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10/13/2011, Jinan, China
Qilu Internation Neuroscience Symposium
Shangdong University School of Medicine
Membrane Trafficking Mechanism
Underlying Neuronal Polarization
Zhen-Ge Luo (罗振革), Ph.D
Institute of Neuroscience
Chinese Academy of Sciences
Shanghai, 200031
Model systems
Parallel between neuronal polarization in vitro and in vivo
Witte and Bradke. 2008 Current Opinion in Neurobiology
Neuronal polarization requires four main steps:
1. Increase in the amount of plasma membrane
(by vesicle recruitment and fusion);
2. Increase in the local concentration and activation of signalling
molecules;
3. Increase in the dynamics of actin filaments;
4. Enhancement of microtubule formation.
Signaling pathways involved in mammalian axon specification
during neuron polarization
Plasmalemmal precursor vesicles during early neurite elongation
Pfenninger KH 2009 Nature Reviews Neuroscience
Red (emission at 620 nm): Golgi-derived vesicles
Green (emission at 515 nm): Plasmalemma
Golgi-derivation of BODIPY-labeled vesicles
Time-dependent accumulation of BODIPY-vesicles in distal axon
BODIPY labeling of Plasmalemmal Precursor Vesicles (PPV)
Wash1
Free BODIPY-ceramide
Golgi-derived vesicles
Wash2
Disappearance of BODIPY-Red signals in neurite growth cones
Membrane fusion activity correlates with axon specification
Question:
--How is the directional membrane
fusion regulated?
Lethal Giant Larvae is Essential for Various
Types of Cell Polarity
Yeast polarized budding
Drosophila neuroblast division
Genetics. 1998; 149:1717-27.
Dev Neurosci. 2006; 28:13-24
Mammalian epithelial apical-basal polarity
J Cell Sci. 2006;119:2107-18.
Mammalian astrocyte
Nat Cell Biol. 2003; 5:301-8.
Expression of Lgl1 in the developing brain
Localization of Lgl1 in cultured hippocampal neurons
Lgl1 is essential for neuronal polarity
a
c
b
Over-expression of Lgl1 promotes axon development
Down-regulation of Lgl1 decreases directional membrane fusion
Q: How does Lgl regulate membrane fusion
and neuronal polarity?
J Cell Biol. 2006; 172: 55-66
Mol Biol Cell. 2006;17:3156-75
RAB10
RAB8a
RAB8b
RAB13
Sec4p
Lgl1 is associated with
mammalian Rab10
Lgl1 is associated with Rab10 in primary neurons
Colocalization of Rab10 with PPV
Mutated forms of Rab10 affect neuronal polarity
Rab10Q58L, GTP-locked active form
Rab10T23N, GDP-locked inactive from
Rab10 is essential for neuronal polarity
Rab10 acts downstream of Lgl1 in regulating
axon development
--Rab10 partially rescues the neuronal polarity in cells with Lgl1 knockdown;
--Lgl1 has no effect on axon defects caused by Rab10DN or Rab10 siRNA;
--Rab10DN or siRNA prevents the enhanced axon development caused by Lgl1.
Role of Rab10 in membrane fusion
--Down-regulation of Lgl1 decreases directional membrane fusion;
--Rab10 rescues directional membrane fusion in cells with Lgl1 knockdown
Conclusion:
Rab10 acts downstream of Lgl1 in polarized membrane
fusion & neuronal polarization
Q: How does Lgl1 regulate Rab10?
Lgl1 is not a GEF for Rab10
Rab10-GDP
GAP
GEF
Rab10-GTP
GEF: Guanine nucleotide exchange factor
GAP: GTPase-activating protein
Lgl1 promotes membrane
association of Rab10 in neurons
The nucleotide and membrane attachment/detachment cycles of Rab GTPases
GDI: GDP dissociation inhibitor
GDF: GDI displacement factor
1.Inactive (GDP-bound) prenylated Rab GTPases are bound to GDI, which masks their
isoprenyl anchor and thereby keeps the Rab in a soluble cytosolic form.
2.Membrane attachment of Rabs requires the function of a GDF that dissociates the
GDI–Rab complex and allows the prenyl anchor to be inserted into the membrane.
3.Specific GEFs exchange the bound GDP for GTP, thereby activating the Rab GTPases.
4.The active, membrane-bound Rabs are then able to fulfill their various functions in
membrane traffic by binding to their specific effector proteins.
5.Finally, specific GAPs inactivate the Rabs by accelerating the hydrolysis of the bound
GTP into GDP.
6.The inactive, GDP-bound Rabs can then be extracted from the membrane by GDI and
recycled for another round of function
Grosshans B L et al. PNAS 2006;103:11821-11827
Sivars U., et al.
Yip3 catalyses the dissociation of
the endosomal Rab-GDI complex
Nature 2003, 425: 856
Yip3 as a GDF for Rab9 and Rab5
Pfeffer S. 2004 Nature Reviews Mol. Cell Biol.
Conclusion: Lgl1 is a GDF for Rab10
Evidence 1: Lgl1 binds to Rab10 directly, mainly GDP-bound form
Evidence 2:
Lgl1 displaces the Rab10-GDI complex in HEK293 cells
GDI
Rab10
Lgl
GDI
Rab10
Lgl
Lgl1 displaces the Rab10-GDI complex in vitro
Evidence 3:
Lgl1 activates Rab10 by releasing GDI
Evidence 4:
Lgl1 recruits Rab10 from the GDI-Rab10
complex into Phosphatidylcholine (PC)
liposomes
GDI
Rab10
GDF
Rab10
GDI
Membrane
vesicles
Vesicle floating assay
Domain mapping for GDF activity
Direct association of Lgl with PPV
Paramagnetic beads coated with purified Lgl1
protein were incubated with cortical neuron
postnuclear supernatant
Identity of Lgl1-associated vesicle
Negative:
Rab5 (early endosome)
Rab11 (recycling endosome)
VAMP2 (synaptic vesicle)
Bip (Endoplasmic Reticulum)
Positive:
TGN38 (trans-golgi network)
TrkB, Frizzled7, IGFR (growth factor receptors)
Dvl, Par3, Par6, aPKC (polarity proteins)
TIRF analysis of PPV fusion
Total internal reflection
fluorescence microscope
TIRF events for
pHLuorin-TrkB
Role of Lgl1/Rab10 in neuronal polarization in vivo
Conclusion
 Both Lgl1 and Rab10 are essential for directional
membrane fusion and axon development
 Lgl1 is a newly identified GDF for Rab10
 Rab10 acts downstream of Lgl1 in regulating
membrane insertion underlying axon
development
Wang T., Liu Y., et al. Dev. Cell 2011, 21: 431
Polarized membrane insertion
effectors
Cytoplasma
Rab10-GTP
Lipid bilayer
Rab10-GDP
GDI
Rab10-GDP
Lgl
Plasmalemmal precursor vesicles
Acknowledgement
Funding support:
Chinese Academy of
Sciences
Natural Science
Foundation of China
Wang Tong(王彤) Liu Yang (刘阳)
Chinese Ministry of
Science and Technology
Thanks for attention !
Role of Lgl1/Rab10 in corticl neurogenesis
Role of Rab8 and Rab13 in neuronal polarity
Lgl1 cannot displace the complex of Rab8/GDI
or Rab13/GDI
Lgl-C is sufficient to recruit Rab10 to membrane fraction
Role of Lgl1/Rab10 in corticl neurogenesis