TITLE: LRRK2RCKW   single molecule kinesin motility assays
AUTHORS: John  Salogiannis

[DESCRIPTION]
This protocol is about LRRK2RCKW single molecule kinesin motility assays.

[BEFORE_START]
Please take notice of the buffer preparation in section 'Materials'.
Make sure that you have labeled taxol-stabilized microtubules available. See the protocol here.

[GUIDELINES]
As used in:
 

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).

[STEPS]
SECTION: Create microscope slides:
1. Adhere Biotin-PEG-functionalized coverslips (Microsurfaces) to a microscope slide using double-sided scotch tape, creating 4 channels per slide.
SECTION: Prepare LRRK2:
6. 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.
SECTION: Next steps:
8. Add LRRK2RCKW sample to the channel (19 µL). Incubate for 5 min. Prepare next step while waiting.
SECTION: Next steps:
9. Wash twice with the motility assay buffer supplemented with 1 mg/mL.
SECTION: Prepare kinesin:
10. Make a 4 nanomolar (nM) solution of K560-GFP in the Motility Assay buffer with casein supplemented with an oxygen scavenger system (0.4 %, 45 µg/mL (Sigma-Aldrich), and 1.15 mg/mL (Sigma-Aldrich)), 71.5 millimolar (mM)  and 1 millimolar (mM). 
SECTION: Next steps:
11. Add 19 µL to each chamber.

SECTION: Next steps:
12. Image slide.
SECTION: Create microscope slides:
2. Add the streptavidin buffer to each channel and incubate for 3 min.
SECTION: Create microscope slides:
3. Wash twice with Motility Assay buffer.
SECTION: Create microscope slides:
4. Add a 1:150 dilution of taxol-stabilized microtubules (19 µL per channel) and incubate for 3 min .
See https://dx.doi.org/10.17504/protocols.io.bp2l6bdedgqe/v1 for making taxol-stabilized microtubules.
SECTION: Create microscope slides:
5. Wash twice with LRRK2 buffer. Add more buffer if necessary to prevent drying out.
SECTION: Prepare LRRK2:
7. 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 10 min at Room temperature.