Dissertation Talk 2009


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  • In my talk, I will describe studies that uncover conserved mechanisms underlying membrane protein targeting. In cell biology, one the fundamental aspects of all eukaryotic cells is cell polarity. What underlies this polarity is that specific proteins and lipids are delivered to specific regions of the plasma membrane, thereby defining and maintaining their unique molecular and functional identities. This is the result of some fundamental mechanisms preserve from yeast to humans. Here I will describe work covering the neuronal trafficking behaviors of two classes of proteins– syntaxins and K+ channels. Functions:Consolidate short-term and long-term memoryEpisodic and contextual memorySpatial Orientation/NavigationDetection of novel stimuliNeurogenesisHere we show that syntaxins 3 and 4 are expressed in hippocampal neurons and that polarized expression of syntaxin 3 confers specificity to axonal protein trafficking. Syntaxin 3 localizes to the axonal plasma membrane, particularly to axonal tips, whereas syntaxin 4 localizes to the somatodendritic plasma membrane. Disruption of a conserved N-terminal targeting motif causes mislocalization of syntaxin 3, resulting in coincident mistargeting of the axonal NgCAM, but not of the somatodendritic transferrin receptor. RNAi-mediated knockdown of endogenous syntaxin 3 leads to partial mistargeting of NgCAM, demonstrating that syntaxin 3 is critical to axonal targeting. Scaffolding proteins at postsynaptic sites are crucial for organizing ion channels into signaling complexes essential for synaptic transmission. Inward rectifier potassium Kir2.3 channels are enriched at spines, when expressed in cultured hippocampal neurons, but the mechanisms underlying channel localization are unclear. We used mutational analysis to determine how protein-interacting motifs in the C-terminus of Kir2.3 influence its localization. We show that Kir2.3 channels traffic to spines and that synaptic localization requires a previously identified C-terminal PDZ (postsynaptic density-95/Discs large/zona-occludent-1)-binding motif. We also identify a polyproline motif in the C-terminus of Kir2.3 that associates with Homer-1 and Homer-2 in brain. Deleting the PDZ-binding motif abolishes binding of PSD-95 with Kir2.3 and dramatically reduces Kir2.3 synaptic localization, whereas mutating residues in the polyproline motif has little effect on channel localization. Using RNA interference, we show that PSD-95 is necessary for localizing Kir2.3 to spines.
  • Based on the trafficking studies using hipp neurons, a variety of mechanisms have been proposed including selective delivery (in which carrier vesicles deliver cargo exclusively to specifically to either the axonal or dendritic membrane); or selective fusion (in which cargo could be delivered to either the axonal or dendritic domains but become expressed exclusively on the surface of one by selective fusion machinery); or the expression of proteins on the correct surface domain could also be achieved by selective endocytic machinery, in which case cargo are first delivered uniformily to the surface but are selective endocytosed and subsequently delivered to the correct domain. Test the hypothesis that syntaxins are involved in specifying the targeting of membrane proteins to specific domainsuse this slide as the opportunity to describe in words what you are going to test (Selective fusion hypothesis), and what your results are going to be (Stx3 specifies selective axonal fusion)
  • Fusion is thought to occur when SNAREs on the vesicle, v-SNAREs, interact with those on the target membranes (t-SNAREs) and based on the zipper model they zip up into a four helical bundle that drives fusion of apposing membranes. The pairing of v-SNAREs expressed on cargo vesicles matching up with a cognate t-SNAREs on the plasma membrane was initially proposed by the SNARE hypothesis from James Rothman to contribute to specificity of membrane trafficking. This was demonstrated by in vitro reconstitution fusion assays that a successful SNARE pair lead to successful membrane fusion.
  • There are primarily three subclass of molecules that make up the SNARE complex. Syntaxin. Fusion. Brain. Synaptic vesicles. Isolated in late 80s and early 90s.
  • Not only are SNAREs important for synaptic transmission but also they are important mediators of fusion along all intracellular trafficking pathways in mammals. How is specificity achieved during membrane trafficking? In other words, Question: What enables cargo-carrying vesicles to target and deliver their cargo to specific domains (axons or dendrites) correctly?From yeast to mammals, SNAREs are required for vesicle fusion along both secretory and endocytic pathways.
  • Different subcellular locations of these syntaxins suggest that there is a specificity of trafficking pathways and perhaps fusion specificity of cargo carring vesicles to a specific membraneSyntaxin 4 knockout is embronic lethal but partial knockout exhibit 50% reduction in whole-body glucose uptake Apical targeting of stx3 depends on an N-terminal domain. Point mutations and chimera studies showed that this domains was indeed necessary and sufficient for apical trafficking of syntaxin 3.Since these syntaxins localize to different subcellular locations, this suggest that they might be involved in specifying fusion of cargo-laden vesicles to specific compartmentsAlthough these findings indicate that plasma membrane syntaxins are important for polarized trafficking pathways in epithelial cells, whether selective fusion of vesicles carrying cargo is involved in neuronal polarity and selective targeting of proteins is unknown. Here I wanted to test the hypothesis that syntaxins are involved in specifying the
  • Before we get to that question, we wanted to first ask whether syn 3 and 4 were expressed in our hipp cutlures. By RT-PCR done by Nikunj as shown here on the left and by western blotting on the right, syntaxin 3 and 4 were found endogenously expressed in our cultured neurons. We found that there were 2 isoforms A and B of syn3 expressed in neurons, whereas only the A isoform was present in normal rat kidney cell lines. Similarly in westerns, both syn 3 and 4 are again expressed from cultured neurons as well as adult hippocampus homogenates similar to syn1Next, we wanted to ask where syn3 and 4 were polarized in neurons?
  • One of first clues came from Nikunj studies in epithelial cells where he showed that an N-terminal motif: FMDEFF was found to be critical for its targeting to the apical membrane in epithelial cells. We thought that would be good place to start so several constructs were made including a mutant where the N-terminus containing this motif was removed and another mutant where there amino acids were individually mutated to alanine. when they mutated the F31, E34, and F36 individually to alanine.
  • When I expressed these syntaxin 3 mutants in neurons I found that
  • Now that we were able to show that Stx3 and Stx4 are polarized in neurons, and that it is possible to mislocalize Stx3, we were able to address the key question of whether selective membrane fusion contributes to polarized neuronal trafficking. The selective fusion model predicts that if the specific SNARE protein is moved to a new location in the cell, then the cargo will fuse at the new location. The overall strategy to test this was to use an axonal cargo and a dendritic cargo, and ask whether these cargoes could be re-routed by mislocalization of syntaxin 3.
  • One attractive candidate as an axonal cargo was NgCAM, Live cell imaging studies done in Gary banker’s lab (Burack 2000) shown NgCAM go to surface but internally was in both axon and dendrites.
  • When we quantified the results by measuring the intensities of surface proteins in axons over dendrites to determine the polarity index, the majority of the cells showed that syn3 mislocalization caused NgCAM to also mislocalize.
  • as a mixture of shRNAs gives effective knockdown
  • Given that SNAREs are involved in the fusion of vesicles, we wanted to test whether overexpression of syntaxins might lead to an increase in delivery of lipid molecules.
  • Transport K+ ions in muscle and kidney
  • Rare genetic disorderInherited autosomal dominant Affects heart, small lower jaw, abnormal curvature of fingers and toes
  • Now that we were able to show that Stx3 and Stx4 are polarized in neurons, and that it is possible to mislocalize Stx3, we were able to address the key question of whether selective membrane fusion contributes to polarized neuronal trafficking. The selective fusion model predicts that if the specific SNARE protein is moved to a new location in the cell, then the cargo will fuse at the new location. The overall strategy to test this was to use an axonal cargo and a dendritic cargo, and ask whether these cargoes could be re-routed by mislocalization of syntaxin 3.
  • Now in my second main project focused on understanding the mechanisms underlying the class of Kir2 potassium channel localization in neurons?Accumulating electrophysiological evidence indicates that Kir2 channels are critical regulators of synaptic integration and dendritic excitability in the forebrain and striatum (Day et al. 2005; Shen et al. reduce opening of Kir2 channels enhances dendritic excitability and synaptic integration. Modulation of these K+ channels can thus alter synaptic signaling events, and defective regulation could lead to neurological disease. Kir2.2/2.3 channel mediated conductance appears to regulate the activity of other types of channels HCN, thereby controlling dendritic excitability. Blocking channels lead to neurons to depolarize which led to deactivation of HCN channelsHold the dendritic membrane near the K+ equilibrium potenital and dampen responsiveness to glutamatergic excitatory synaptic transmission. Given the significance of these channels functional roles in neurotransmission, it is therefore important to understand how these channels are regulated in neurons. In the lab, we have focused on understanding just that.It is therefore not hard to imagine that improper channel localization could cause communication defects in neural circuitry underlying proper brain function. Correct location of channels at specific subcellular sites
  • Kir2.3 highly localized as puncta along dendrites.Co-labeling with post-synaptic marker, PSD-95, bright Kir2.3 puncta concentrated at spines.
  • Previous unpublished from Zach’s work in the Jan lab had already identified a Homer binding motif that is exclusively found in Kir2.3 and not in the other Kir2 isoforms.
  • A similar pattern of co-localization was found with Kir2.3-H, which lacks the polyproline motif, and PSD-95. By contrast, Kir2.3 constructs that lacked the PDZ binding motif (Kir2.3Δ3 and Kir2.3-HΔ3) were not recruited to synaptic sites in the presence of overexpressed PSD-95. Homer mutant (-H)PDZ mutant (delta 3)this high correlation coefficient is numerical evidence for the colocalization of the two proteins
  • Summary slides part 1—syntaxins and part 2—Kir2.3 and PSD-95
  • Here I showed you the importance of these scaffolding interactions of K+ channels in recruiting these chanenls to spines and making it possible to bring them in close proximity with other signaling molecules to regulate synaptic transmission.
  • Dissertation Talk 2009

    1. 1. Trafficking of Syntaxins and Kir2 Potassium Channels in Neurons<br />Linda SooHoo<br />University of California, Santa Barbara<br />
    2. 2. The Meaning of Life<br />
    3. 3. Overview<br />Introduction<br />Neuronal polarity and polarized trafficking pathways<br />Parts 1 & 2:<br />Polarized protein trafficking: Are SNARE proteins polarized in neurons, and are they involved in polarized trafficking of neuronal membrane proteins?<br />Ion channel localization: Are trafficking proteins associated with ion channels, and do they play a role in their localization and clustering? <br />
    4. 4. Neurons are polar and <br />need to traffic cargoes<br />asymmetrically<br />Cell<br />Body <br /><ul><li>Communication
    5. 5. Directional transmission of information
    6. 6. Compartments
    7. 7. Axons</li></ul>(e.g. Kv1.4, Nav1.2, GAP-43, etc.)<br /><ul><li>Dendrites</li></ul>(e.g. GluR1-4, mGluRα, transferrin receptors, Kv4.2, etc.)<br />Dendrites<br />Axon<br />
    8. 8. Neuronal signaling occurs via dendritic spines<br />AXON<br />DENDRITE<br />
    9. 9. Synaptic Transmission<br />Communication between neurons at synapses<br />Changes to strength of this communication defines synaptic plasticity<br />Mechanisms underlying synaptic plasticity may include:<br />Formation or loss of synaptic contacts (developmental)<br />Physiological changes:<br />Science 2006<br /><ul><li>Changes in quantity of neurotransmitters release and response by receptors
    10. 10. Changes in electrical signaling driven by ionotropic receptors or ion channels
    11. 11. Changes in cell surface localization of ion channels and receptors </li></li></ul><li>Cultured Hippocampal Neurons (Banker)<br />One process becomes the axon and grows dramatically, while the other processes become dendrites.<br />Craig and Banker (1994) Annu. Rev. Neurosci. <br />Question: How are the axonal and dendritic compartments maintained—vesicle targeting specificity?<br />
    12. 12. Polarized Vesicle Targeting in Neurons<br />Polarized delivery<br />Selective membrane fusion<br />Selective endocytosis<br />Potential Players Involved:<br />Motor Proteins<br />SNARE Complex<br />Endocytic Machinery<br />Horton and Ehlers (2003) Neuron<br />
    13. 13. Making Fusion Possible<br />SNARE protein superfamily<br />(Soluble-N-ethylmaleimide-sensitive factor Attachment protein REceptor)<br />Characteristic 60-70 aa cytoplasmic coiled-coil domain (SNARE motif)<br />Vesicle membrane fusion<br />V-SNARE targets vesicle to its correct membrane fusion partner T-SNARE (target)<br />SNARE core complex, tight and stable 4 helical bundle<br />Membranes merge<br />Target membrane<br />
    14. 14. SNARE Core Complex<br />SNAPs<br />VAMPs<br />Syntaxins<br /> Cargo Vesicle<br />Target Membrane<br />
    15. 15. SNAREs mediate membrane fusion along intracellular trafficking pathways<br />Jahn & Scheller (2006), Nat. Rev. Mol. Cell. Biol.<br />
    16. 16. MT<br />Golgi<br />Nucleus<br />Plasma membrane syntaxins<br />MDCK polarized epithelial cells:<br />Syntaxin 3: apical membrane.<br />Syntaxin 3 F31A mutant: non-polarized.<br />Syntaxin 4: basolateral membrane.<br />(Low et al., 1998; Low et al., 2006; Sharma et al.,2006)<br />Apical<br />Different syntaxin distribution suggests that these syntaxins may participate in specifying fusion of vesicles to specific compartments.<br />Question: Are these syntaxins involved in specifying the targeting of selective proteins to specific neuronal domains?<br />
    17. 17. Syntaxins 3 and 4 are expressed in hippocampal neurons<br />NRK = Normal rat kidney cells<br />Syntaxin 4 <br />Syntaxin 3 <br />Question: Are the plasma membrane syntaxins<br />Stx3 and Stx4 polarized in neurons?<br />
    18. 18. Syntaxin 3 Localizes to Axon and Neurite Tips<br />Total Syntaxin 3<br />Total Syntaxin 3<br />GFP<br />
    19. 19. Syntaxin 4 Localizes to Dendrites<br />Axon<br />Total Syntaxin 4<br />Total Syntaxin 4<br />GFP<br />
    20. 20. N-terminal trafficking motif:<br />Syntaxin 3 mutant constructs<br />FMDE<br />Cytoplasmic<br />Extracellular<br />
    21. 21. N-terminal targeting motif is required for polarized syntaxin 3 trafficking<br />Stx3Δ38<br />GFP<br />Stx3AAA<br />GFP<br />axon<br />dendrites<br />axon<br />axon<br />
    22. 22. Is syntaxin 3 responsible for polarized trafficking of vesicles carrying cargo to the axons?<br />
    23. 23. NgCAM (L1)—an axonal targeted cargo <br />NgCAM (L1) is a neural cell adhesion molecule, a member of the immunoglobulin superfamily, that is expressed in axons and growth cones.<br />Intracellular NgCAM is expressed in a non-polarized manner in hippocampal neurons.<br />NgCAM surface expression is highly polarized to the axon.<br />This suggests that selective fusion is the mechanism involved in axonal polarity of NgCAM.<br />
    24. 24. Syntaxin 3 localizes NgCAM to axon<br />
    25. 25. Syntaxin 3 mutants mislocalize NgCAM<br />
    26. 26. Syntaxin 3 does not affect trafficking of a dendritic cargo transferrin receptor<br />
    27. 27. Mislocalization of syntaxin 3 leads to loss of NgCAM polarity<br /><ul><li>Axonal proteins have high Polarity Index Ratios
    28. 28. Dendritic proteins have low Polarity Index Ratios</li></ul>N =25-30 cells<br />Polarity Index: Ratio of surface NgCAM (axon)<br /> surface NgCAM (dendrite)<br />Conclusion: Syntaxin 3 is required for mediating targeting of axonal cargo protein and not dendritic cargo protein<br />
    29. 29. H1<br />Stx3<br />CMV<br />GFP<br />Is syntaxin 3 necessary for localizing NgCAM to axon?<br /><ul><li>Cos cell expression</li></ul>shSyntaxin 3<br />shNontarget<br />Syntaxin 3<br />GAPDH<br />
    30. 30. Surface NgCAM<br />GFP/Surface NgCAM<br />shNontarget<br />axon<br />shSyntaxin 3<br />
    31. 31. Syntaxin 3 is necessary for NgCAM axonal polarization<br />Surface NgCAM polarity measurements<br />p ≤ 0.001<br />**<br />
    32. 32. Stx 4<br />Stx 3<br />Control<br />Syntaxin 3 involved in axonal outgrowth<br />
    33. 33. Summary: Syntaxin 3 & NgCAM<br /><ul><li>Syntaxin 3 localizes to axons, whereas syntaxin 4 localizes to dendrites
    34. 34. Mislocalization of syntaxin 3 leads to mislocalization of axonal protein NgCAM and not dendritic protein transferrin receptor
    35. 35. Necessity: Knockdown of endogenous syntaxin 3 leads to misloclalization of NgCAM.
    36. 36. Syntaxin 3 is involved in axonal outgrowth in early neurons.
    37. 37. Results demonstrate that selective fusion contributes to specifying polarized protein trafficking in neurons.</li></li></ul><li>Neuronal Polarity & Precise Localization of Ion Channels are Key to Neuronal Function<br />Potassium Channels<br />Sodium Channels<br />Calcium Channels<br />Neurotransmitter <br />Channels<br />
    38. 38. Potassium Channels<br />Adapted from Lily Jan’s lab, UCSF<br /> Kir2 subclass of K+ channels<br /><ul><li>Involved in action potential repolarization
    39. 39. Set and maintain the resting potential</li></li></ul><li>Kir 2 Channel Physiology<br />Restore resting membrane potential in heart<br />Regulate neuronal excitability<br />Maintain skeletal muscle excitability<br />Mediate K+ buffering by glia<br />Involved in dilation of blood vessels<br />Salt reabsorption in kidney<br />
    40. 40. Short QT-Syndrome<br /><ul><li>Cardiac Arrhythmias
    41. 41. Ventricular Fibrillation
    42. 42. Sudden Death</li></ul>ir2.1<br />Kir2 Channel Diseases<br />Mutations in Kir2.1 Cause Andersen-Tawil syndrome<br />Cardiac Arrhythmias<br />Periodic Paralysis<br />Developmental Alterations<br />Andelfinger et al., 2002<br />Plaster et al., 2001;<br />
    43. 43. Questions<br />Are Kir2 channels associated with trafficking complexes?<br /><ul><li> Proteomic identification of interacting proteins</li></ul>How do these complexes affect:<br /><ul><li>Channel targeting & clustering in neurons</li></li></ul><li>PDZ Domain<br />Purification of Channel-interacting Complexes<br />RRESEI<br />(Leonoudakis et al., JBC, 2004)<br />
    44. 44. Kir2-Associated Proteins identified by Mass Spectrometry<br />Brain:<br />Interacting scaffolding/adaptor proteins include:<br /><ul><li> SAP102
    45. 45. Chapsyn-110/PSD-93
    46. 46. PSD-95
    47. 47. SAP97</li></li></ul><li>How are Kir2 channels localized in neurons?<br />?<br /><ul><li>Regulating neuronal excitability
    48. 48. Involved in action potential repolarization
    49. 49. Setting and restoring the cell’s resting membrane potential</li></ul>?<br /><ul><li>Kir2 channels are critical regulators of dendritic excitability and synaptic integration in the forebrain and striatum.</li></li></ul><li>Factors critical for channel function<br />Correct location of channels at specific subcellular sites<br />Right number of channels<br />Channels open at the right time<br />Regulation of Kir2 channel trafficking<br />
    50. 50. Kir2.3 is clustered in dendritic spines of hippocampal neurons<br />HA-Kir2.3 (red)<br />and <br />GFP(green) transfected into neurons<br />
    51. 51. Kir2.3 clustering in spines depends on PDZ interaction<br />HA-Kir2.3 is clustered<br />HA-Kir2.3D3 is not clustered<br />
    52. 52. GFP-PSD-95 Recruits Kir2.3 Channels to Clusters<br />HA-Kir2.3 (red)<br />GFP-PSD-95 (green)<br />Merge<br />HA-Kir2.3 & GFP-PSD-95<br />HA-Kir2.3Δ3 (red)<br />GFP-PSD-95 (green)<br />Merge<br />HA-Kir2.3D3 & GFP-PSD-95<br />
    53. 53. Mutations in Homer-binding domain of Kir2.3<br />
    54. 54. GST-pulldowns from rat brains<br />Kir2.3 C-terminal domain interacts with MAGUK PSD-95 and Homer proteins<br />Dmitri Leonoudakis, unpublished<br />Question: Does the synaptic localization of Kir2.3 channels depend on the PDZ interaction or Homer interaction?<br />
    55. 55. Kir2.3 clustering in spines depends on PDZ interaction<br />Colocalization with PSD-95<br />n = 14-15 cells<br />
    56. 56. H1<br />si-PSD-95<br />CMV<br />GFP<br />shPSD95 effectively knocks down PSD-95<br />
    57. 57. si-PSD-95 Eliminates PSD-95 Clustering in Spines<br />PSD-95 (endogenous)<br />PSD-95 (endogenous)<br />GFP<br />GFP<br />Uninfected<br />Infected with Adeno-si-PSD-95 GFP<br />
    58. 58. PSD-95 is Required for Channel Clustering in Dendritic Spines<br />PSD-95<br />HA-Kir2.3<br />HA-Kir2.3<br />PSD-95<br />GFP<br />Transfected with si-PSD-95 GFP and HA-Kir2.3<br />Untransfected<br />
    59. 59.
    60. 60. Conclusions<br />PDZ-binding motif is required for potassium channel clustering to dendritic spines.<br />PSD-95 is required for targeting and clustering Kir2 channels to dendritic spines<br />These studies demonstrate that Kir2 channel localization to spines depend on its association with PSD-95 via the PDZ-binding motif. <br />
    61. 61. Summary<br />What enables proteins to be expressed correctly at specific subcellular locations?<br /><ul><li>Syntaxin 3-dependent membrane fusion of NgCAM to axons.</li></li></ul><li><ul><li>Potassium channel clustering to dendritic spines requires PDZ-mediated dependent interactions by PSD-95.</li></ul>Kir2<br />Postsynaptic density<br />NH3<br />RRESAI<br />Spine<br />Dendritic shaft<br />
    62. 62. Acknowledgements<br />Neuroscience Research Institute<br />Lab Members:<br />Chris D. Banna Dmitri Leonoudakis<br />Carolyn M. Radeke Melanie Williams<br />Lior Dessau Lisa Conti<br />Advisor: Dr. Carol Vandenberg<br />Funding:<br />Cottage Hospital Grant<br />Muscular Dystrophy<br />American Heart Association<br />Collaborators:<br />Thomas Weimbs<br />Seng Wei Low<br />Nikunj Sharma<br />(UCSB)<br />