Defining the α-synuclein interactome

Click here to load reader

  • date post

    13-Apr-2017
  • Category

    Documents

  • view

    346
  • download

    0

Embed Size (px)

Transcript of Defining the α-synuclein interactome

  • Defining the -synuclein interactome In situ

    Xiuqi Cao

    Undergraduate Student at Yale University B.S. in Molecular, Cellular & Developmental Biology, 2017

    Mentor: Karina Vargas

    PI: Sreeganga S. Chandra

  • Clayton DF, George JM.

    J Neurosci Res. 1999

    Background

    Localized in presynaptic terminal and implicated in many parts of synaptic vesicle cycle

    The function of -synuclein

    is poorly understood because of its conformational plasticity

    Previous -synuclein overexpression experiments may have lead to pathological rather than physiological response

    Chandra S. (2009) Encyclopedia of Neuroscience

    Unfolded -Synuclein Oligomeric -Synuclein

    Helical Conformation -Synuclein Fibrils

  • Using abg-Synuclein Triple-Knockout (TKO) Mice to Pinpoint Synuclein Function

    Greten-Harrison et al. (2010) PNAS 107:19573-78

  • vGlut-pHluorin Imaging To Monitor Synaptic Vesicle Cycling

    Karina Vargas

  • TKO Leads to Slowed Endocytosis that can be Rescued via Synuclein Expression

    Karina J. Vargas et al. J. Neurosci. 2014;34:9364-9376

  • Synucleins Implicated in Clathrin Mediated Endocytosis

    Karina J. Vargas et al. J. Neurosci. 2014;34:9364-9376

  • -Synuclein Is Recruited to Clathrin Coated Pits Prior to Dynamin

    0 min

    15 min

    23 min

    30 min

  • Proposed Model of Synuclein Function

    Karina J. Vargas et al. J. Neurosci. 2014;34:9364-9376

    Dynamin

  • Synucleins Act Early On In SV Endocytosis

    Karina J. Vargas et al. J. Neurosci. 2014;34:9364-9376

  • Proximal Protein Labeling

    Purpose: Define the normal function of -synuclein through determining its protein interactome

  • Proximity-Dependent Biotin Identification via BirA* Fusion

    Kyle J. Roux et al. J Cell Biol 2012;196:801-810

  • Synuclein-BirA* has Limited Biotinylation in Differentiated SH-SY5Y Cells

    -synuclein

    biotin

    55 kDa

    55 kDa

    55 kDa myc

    Wang et al, Curr. Biol 2014

    Burre et al, J Neurosci 2015

  • Proximity-Dependent Biotin Identification via APEX2 Fusion

    Stephanie S. Lam et al. Nature Methods 2015;12:51-54

    APEX2 biotinylates stronger and faster in 1 minute than BirA* in 24 hours

  • Ongoing Studies

    We will be reconstituting the APEX2-synuclein fusion protein with isolated PTK2 membrane and whole brain cytosol to attempt biotinylation of endocytic proteins.

  • Synuclein: a protein-chameleon

  • Acknowledgements

    PI: Dr. Sreeganga Chandra Dr. Karina Vargas Dr. Yong-Quan Zhang Aurelie Nardin Yumi Taguchi Greg Wirak Erica Gorenberg Wilhemina Koomson Vicky Chou Justin Abbasi Christina Chen Funding: NINDS (R01NS083846; R01NS064963)