Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins

Abstract

The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. Kinesins display differences across the superfamily both in ATP turnover and in microtubule interaction. These differences tailor specific kinesins to various functions such as cargo transport, microtubule sliding, microtubule depolymerization and microtubule stabilization. To understand the mechanism of action of a kinesin it is important to understand how the chemical cycle of ATP turnover is coupled to the mechanical cycle of microtubule interaction. To dissect the ATP turnover cycle, one approach is to utilize fluorescently labeled nucleotides to visualize individual steps in the cycle. Determining the kinetics of each nucleotide transition in the ATP turnover cycle allows the rate-limiting step or steps for the complete cycle to be identified. For a kinesin, it is important to know the rate-limiting step, in the absence of microtubules, as this step is generally accelerated several thousand fold when the kinesin interacts with microtubules. The cycle in the absence of microtubules is then compared to that in the presence of microtubules to fully understand a kinesin's ATP turnover cycle. The kinetics of individual nucleotide transitions are generally too fast to observe by manually mixing reactants, particularly in the presence of microtubules. A rapid mixing device, such as a stopped-flow fluorimeter, which allows kinetics to be observed on timescales of as little as a few milliseconds, can be used to monitor such transitions. Here, we describe protocols in which rapid mixing of reagents by stopped-flow is used in conjunction with fluorescently labeled nucleotides to dissect the ATP turnover cycle of a kinesin.

The family-specific α4-helix of the kinesin-13, MCAK, is critical to microtubule end recognition

“…Thus, the ability of microtubule ends to accelerate ADP dissociation should also be impaired by these mutations. To test this, we measured the kinetics of ADP dissociation by preloading the nucleotide-binding site with a fluorescent ADP analogue (mantADP) and using rapid mixing to observe the fluorescence decrease associated with its dissociation from the motor domain (figure 4 a – c ) [18,19]. The rate constant for mantADP dissociation from MCAK in solution and in the presence of unpolymerized tubulin is unchanged by the mutations (figure 4 d and table 1).…”

“…MCAK or MCAK variants were preloaded with mantADP as described in [18,19]. The kinetics of dissociation of mantADP from MCAK were measured by rapidly mixing 1 : 1 v/v, using an SX20 stopped-flow fluorimeter (Applied Photophysics) with an excess of unlabelled ATP at 25°C.…”

“…However, microtubules break when pushed through the stopped-flow, resulting in microtubules with an average length of 1.2 µm [8]. The resulting fluorescent transients were fitted using IGOR P ro (Wavemetrics, Lake Oswego, OR, USA) to a single or a double exponential function plus a line of constant negative slope to account for photobleaching of the mant group, as described in [19]. …”

A Cdk1 phosphomimic mutant of MCAK impairs microtubule end recognition

“…The dissociation of mantADP from MCAK was measured as described previously ( Patel et al, 2014 ), in solution or with the addition of 10 µM tubulin or 5.7 µM double cycled microtubules (chosen to have a comparable number of microtubule ends as the ATPase assay with microtubules). The fluorescence traces were fitted to a single exponential, or double exponential if required, with an additional linear function to account for the photobleaching of mant.…”

Peer Review #2 of "A Cdk1 phosphomimic mutant of MCAK impairs microtubule end recognition (v0.1)"

“…88 ADP dissociation assays 89 The dissociation of mantADP from MCAK was measured as described previously (Patel et al 2014), in 90 solution or with the addition of 10 µM tubulin or 5.7 µM double cycled microtubules (chosen to have a 91 comparable number of microtubule ends as the ATPase assay with microtubules). The fluorescence 92 traces were fitted to a single exponential, or double exponential if required, with an additional linear 93 function to account for the photobleaching of mant.…”