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    Compl Compl Presentation Transcript

    • The protein complexin and its role in coupling secretory vesicles and Ca2+ channels
      Quintana J., Rohan J., Chow R.H.
      Department of Physiology & Biophysics and Zilkha Neurogenetic Institute,
      Keck School of Medicine, University of Southern, California, Los Angeles
    • Big Picture
      understand better how secretion is regulated
      use the acquired knowledge to create a solution against diseases which there is not enough secretion
      Example: type 2 diabetes
    • Background
    • Hormones Exocytosis
      signaling molecules
      stored in vesicle
      example: insulin
      process by which the membrane of a vesicle fuses with the cell plasma membrane, and the vesicle contents are secreted
      Vesicles
      • to be readily releasable, they have to be docked and primed
    • SNARE Complex
      It assist exocytosis
      through “zippering”
      Complexin
      more secretion when present
      cartoon
    • RRP & IRP
      • Readily Releasable Pool (RRP):primed vesicles both near and far from Ca2+ channels
      • Immediately Releasable Pool (IRP): primed vesicles near Ca2+ channels
      Calcium & Ca2+ Channels
      triggers exocytosis
      vesicles nearer to Ca2+ channels secrete more reliably
    • Question
      Does the protein complexin help vesicles to associate with calcium channels?
    • Hypothesis
      Cplx facilitates co-localization of vesicles and Ca2+ channels.
      We expect to see a change in the IRP if a cell is missing Cplx (i.e. Cplx knock out)
      Wild Type
      Knock Out
      IRP (WT)IRP (KO)
      >
      RRP IRP
      RRP IRP
      RRP RRP
    • Methods
    • Genotyping
      KO mice
      Adrenal chromaffin cells from litter mates of Cplx +/-(hemizygous) mice
      L 1 2 3 4 5 6 7 8 9 10 11
      KO Bands
      WT Bands
      HZ
      WT
      KO
    • Patch Clamp
      biophysical tool
      Capacitance (C): electrical measurement that is proportional to the area of the cell membrane
      IRP = ∑∆C short
      RRP = ∑∆C short + ∑∆C long
      Modified from Sakmann and Neher, Sci. Am. 1991
    • Depolarization
      short depolarization protocol
      long depolarization protocol
    • How to measure the IRP & RRP
      ∑∆C long
      ~RRP
      ∑∆C short
      ~IRP
      Adapted from Voets et al., Neuron 23, 607 (1999).
    • Capacitance Data
    • Conclusion
      Our preliminary data supports our hypothesis that Cplx helps co-localize vesicles and Ca2+ channels.
      Data needs to be replicated and statistically validated.
      If validated, then Cplx serves not only to help prime vesicles, but also to increase reliability of stimulus-secretion coupling.
    • Acknowledgements
      ZNI
      Dr. Robert Chow
      Chow’s Lab
      Joyce G. Rohan
      Madison Zitting
      Alan Okada
      Dr. Emil Bogenmann
      NIH
      Step Up Program
    • Question