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  • 1. Plasma membranes are fluid: membrane-spanning proteins rapidly diffuse unless restrained
  • 2. Membrane-spanning proteins are precisely localized at sites of physiological function: what is the code? NaV CNG- CHANNEL INITIAL SEGMEN T NODE OUTER SEGMENT Na/K ATPase E-CADHERIN INITIALSEGMENT NODE DYSTROGLYCAN ATAXIA ARRHYTHMIA MUSCULAR DYSTROPHY BLINDNESS
  • 3. Agre, P., Orringer, E., Bennett, V., N. Eng. J. Med. 1982;306:1155-61
  • 4. Channels/transporters Anion exchanger Na/K ATPase Volt. Sens. Na channel Na/Ca exchanger IP3 receptor RhBG NH3 transporter KCNQ2/3 Kv3.1b CNG-beta Kir6.2 (K (ATP) channel) Cell adhesion molecules CD44 L1 CAMs E, N-cadherins dystroglycan Ankyrins-G, -B and-R and their partners Recognition by ANK repeats developed independently multiple times in evolution ANK repeats
  • 5. 480/270 kDa Ankyrin-G co-clusters with volt.-gated Na channels and 186 kDa neurofascin at axon initial segments and nodes of Ranvier JCB 110:1341-52 (1990) JCB 114:1243-59 (1991) JBC 270:2352-59 (1995) JCB 135:1355-67 (1996) JNeurosci.17:7025-36 (1997) Steve Lambert Katya Kordeli
  • 6. Cerebellar knockout of ankyrin-G: severe ataxia and loss Of ability of Purkinje neurons to fire action potentials Zhou et al., J. Cell Biol. 143: 1295-1304 ( 1998)
  • 7. Ankyrin-G (-/-) Purkinje neuron initial segments: coordinate loss of Nav1.6, beta-4 spectrin, and neurofascin +/+ -/- -/- Jenkins and Bennett, JCB 155: 739-46 (2001)
  • 8. Josh Huang: Ango et al. Cell 119:257 (2004) An ankyrin-G-based Gradient of Neurofascin directs Interneuron synapses At Purkinje neuron Axon initial segments
  • 9. KCNQ 2/3 require ankyrin-G to co-localize with Na channels at axon initial segments Ed Cooper: Pan et al. (2006) J. Neurosci.26:2599 Wildtype AnkG null
  • 10. Christian Shultz: Sobotznik et al. PNAS 106:17564 (2009) Ankyrin-G-null axon initial segments become dendrites
  • 11. Loss of fasciculated microtubules in ankG-null AIS: cytoplasmic role for 480/270 kDa ankG inserted residues
  • 12. ankyrin-G/beta-4 spectrin as a common element in establishing axon initial segment synapses with interneurons Howard et al., (2005) Trends Neuroscience 28:310
  • 13. Chandelier interneuron synapses at axon initial Segments: final stage of development Howard et al., (2005) Trends Neuroscience 28:310
  • 14. Selective alterations in postsynaptic markers of chandelier cell inputs to cortical pyramidal neurons in subjects with schizophrenia. Cruz et al. (2009) Neuropsychopharmacology 34:2112-24. Linkage, association, and gene-expression analyses identify CNTNAP2 as an autism-susceptibility gene. Alarcón et al. (2008) Am J Hum Genet. 82:150-9. Two variants in Ankyrin 3 (ANK3) are independent genetic risk factors for bipolar disorder. Schulze et al. (2008) Mol Psychiatry 14:487-91. Collaborative genome-wide association analysis supports a role for ANK3 and CACNA1C in bipolar disorder.Ferreira et al. (2008) Nat Genet. 40:1056-8. Axon initial segments: Center stage as substrates for Psychiatric disease
  • 15. L1 CAMs: -QFNEDGSFIGQY- - nematode/fly NaV: -VPIAVGESDFE- fruit fly zebrafish KCNQ2: -PYIAEGESDTDSD- dog fish zebrafish KCNQ3: -RYLAEGETDTDTD- dog fish zebrafish ? How difficult is it to evolve ankyrin-binding activity Convergent evolution of ankyrin-binding motifs in components of axon initial segments Family motif absent present
  • 16. Ankyrin-G and Nav1.5 in cardiomyocytes: co-localize at intercalated discs and T-tubules and co-immunoprecipitate Mohler et al. PNAS 101:17533-8 (2004)
  • 17. E1053K mutation of Nav1.5: cardiac arrhythmia (Brugada syndrome); loss of both ankyrin-binding and delivery of Nav1.5 to the cardiomyocyte cell surface Mohler et al., PNAS 101:17533-8 (2004) Imperm. Perm
  • 18. Ankyrin-G co-localizes with E-cadherin in early mouse embryos and is required for compaction Kizhatil et al. J. Biol. Chem. 282:26552 (2007)
  • 19. Ankyrin-G binds to the cytoplasmic domain of E-cadherin and Is required for E-cadherin to accumulate at the cell surface E-cadherin localizes in An intracellular compartment In ankyrin-G-depleted Bronchial epithelial cells
  • 20. Knockdown of ankyrin-G blocks biogenesis of the lateral membrane in telophase (same result with beta2-spectrin)
  • 21. (Ervasti JM. JBC 268(16); 2003) The dystrophin-glycoprotein complex transmits contractile force to the extracellular matrix at costameres DGC is missing from the Sarcolemma in Duchenne muscular dystrophy
  • 22. Ankyrin-B knockout mice: Congenital myopathy and loss of sarcolemmal dystrophin and dystroglycan Tuvia et al. (1999) J. Cell Biol. 147:995 Ayalon et al. (2008) Cell 135:1189
  • 23. Knockdown of ankyrin-B: Intracellular retention of beta-dystroglycan in the juxta-TGN Adult muscle ankB siRNA
  • 24. Control ankG siRNA Ankyrin-G is required to retain the DGC at costameres Gai Ayalon
  • 25. Ankyrins and the DGC: Dp71 dystrophin binds to ankyrin-B and Ankyrin-G; beta-dystroglycan cyt. domain binds to ankyrin-G Dp71 forms a Ternary complex With ankyrins and Beta-dystroglycan
  • 26. Mutation of DP71 causing Becker’s muscular dystrophy reduces binding to ankyrin and localization at costameres
  • 27. Ankyrin-G and dystrophin Dp71 bind to distinct sites on dystroglycan and can form a ternary complex
  • 28. A-B A-G Dystrophin (Dys) Beta-Dystroglycan (DG) ?Transport from TGN to costameres TGN A-B DG Dys
  • 29. Phototransduction requires segregation of the cyclic nucleotide-gated channel to outer segments of photoreceptors Burns and Arshavsky, Neuron 48:387 (2005) spectrin in the inner segment: Madreperla et al., JCB 1989 ? Are ankyrins involved in photoreceptor polarity CNG1
  • 30. Ankyrin-G segregates to the outer segments of rod photoreceptors and ankyrin-B to the inner segments Krish Kizhatil: Science 323:1614 (2009)
  • 31. shRNA-knockdown of ankyrin-G in the neonatal mouse retina eliminates CNG channel subunits in rod outer segments but not rhodopsin Injected-P0; Sacrificed-P 21
  • 32. CNGB1 channel has a C-terminal ankyrin-G-binding site that is eliminated by a retinitus pigmentosa mutation Co-IP from bovine photoreceptor outer segment extracts 1= full length Nf 2= Nf-CNG N-term 3= Nf-CNG C-term 4= Nf-CNG C-term RP
  • 33. Ankyrin-G-binding is both necessary and sufficient to Target human CNG-beta1 to rod outer segments in transgenic tadples
  • 34. Independently evolved ankyrin-binding sites share an unstructured conformation and include tyrosine and dileucine “motifs” RhBG KLP FLD SPP AE1 PAVLTRSGDPS KCNQ2 PYIAEGESDTDSD NaV VPIAG E SDFE Kir6.2 VPIVAE E D E-cadherin KEPLLPPEDDTRDNVYYYDEEGGGEED NF/L1CAMs QFNEDGSFIGQ Y Dystroglycan GVP IIF ADELDDSK CNG beta PEPGEQ IL SVKMPE Hypothesis: ankyrin-G is a general adaptor that reads diverse natively unstructured motifs in polarized cells ANK repeat solenoid: ? Extended groove binds peptides
  • 35. A conserved ankyrin-based mechanism for formation and maintenance of diverse specialized membrane domains that have recently evolved in vertebrates: -Ankyrins function as adaptors for functionally related membrane proteins through easily accessed recognition code Future Questions: What specifies ankyrin localization? What are the effectors that translate ankyrin-binding into cellular localization and domain assembly? Role of ankyrins in cellular organization of signaling pathways Ex TGF-beta and EGF receptors
  • 36. Knockdown of ankyrin-B eliminates a subset of Microtubules associated with costameres Control -Ank-B Green= MT Red= Ank-B
  • 37. Dyn4 Ankyrin-B binds to dyn4/p62 of the dynactin complex and is required for association of dyn4/p62 with costameres: Possible mechanism for costamere localization of microtubules
  • 38. Ankyrin-B-binding activity of Dynactin-4 is required for costamere localization of microtubules and the DGC Dynactin-4/HA  -Tubulin Dyn4 siRNA+ HA-Dyn4 Dyn4 siRNA Dyn4 siRNA+ HA-Dyn4 N331A  -DG Dystrophin Ankyrin-B Dyn-4 N331A= loss of ankyrin-B binding mutation
  • 39. Acknowledgements
    • Jonathan Davis
    • Lydia Davis
    • Jan Hoffman
    • Jane Healy
    • Khadar Abdi
    • Kent Nilsson
    • Yiding Wang
    • Gai Ayalon
    Ed Otto Katya Kordeli Steve Lambert Peter Michaely Daixing Zhou Shmuel Tuvia Mona Bihusi Lihsia Chen Scott Jenkins Tony Gramolini Woohyun Yoon Peter Mohler Krish Kizhatil Piotr Marszelak Josh Huang Silvia Priori Mark Keating Ed Cooper Steve Scherer Christian Schultz Vadim Arshavsky& Sheila Baker Former lab members Laboratories