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Slide2: Membrane Protein Targeting in Cultured Hippocampal Neurons Bernard Sampo Laboratory of Gary Banker – CROET Oregon Health & Science University


Slide3: 40 um


Slide4: Preparing hippocampal cultures


Slide5: Hippocampal neurons develop a polarized form in culture


Slide7: soluble GFP


Slide9: Some proteins are polarized to the axonal domain


Slide10: Some proteins are polarized to the somatodendritic domain


Slide11: GFP-Kv2.1 soluble Tomato The potassium channel Kv2.1 is restricted to the cell body and proximal dendrites


Slide12: A barrier at the axon initial segment limits diffusion between domains


Slide13: 9x Movie 1: Please go to http://www.vimeo.com/887388 to play this movie


Where Along the Secretory Pathway does the Trafficking of Axonal and Dendritic Membrane Proteins Diverge?: Where Along the Secretory Pathway does the Trafficking of Axonal and Dendritic Membrane Proteins Diverge?


Slide16: 9X SS(NPY)-GFP Imaging set-up widefield microscope 50-100 frames 600 ms/frame 20 ms frame interval Movie 2: Please go to http://www.vimeo.com/893710 to play this movie


Slide17: time 1 2 3 proximal distal Kymograph number of moving carriers number of anterograde and retrograde events transport velocity run length (distance traveled without pausing) 5 4 3 2 1 3 2 1


Slide18: Movie 3: Please go to http://www.vimeo.com/888377 to play this movie


Slide19: Transfection Accumulation in the TGN 37°C 34°C - 210 / 270 min 0 min 60 min Imaging Transport Assay


Slide20: 9X GFP-LDLR Movie 4: Please go to http://www.vimeo.com/888392 to play this movie Axon Dendrite


Slide21: 9X GFP-LDLR Movie 5: Please go to http://www.vimeo.com/888428 to play this movie Axon Dendrite


Slide22: NgCAM-GFP 9X Movie 6: Please go to http://www.vimeo.com/888711 to play this movie Axon Dendrite


Slide23: axon dend1 dend2 dend3 dend4 dend5 axon dend1 dend2 dend3 dend4 dend5 proximal distal 36 sec


Slide24: Conclusion Trafficking of dendritic and axonal membrane proteins diverges at the level of transport.


Slide25: GFP-LDLR NgCAM-mCherry 478 ms 478 ms x60 1st 2nd spinning disk confocal Movie 8: Please go to http://www.vimeo.com/888725 to play this movie


Slide26: GFP-Kv2.1 mCherry-LDLR Movie 9: Please go to http://www.vimeo.com/890024 to play this movie


Slide27: VAMP2-GFP NgCAM-mCherry Movie 10: Please go to http://www.vimeo.com/891722 to play this movie


Slide28: Conclusions Trafficking of dendritic and axonal membrane proteins diverges at the level of sorting and transport. Axonal and dendritic proteins are sorted at the Golgi into different transport vesicles. A common carrier may transport and deliver axonal proteins to the axonal surface. More than one vesicle population both transport and deliver dendritic proteins to the dendritic surface. Both sorting and transport account for the selective distribution of dendritic proteins.


Slide29: Polarization of axonal membrane proteins may depend on additional events downstream of transport


Slide30: Two Mechanisms for the Polarization of Axonal Membrane Proteins Selective Fusion


Slide31: Two Mechanisms for the Polarization of Axonal Membrane Proteins Selective Endocytosis


Slide33: cell surface NgCAM-CD8a soluble GFP


Slide34: Dendritic Cell Surface Axonal Cell Surface Dendritic Protein VAMP2 NgCAM Selective Fusion Selective Endocytosis Two Mechanisms for the Polarization of Axonal Membrane Proteins


Slide35: Dendritic Cell Surface Axonal Cell Surface NgCAM Selective Fusion What cellular mechanisms ensure that exocytosis of NgCAM-containing carriers occurs only in the axon?


Slide37: GFP-RalA Ng-mCherry Movie 11: Please go to http://www.vimeo.com/891861 to play this movie


Slide38: GFP-RalA Ng-mCherry Movie 12: Please go to http://www.vimeo.com/893568 to play this movie


Slide39: GFP-RalA mCherry-LDLR Movie 13: Please go to http://www.vimeo.com/891784 to play this movie


Slide41: NgCAM NgCAM + RBD-Sec5 1000 Normalized Cell Surface Staining (AIU) NgCAM in axons 0 (GFP) (GFP) (GFP) (GFP)


Slide42: LDLR LDLR + RBD-Sec5 Normalized Cell Surface Staining (AIU) (GFP) (GFP)


Slide43: GFP-LDLR + RBD-Sec5 Movie 14: Please go to http://www.vimeo.com/889877 to play this movie


Slide44: GFP-LDLR Movie 15: Please go to http://www.vimeo.com/893464 to play this movie


Slide45: GDP Dendritic Cell Surface Axonal Cell Surface Exocyst Exocyst GDP GTP Domain-Specific Activation of RalA on NgCAM Carriers May Regulation its Selective Delivery to the Axonal Surface Selective Activation


Slide47: Axon Axon


Slide48: Conclusions RalA is associated with post-Golgi carriers that transport NgCAM and LDLR to axons and dendrites. RalA-mediated activation of the exocyst complex is required for the delivery of both NgCAM and LDLR to the cell surface. Constitutively active RalA increases the delivery of LDLR to the dendritic cell surface, but does not increase its delivery to the axonal membrane because LDLR carriers are excluded from the axon. Constitutively active RalA disrupts the polarity of NgCAM by enhancing its delivery to the dendritic plasma membrane. Domain-specific activation of RalA may ensure the selective delivery of NgCAM to the axonal surface.


Slide49: Where Along the Secretory Pathway does the Trafficking of Axonal and Dendritic Membrane Proteins Diverge?


Slide50: Cellular Events Underlying the Polarization of Membrane Proteins


Slide51: Cellular Events Underlying the Polarization of Membrane Proteins


Slide52: Cellular Events Underlying the Polarization of Membrane Proteins


Slide53: 3 1 2 1 - Sorting Cellular Events Underlying the Polarization of Membrane Proteins 2 - Transport 3 - Fusion


Slide54: Lab Members Gary Banker Stefanie Kaech Greta Glover Julie Luisi Harp Barbara Smoody