Aug 25, 2023

Public workspaceLiposome tubulation

DOI
dx.doi.org/10.17504/protocols.io.e6nvwkx2dvmk/v1
  • 11. Departments of Neuroscience and of Cell Biology, Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 22. Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
xinbo.wang
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DOI: dx.doi.org/10.17504/protocols.io.e6nvwkx2dvmk/v1
Protocol Citation: Xinbo Wang, Pietro De Camilli 2023. Liposome tubulation. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwkx2dvmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 19, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 68886
Keywords: Liposome tubulation, LRRK2, Electron microscopy, ASAPCRN
Abstract
This protocol details methods for the LRRK2-induced liposome tubulation experiment and its analysis by confocal fluorescence microscopy and negative stained electron microscopy.
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Confocal fluorescence microscopy analysis
Confocal fluorescence microscopy analysis
30m
30m
Prepare the samples in a PCR tube with Concentration300 nanomolar (nM) LRKK2 proteins (WT or mutant full length LRRK2 or RCKW), Concentration20 micromolar (µM) liposomes with or without Concentration1 millimolar (mM) GMPPNP (or other guanylnucleotides).
Note
Note: Liposome tubulation is sensitive to LRRK2 concentration. Too much protein results in more liposome aggregates.

Pipetting
Immediately deposit Amount6 µL -Amount10 µL samples of step 1 on a Thikness35 mm glass bottom dish and incubate at Temperature37 °C for Duration00:30:00 .
Note
Note: Drop some buffer in the dish to prevent samples from drying out due to evaporation during incubation.

30m
Incubation
After incubation, capture images with a Spinning disk confocal (SDC) microscopy at TemperatureRoom temperature on a Nikon Ti-E inverted microscope using the Improvision UltraVIEW VoX system (Perkin-Elmer).
Note
Note: Movies were collected from time Duration00:00:00 .

Imaging
Negative stained electron microscopy (EM) analysis
Negative stained electron microscopy (EM) analysis
31m 25s
31m 25s
Glow-discharge carbon-coated grids (25 mA, Duration00:00:45 ).
45s
Place the discharged grids into a Thikness35 mm glass bottom dish.
Prepare samples in a PCR tube with Concentration300 nanomolar (nM) LRKK2, Concentration80 micromolar (µM) liposomes and Concentration1 millimolar (mM) GMPPNP.
Pipetting
PCR
Immediately apply Amount6 µL of the mixture to the grid and incubate the mixture at Temperature37 °C for Duration00:30:00 .
Note
Note: Drop some buffer in the dish to prevent samples from drying out due to evaporation during incubation.

30m
Incubation
Pipetting
Blot the grid with filter paper after incubation and stain samples with 2% uranyl acetate for Duration00:00:40 .
40s
Dry the grid with filter paper.
Take images using a Talos L 120C TEM microscope at 80 kV with Velox software and a 4k × 4K Ceta CMOS Camera (Thermo Fisher Scientific).
Imaging