN
migrating cultured muscle cells
Barlovatz Meimon & Pouchelet, INSERM, FRANCE
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Cell Adhesion ReceptorsCADHERIN INTEGRIN IgCAM
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Dr. Lorene LanierUMN Neuroscience
Cultured rat hippocampal neuronsIgCAM
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mammalian L1CAMs
L1 NrCAM CHL1 neurofascin
FNIII
Ig
binding sitesto
actin-linkers
cell-cell and cell-ECM adhesion
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L1 NrCAM CHL1 neurofascin
Corpus callosum hypoplasiaRetardation/intellectual disabilityAdducted thumbsSpastic paraplegiaHydrocephalus
Fransen et al., 1994. Hum. Mol. Gen.Vits et al., 1994. Nat. Genetics
Neuronal role for mammalian L1CAMs
L1 syndrome
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L1 NrCAM CHL1 neurofascin
Corpus callosum hypoplasiaRetardation/intellectual disabilityAdducted thumbsSpastic paraplegiaHydrocephalus
autism addiction
Sakurai et al., 2006. Psych. GeneticsYoo et al., 2012. Psych. Invest.
Fransen et al., 1994. Hum. Mol. Gen.Vits et al., 1994. Nat. Genetics
Neuronal role for mammalian L1CAMs
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L1 NrCAM CHL1 neurofascin
Corpus callosum hypoplasiaRetardation/intellectual disabilityAdducted thumbsSpastic paraplegiaHydrocephalus
autism addiction
Sakurai et al., 2006. Psych. GeneticsYoo et al., 2012. Psych. Invest.
schizophrenia
Chu & Liu, 2010. J. Human Genetics
Fransen et al., 1994. Hum. Mol. Gen.Vits et al., 1994. Nat. Genetics
Neuronal role for mammalian L1CAMs
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L1 NrCAM CHL1 neurofascin
Corpus callosum hypoplasiaRetardation/intellectual disabilityAdducted thumbsSpastic paraplegiaHydrocephalus
autism addiction
Sakurai et al., 2006. Psych. GeneticsYoo et al., 2012. Psych. Invest.
schizophrenia
Chu & Liu, 2010. J. Human Genetics
Neurodevelopmentneurite outgrowthaxon guidanceMyelination
Neuronal role for mammalian L1CAMs
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1. External embryonic development2. Large embryos3. Amenable to manipulation4. Early chick embryonic structures are
very similar to those of the human embryo.
1. Mammal 2. Reverse genetics
1. External embryonic development2. Large number of transparent embryos3. generation time ~3 months but quick
embryonic development
1. Genetically accessible2. Short generation time
~ 2 weeks, 3.5 days3. Small genomes4. Small size
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Caenorhabditis elegans
Sydney Brenner
Robert Horvitz
John Sulston
Nobel Prize in Physiology or Medicine 2002
for their discoveries concerning “genetic regulation of organ development and programmed cell death
How does the nervous systemdevelop?function?
control behavior?
Identified all cellsInvariant cell lineage: 302 neurons Transparent
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zygote
Invariant cell lineage of 959 somatic cells
302 neurons
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1994: 1st showing of a GFP reporter in a multicellular organism
mechanosensory cells
Osamu Shimomura
Martin Chalfie
Roger Y. Tsien
2008 recipients of the Nobel Prize in Chemistry for the discovery and development of GFP
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Importance of fluorescence proteins in C. elegans research
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Advantages of C. elegans as model to study nervous system development & function
• Invariant cell lineage 302 neurons • Transparent• Neural circuitry completely mapped:
serial electron micrographsLight microscopy
WiringChemical and electrical synapses
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SAX-7
C. elegans L1CAMs
SAX-7 LAD-2
Wang et al., 2008. J. Cell Biol.Chen et al., 2001. J. Cell Biol.
expressed in 14 neuronsexpressed ubiquitously
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lad-2 animals show axon guidance defects
A/P
SDQL
D/V
Wang et al., 2008. J. Cell Biol.
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Which axon guidance pathway does LAD-2 mediate?
NetrinsSlits
SemaphorinsEphrins
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Semaphorin family and Receptor
Adapted from Pasterkapm and Kolodkin, 2003. Curr. Opin. Neurobiol.
MAB-20PLEXIN RECEPTOR
PLX-2
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mab-20/Sema2 & plx-2 have axon guidance roles
Wang et al., 2008. J. Cell Biol.
SDQL SDQL
PLX-2
MAB-20
actin reorganization
Axon guidance
X X
X
X
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PLX-2
MAB-20
actin reorganization
Axon guidance
plexin
MAB-20
PLX-2
Sema
Axon guidance
actin reorganization
X X
Xno enhanced defects
Possible semaphorin pathways
X X
X
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mab-20/Sema2 & plx-2 have axon guidance roles
Wang et al., 2008. J. Cell Biol.
SDQL SDQL
XXMAB-20 PLX-2no
enhanced defects
XX axon
guidance
MAB-20 receptor X
ligand Y PLX-2
guidancedefects
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mab-20/Sema2 & plx-2 have axon guidance roles
Wang et al., 2008. J. Cell Biol.
SDQL
XXMAB-20 PLX-2no
enhanced defects
XXMAB-20 protein X
protein Y PLX-2
enhanced defects
Does LAD-2 mediate axon guidance via MAB-20/Sema2?
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Does LAD-2 mediate axon guidance via MAB-20/Sema2?
XXMAB-20 PLX-2no
enhanced defects
XXMAB-20 PLX-2
Ligand Y LAD-2
LAD-2X
X enhanced defects
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Wang et al., 2008. J. Cell Biol.
LAD-2 functions in MAB-20/Sema2-mediated axon guidance
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Does MAB-20/Sema2 act as a repellent to SDQL?
mab-20
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Does MAB-20/Sema2 act as a repellent to SDQL?
mab-20
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Does MAB-20/Sema2 act as a repellent to SDQL?
mab-20
Ectopic ventral MAB-20 expression
mab-20mab-20
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MAB-20/Sema2 acts as a repellent
mab-20
Wang et al., 2008. J. Cell Biol.
Ex[mab-20]
mab-20; Ex[mab-20]P < 0.01
P < 0.01
P < 0.05
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WT
Can dorsal MAB-20/Sema2 induce ventral migration of SDQL axon?
P < 0.01
P < 0.01
P < 0.01
Ex[mab-20]
lad-2; Ex[mab-20]
Wang et al., 2008. J. Cell Biol.
WT
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WT
P < 0.01
P < 0.01
P < 0.01
Ex[mab-20]
lad-2; Ex[mab-20]
Wang et al., 2008. J. Cell Biol.
MAB-20/Sema2 acts as a repellent
WT
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P < 0.01
P < 0.01
P < 0.01
Ex[mab-20]
lad-2; Ex[mab-20]
Wang et al., 2008. J. Cell Biol.
Is LAD-2 required to mediate MAB-20/Sema2 signal?
lad-2
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P < 0.01
P < 0.01
P < 0.01
Ex[mab-20]
lad-2; Ex[mab-20]
LAD-2 is required to mediate MAB-20/Sema2 signal!
Wang et al., 2008. J. Cell Biol.
lad-2
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Can MAB-20/Sema2 interact with PLX-2?
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MAB-20/Sema2 interacts with PLX-2
Wang et al., 2008. J. Cell Biol.
Myc::PLX-2FLAG::MAB-20 +
++
Myc::PLX-2
FLAG::MAB-20
LYSATE IP
++
+_
3 4 3 4
_
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Can LAD-2 interact with MAB-20 & PLX-2?
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LAD-2 interacts with MAB-20/Sema2 & PLX-2
Wang et al., 2008. J. Cell Biol.
LAD-2Myc::PLX-2
FLAG::MAB-20
_
++
_
++
LAD-2
Myc::PLX-2
FLAG::MAB-20
IPLYSATE
+++ _
++
_
+_
+++
LAD-2 SAX-3
1 3 4 1 3 4
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LAD-2 secures the MAB-20/Sema2 - PLX-2 interaction
LAD-2
Myc::PLX-2
FLAG::MAB-20
SAX-3
SAX-3LAD-2
Myc::PLX-2FLAG::MAB-20
_
++
_
++
++
+++_ __+ _
++
_
+_
++
_ _ _
+++
+
1 2 3 4 1 2 3 4
IPLYSATE
Wang et al., 2008. J. Cell Biol.
_ _
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Model for how LAD-2 mediates MAB-20/Sema2 function
Wang et al., 2008. J. Cell Biol.
LAD-2
L1
nematostella Vectensis
Neuropilin present (Putnam et al., 2007)No L1CAMs (C. Magie, Pers. comm)