Kinesin Density and Landing Rate Assay

Kinesin landing rates and surface density calculations were performed using a previously established protocol by Katira et al.^{6 (link)}. Briefly, a flow cell filled with a solution of kinesin diluted in immobilized microtubule BRB80CAT was incubated for 5 min. Next, the flow cell was washed with a 4 µg/mL solution of MTs (sheared three times using a 21 G needle from Becton Dickinson, Franklin Lakes, NJ) in imaging solution with AMP-PNP. The flow cell was sealed with VALAP then immediately imaged. Images were collected every 10 seconds, and the time between MT injection and image collection was measured using a digital stopwatch. The number of landed MTs for each frame were counted using ImageJ RidgeDetection then plotted against the time elapsed after injection of MTs. The kinesin landing rate was determined by fitting the following equation: $$N(t)=N_{Max}\left[1-e^{\left(-\frac{Rt}{N_{Max}}\right)}\right]$$ where N(t) is the number of landed MTs, t is time elapsed after MT injection and R is the kinesin landing rate. Kinesin surface density was calculated using the following equation: $$\rho =-\frac{\ln \left(1-\frac{R}{Z}\right)}{A}$$
where $\rho$ is kinesin surface density, R is kinesin landing rate, Z is the diffusion limited kinesin landing rate assumed to be equal to the landing rate observed at 10-fold dilution from kinesin stock solution, and A is MT area, $A=Lw$ assuming a width w of 25 nm and average length L measured from microtubule images.