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Biophysical studies of the mechanochemical cycle of kinesin motors are essential for understanding the mechanism of energy conversion. Here, we report a systematic study of the impact of temperature on velocity and run length of homodimeric Drosophila kinesin-1, homodimeric C. elegans OSM-3 and heterodimeric C. elegans kinesin-II motor proteins using in vitro single-molecule motility assays. Under saturated ATP conditions, kinesin-1 and OSM-3 are fast and processive motors compared to kinesin-II. From in vitro motility assays employing single-molecule fluorescence microscopy, we extracted single-motor velocities and run lengths in a temperature range from 15 °C to 35 °C. Both parameters showed a non-Arrhenius temperature dependence for all three motors, which could be quantitatively modeled using a simplified, two-state kinetic model of the mechanochemistry of the three motors, providing new insights in the temperature dependence of their mechanochemistry.

Intracellular transport takes place in the crowded environment of the cell, densely packed with proteins, cytoskeletal filaments, vesicles and organelles. In most transport systems, multiple motors team up to transport a single cargo, resulting in collective motor behavior that cannot be explained only from single-motor properties. Intraflagellar transport (IFT) in C. elegans cilia is an example of functional coordination and cooperation of two motor proteins with distinct motility properties operating together in groups to transport a single cargo: homodimeric OSM-3 (fast and processive) and heterotrimeric Kinesin-II (slow and less processive). To study the mechanism of the collective dynamics of OSM-3 and kinesin-II in cilia, we use fluorescence microscopy and high-density in-vitro motility assays. Using an advanced analysis technique based on correlation of the fluorescence intensities, we extracted the quantitative motor parameters (such as motor density, velocity and average run length) from the image sequences in an automated way with a high level of precision. Our experiments and analyses show that OSM-3 and Kinesin-II motors are affected differently by the presence of the other motor. For example, OSM-3 velocity and run length are substantially reduced in the presence of the Kinesin-II. Numerical calculations based on TASEP-LK model (Totally Asymmetric Simple Exclusion Process-Langmuir Kinetics) underline the importance of intermolecular interactions, such as the change of the OSM-3 affinity to the microtubule dependent on the Kinesin-II density. Based on this approach, a general picture of collective dynamics of OSM-3 and Kinesin-II motor proteins in IFT is formulated.