Jun 06, 2025
Version 2
LRRK2RCKW single molecule kinesin motility assays_2024 V.2
Version 1 is forked from LRRK2RCKW single molecule kinesin motility assays
- John Salogiannis1,
- Katherine Surridge2
- 1Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093;
- 2UCSD
External link: https://doi.org/10.1126/sciadv.adt2050
Protocol Citation: John Salogiannis, Katherine Surridge 2025. LRRK2RCKW single molecule kinesin motility assays_2024. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvok4dzl4o/v2Version created by Katherine Surridge
Manuscript citation:
Raig ND, Surridge KJ, Sanz-Murillo M, Dederer V, Krämer A, Schwalm MP, Lattal NM, Elson L, Chatterjee D, Mathea S, Hanke T, Leschziner AE, Reck-Peterson SL, Knapp S Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2. Science Advances 11(23). doi: 10.1126/sciadv.adt2050
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: December 10, 2024
Last Modified: June 06, 2025
Protocol Integer ID: 114635
Keywords: LRRK2, motility assay, kinesin, imaging, single-molecule, ASAPCRN, lrrk2rckw single molecule kinesin motility assay, single molecule kinesin motility assays-2024 this protocol, single molecule kinesin motility assays-2024, single molecule kinesin motility assay, lrrk2rckw, assay
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000519
Abstract
This protocol is about LRRK2RCKW single molecule kinesin motility assays. Forked from dx.doi.org/10.17504/protocols.io.ewov14qykvr2/v1
Attachments
Guidelines
Image Recommendation:
Settings will vary per microscope. We imaged K560-GFP every 500 msecs for 2 mins with 20% laser (488) power at 150 ms exposure time.
Image using the 405 nm laser to determine the locations of the microtubules. Preferably at the start and end of the experiment.
Single-molecule motility assay analysis recommendation:
Kymographs were generated from motility movies and quantified for percent motility, using ImageJ (NIH). Specifically, maximum-intensity projections were generated from time-lapse sequences to define the trajectory of particles on a single microtubule. The segmented line tool was used to trace the trajectories and map them onto the original video sequence, which was subsequently re-sliced to generate a kymograph. Motile and immotile events were manually traced. For percent motility per microtubule measurements, motile events (> 1 sec and > 1µm) were divided by total events per kymograph. Statistical analyses were performed in Prism8 (Graphpad).
Materials
Recommended Equipment and Setup:
This single-molecule imaging experiment was originally performed using total internal reflection fluorescence (TIRF) microscopy with an inverted microscope (Nikon, Ti-E Eclipse) equipped with a 100x 1.49 N.A. oil immersion objective (Nikon, Plano Apo), and a MLC400B laser launch (Agilent), with 405 nm, 488 nm, 561 nm and 640 nm laser lines (561 and 640 nm laser lines are not needed for this version of the experiment). Excitation and emission paths were filtered using single bandpass filter cubes (Chroma), and emitted signals were detected with an electron multiplying CCD camera (Andor Technology, iXon Ultra 888). Illumination and image acquisition were controlled with NIS Elements Advanced Research software (Nikon), and the xy position of the stage was controlled with a ProScan linear motor stage controller (Prior).
Required Buffers:
Streptavidin/Biotin Buffer:
- 1 mg/mL Streptavidin
- 30 millimolar (mM) HEPES pH 7.4
- 2 millimolar (mM) MgOAc
- 1 millimolar (mM) EGTA
- 10 % Glycerol
Motility Assay Buffer:
- 30 millimolar (mM) HEPES pH 7.4
- 50 millimolar (mM) KOAc
- 2 millimolar (mM) MgOAc
- 1 millimolar (mM) EGTA
- 10 % Glycerol
- 1 millimolar (mM) DTT
- 20 micromolar (µM) Taxol
Motility Assay Buffer with casein:
- 30 millimolar (mM) HEPES pH 7.4
- 50 millimolar (mM) KOAc
- 2 millimolar (mM) MgOAc
- 1 millimolar (mM) EGTA
- 10 % Glycerol
- 1 millimolar (mM) DTT
- 20 micromolar (µM) Taxol
- 1 mg/mL casein
LRRK2 Buffer:
- 20 millimolar (mM) HEPES pH 7.4
- 80 millimolar (mM) NaCl
- 0.5 millimolar (mM) TCEP
- 5 % Glycerol
- 2.5 millimolar (mM) MgCl2
- 20 micromolar (µM) GDP
Troubleshooting
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Before start
Please take notice of the buffer preparation in section 'Materials'.
Create microscope slides:
1h 11m
Adhere coverslips (Corning) to a microscope slide using double-sided scotch tape, creating 4 channels per slide.
Add 1 mg/mL biotin-BSA (Sigma) to each channel and incubate for 00:03:00
3m
Wash twice with Motility Assay buffer.
Add 0.5 mg/mL Strepdavidin to each channel and incubate for 00:03:00 .
3m
Wash twice with Motility Assay buffer.
Add a 1:100 dilution of taxol-stabilized microtubules (18 µL per channel) and incubate for 00:03:00 .
See https://dx.doi.org/10.17504/protocols.io.bp2l6bdedgqe/v1 for making taxol-stabilized microtubules.
3m
Wash twice with 18 µL LRRK2 buffer. Add more buffer if necessary to prevent drying out throughout experiment.
Prepare LRRK2:
1h 11m
Prepare a 1 micromolar (µM) solution of LRRK2RCKW in a cold LRRK2 buffer. Centrifuge through a 0.1 μm PVDF filter to remove aggregates. Calculate the new effective concentration. Usually around 500 nanomolar (nM) -700 nanomolar (nM) after centrifugation.
Create a working aliquot of LRRK2 in the desired concentration (ex. 25 nanomolar (nM) -100 nanomolar (nM) ) in the LRRK2 buffer at Room temperature (recommended volume of 25 µL ). If adding inhibitors, add them now with DMSO. Incubate for 00:10:00 at Room temperature .
10m
Next steps:
5m
Add LRRK2RCKW sample to the channel (25 µL ). Incubate for 00:05:00 . Prepare next step while waiting.
5m
Wash twice with the motility assay buffer supplemented with 1 mg/mL casein .
Prepare kinesin:
Make a 1.9 nanomolar (nM) solution of K560-GFP in the Motility Assay buffer with casein supplemented with an oxygen scavenger system (0.4 % glucose , 45 Mass Percent glucose catalase (Sigma-Aldrich), and 1.15 mg/mL glucose oxidase (Sigma-Aldrich)), 71.5 millimolar (mM) beta-mercaptoethanol and 1 millimolar (mM) ATP .
Next steps:
Add 18 µL kinesin mixture to each chamber.
Image slide.