Alternate header for print version


Reconstruction
Display image description
Triple tilt electron tomographic reconstruction of LRRK2 filament bundles in HEK293T cells transfected with YFP-LRRK2-IT. Use the WIB contrast and brightness adjustments to improve viewing.
Full resolution image description
Volume reconstruction in MRC format
File format
MRC
Volume_dimension
1504, 2644, 220

Image 2D
Display image description
Semi-aligned tilt series (one of three) of LRRK2 filament bundles in 250 nm sections of HEK293T cells transfected with YFP-LRRK2-IT. Use the WIB contrast and brightness adjustments to improve viewing. Section was coated with 10 nm gold particles on both sides to serve as fiducial cues for alignment.
Full resolution image description
Tar file containing 3 aligned tilt series.

License
Attribution Only: This image is licensed under a Creative Commons Attribution License. View License Deed | View Legal Code

CCDB:86716*  Cite 
Project: P20090
Project name
Multiple Parkinson disease mutations cause LRRK2 to interact closely with microtubules.
Description
Dominant missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson disease (PD) and genome wide association studies identify LRRK2 sequence variants as risk factors for sporadic PD. Intact kinase function appears critical for the toxicity of LRRK2 PD mutants, yet our understanding of how LRRK2 causes neurodegeneration is limited. We will examine how LRRK2 mutations affect the ultrastructure of cells using electron tomography.
Funding agency
NIH, Branfman Family Foundation, and Parkinson Disease Foundation.
Leader(s)
Boassa
Daniela
Collaborator(s)
Junru Hu
Tomas Molina
William Dauer
Masako Terada
Mark Ellisman
Start date
06-01-2010
End date
unspecified
 
Experiment
Experiment ID
40706
Experiment date
06-17-2012
Title
Ultrastructural analysis of LRRK2-microtubule filaments.
Purpose
Study LRRK2-microtubule association by high resolution electron tomography.
Experimenter(s)
Boassa
Daniela
Microscopy product
Microscopy product ID
86716
Instrument
JEOL 4000EX
Microscopy type
IVEM
Product type
MULTIPLE TILT
Image basename
Dauertomo1
Spatial Axis Image Size Pixel Size
X 4000px 0.75 nm/pixels
Y 4000px 0.75 nm/pixels
Subject
Species
Human
Scientific name
Homo sapiens
Strain
sapiens
Group by
Transfection with YFP-LRKK2-IT
Treatment
Transfected using Lipofectamine 2000 (Invitrogen)
Age class
embryonic
Tissue section
Thickness
0.25 µm
Specimen description
Organ
kidney
Structure
microtubule bundle
Cell type
HEK293T
Imaging parameters
Type
Electron microscopy product
Recording medium
4K CCD
Magification
0
Accelerating voltage
400 KeV
Specimen preparation
Protocol used
HEK293T cells transfected with YFP-LRRK2-IT were fixed for 30 min in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) on ice. After washes in 0.1 M cacodylate buffer, cells were post-fixed with 1% osmium tetraoxide for 30 min, stained with 2% uranyl acetate and then dehydrated and embedded in Durcupan epoxy resin. Sections were cut at a thickness of 250 nm. Post-staining of grids was done with 2% uranyl acetate for 5 min and lead citrate for 1 min. Colloidal gold particles (10 nm diameter) were deposited on opposite sides of the thick epoxy sections to serve as fiducial markers.
Imaging product type
Type
Single tilt
Description
singlet_desc
Min range
-60 degrees
Max range
60 degrees
Tilt increment
2 degrees
Notes
Three tilt series were acquired. Current CCDB schema does not support more than 2 tilts.