Sep 12, 2024
LRRK2RCKW single molecule kinesin motility assays_2024
- 1Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093;
- 2UCSD
Protocol Citation: John Salogiannis, Katherine Surridge 2024. LRRK2RCKW single molecule kinesin motility assays_2024. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvok4dzl4o/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: September 12, 2024
Last Modified: September 12, 2024
Protocol Integer ID: 107540
Keywords: LRRK2, motility assay, kinesin, imaging, single-molecule, ASAPCRN
Funders Acknowledgement:
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
As used in:
CITATION
Image Recommendation:
Settings will vary per microscope. We imaged K560-GFP every 500 msecs for 2 mins with 25% laser (488) power at 150 ms exposure time. Each sample was imaged no longer than 15 mins. Each technical replicate recommended to consist of movies from at least two fields of view containing between 5 and 10 microtubules each.
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 run lengths, percent motility, and velocity 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 (> 1 sec) were manually traced. Bright aggregates, which were less than 5% of the population, were excluded from the analysis. Run length measurements were calculated from motile events only. For percent motility per microtubule measurements, motile events (> 1 sec and > 1µm) were divided by total events per kymograph. Velocity measurements were calculated from the inverse slopes of the motile event traces (> 1 sec and > 1µm) only. 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
- 150 millimolar (mM) NaCl
- 1 millimolar (mM) TCEP
- 5 % Glycerol
- 2.5 millimolar (mM) MgCl2
- 20 micromolar (µM) GDP
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:
Create microscope slides:
1h 11m
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:
Prepare LRRK2:
1h 11m
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:
Next steps:
5m
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:
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 µg/mL 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:
Next steps:
Add 18 µL kinesin mixture to each chamber.
Image slide.