Defining the α-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 α-synuclein

is poorly understood because of its conformational plasticity

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

Chandra S. (2009) Encyclopedia of Neuroscience

Unfolded α-Synuclein Oligomeric α-Synuclein

Helical Conformation α-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

α-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 α-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

α-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)