Dataset Information

Single-molecule motility: statistical analysis and the effects of track length on quantification of processive motion.

ABSTRACT: In vitro, single-molecule motility assays allow for the direct characterization of molecular motor properties including stepping velocity and characteristic run length. Although application of these techniques in vivo is feasible, the challenges involved in sample preparation, as well as the added complexity of the cell and its systems, result in a reduced ability to collect large datasets, as well as difficulty in simultaneous observation of the components of the motility system, namely motor and track. To address these challenges, we have developed simulations to characterize motility datasets as a function of sample size, processive run length of the motor, and distribution of track lengths. We introduce the use of a simple bootstrapping technique that allows for the quantification of measurement uncertainty and a Monte Carlo permutation resampling scheme for the measurement of statistical significance and the estimation of required sample size. In addition, we have found that, despite conventional wisdom, the measured characteristic run length is directly coupled to the characteristic track length that describes the microtubule length distribution. To be able to make comparisons between motility experiments performed on different track populations as well as make measurements of motility when motors and tracks cannot be simultaneously resolved, we have developed a theoretical framework for the determination of the effect that track length has on observed characteristic run lengths. This shows good agreement with in vitro motility experiments on two kinesin constructs walking on microtubule populations of different characteristic track lengths.

SUBMITTER: Thompson AR

PROVIDER: S-EPMC3686350 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Publications

Single-molecule motility: statistical analysis and the effects of track length on quantification of processive motion.

Thompson Andrew R AR Hoeprich Gregory J GJ Berger Christopher L CL

Biophysical journal 20130601 12

In vitro, single-molecule motility assays allow for the direct characterization of molecular motor properties including stepping velocity and characteristic run length. Although application of these techniques in vivo is feasible, the challenges involved in sample preparation, as well as the added complexity of the cell and its systems, result in a reduced ability to collect large datasets, as well as difficulty in simultaneous observation of the components of the motility system, namely motor a ...[more]

PMID: 23790373

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Project description:Cytoskeletal motor proteins are essential to the function of a wide range of intracellular mechano-systems. The biophysical characterization of their movement along their filamentous tracks is therefore of large importance. Toward this end, single-molecule, in vitro stepping-motility assays are commonly used to determine motor velocity and run length. However, comparing results from such experiments has proved difficult due to influences from variations in the experimental conditions and the data analysis methods. Here, we investigate the movement of fluorescently labeled, processive, dimeric motor proteins and propose a unified algorithm to correct the measurements for finite filament length as well as photobleaching. Particular emphasis is put on estimating the statistical errors associated with the proposed evaluation method, as knowledge of these values is crucial when comparing measurements from different experiments. Testing our approach with simulated and experimental data from GFP-labeled kinesin-1 motors stepping along immobilized microtubules, we show 1) that velocity distributions should be fitted by a t location-scale probability density function rather than by a normal distribution; 2) that the impossibility to measure events shorter than the image acquisition time needs to be taken into account; 3) that the interaction time and run length of the motors can be estimated independent of the filament length distribution; and 4) that the dimeric nature of the motors needs to be considered when correcting for photobleaching. Moreover, our analysis reveals that controlling the temperature during the experiments with a precision below 1 K is of importance. We believe our method will not only improve the evaluation of experimental data, but also allow for better statistical comparisons between different populations of motor proteins (e.g., with distinct mutations or linked to different cargos) and filaments (e.g., in distinct nucleotide states or with different posttranslational modifications). Therefore, we include a detailed workflow for image processing and analysis (including MATLAB code), serving as a tutorial for the estimation of motility parameters in stepping-motility assays.

Dataset Information

Single-molecule motility: statistical analysis and the effects of track length on quantification of processive motion.

Publications

Single-molecule motility: statistical analysis and the effects of track length on quantification of processive motion.

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