1. Membrane Trafficking Mechanism Underlying Neuronal Polarization Zhen-Ge Luo ( 罗振革 ), Ph.D Institute of Neuroscience Chinese Academy of Sciences Shanghai, 200031 10/13/2011, Jinan, China Qilu Internation Neuroscience Symposium Shangdong University School of Medicine

2. Parallel between neuronal polarization in vitro and in vivo Model systems

3. 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.

4. Signaling pathways involved in mammalian axon specification during neuron polarization

5. Plasmalemmal precursor vesicles during early neurite elongation Pfenninger KH 2009 Nature Reviews Neuroscience

6. Red (emission at 620 nm): Golgi-derived vesicles Green (emission at 515 nm): Plasmalemma

7. Golgi-derivation of BODIPY-labeled vesicles Time-dependent accumulation of BODIPY-vesicles in distal axon

8. Wash1 Wash2 Free BODIPY-ceramide Golgi-derived vesicles BODIPY labeling of Plasmalemmal Precursor Vesicles (PPV)

9. Disappearance of BODIPY-Red signals in neurite growth cones

10. Membrane fusion activity correlates with axon specification

12. Question: -- How is the directional membrane fusion regulated?

13. Lethal Giant Larvae is Essential for Various Types of Cell Polarity J Cell Sci. 2006;119:2107-18. Mammalian epithelial apical-basal polarity Mammalian astrocyte Nat Cell Biol. 2003; 5:301-8. Drosophila neuroblast division Dev Neurosci. 2006; 28:13-24 Yeast polarized budding Genetics. 1998; 149:1717-27.

14. Expression of Lgl1 in the developing brain

15. Localization of Lgl1 in cultured hippocampal neurons

16. Lgl1 is essential for neuronal polarity a b c

17. Over-expression of Lgl1 promotes axon development

18. Down-regulation of Lgl1 decreases directional membrane fusion

19. Q: How does Lgl regulate membrane fusion and neuronal polarity? Mol Biol Cell. 2006;17:3156-75 J Cell Biol. 2006; 172: 55-66

20. RAB10 RAB8a RAB8b RAB13 Sec4p Lgl1 is associated with mammalian Rab10

21. Lgl1 is associated with Rab10 in primary neurons

22. Colocalization of Rab10 with PPV

23. Mutated forms of Rab10 affect neuronal polarity Rab10Q58L, GTP-locked active form Rab10T23N, GDP-locked inactive from

24. Rab10 is essential for neuronal polarity

25. Rab10 acts downstream of Lgl1 in regulating axon development --Lgl1 has no effect on axon defects caused by Rab10DN or Rab10 siRNA; --Rab10 partially rescues the neuronal polarity in cells with Lgl1 knockdown; --Rab10DN or siRNA prevents the enhanced axon development caused by Lgl1.

26. --Rab10 rescues directional membrane fusion in cells with Lgl1 knockdown --Down-regulation of Lgl1 decreases directional membrane fusion; Conclusion: Rab10 acts downstream of Lgl1 in polarized membrane fusion & neuronal polarization Role of Rab10 in membrane fusion

27. Q: How does Lgl1 regulate Rab10? Lgl1 is not a GEF for Rab10 Rab10-GDP Rab10-GTP GEF GAP GEF: Guanine nucleotide exchange factor GAP: GTPase-activating protein

28. Lgl1 promotes membrane association of Rab10 in neurons

29. The nucleotide and membrane attachment/detachment cycles of Rab GTPases Grosshans B L et al. PNAS 2006;103:11821-11827 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 GDI: GDP dissociation inhibitor GDF: GDI displacement factor

30. 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.

31. Evidence 1: Lgl1 binds to Rab10 directly, mainly GDP-bound form Conclusion: Lgl1 is a GDF for Rab10

32. Evidence 2: Lgl1 displaces the Rab10-GDI complex in HEK293 cells GDI Rab10 Lgl GDI Rab10 Lgl

33. Lgl1 displaces the Rab10-GDI complex in vitro

34. Evidence 3: Lgl1 activates Rab10 by releasing GDI

35. GDI Rab10 GDF GDI Rab10 Membrane vesicles Evidence 4: Lgl1 recruits Rab10 from the GDI-Rab10 complex into Phosphatidylcholine (PC) liposomes Vesicle floating assay

36. Domain mapping for GDF activity

37. 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)

38. TIRF analysis of PPV fusion TIRF events for pHLuorin-TrkB Total internal reflection fluorescence microscope

39. Role of Lgl1/Rab10 in neuronal polarization in vivo

40. 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

41. Polarized membrane insertion Lgl Rab10-GDP Rab10-GTP GDI Plasmalemmal precursor vesicles effectors GEF GAP Rab10-GDP Lipid bilayer Cytoplasma Wang T., Liu Y., et al. Dev. Cell 2011, 21: 431

42. Acknowledgement Funding support: Chinese Academy of Sciences Natural Science Foundation of China Chinese Ministry of Science and Technology Thanks for attention ! Wang Tong( 王彤 ) Liu Yang ( 刘阳 )

43. Role of Lgl1/Rab10 in corticl neurogenesis

44. Role of Rab8 and Rab13 in neuronal polarity

45. Lgl1 cannot displace the complex of Rab8/GDI or Rab13/GDI

46. Lgl-C is sufficient to recruit Rab10 to membrane fraction

47. Role of Lgl1/Rab10 in corticl neurogenesis