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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.

Intraflagellar transport (IFT) in C. elegans chemosensory cilia is an example of functional coordination and cooperation of two motor proteins with distinct motility properties operating together in large groups to transport cargoes: a fast and processive homodimeric kinesin-2, OSM-3, and a slow and less processive heterotrimeric kinesin-2, kinesin-II. To study the mechanism of the collective dynamics of kinesin-II of C. elegans cilia in an in vitro system, we used Total Internal Reflection Fluorescence microscopy to image the motility of truncated, heterodimeric kinesin-II constructs at high motor densities. Using an analysis technique based on correlation of the fluorescence intensities, we extracted quantitative motor parameters, such as motor density, velocity and average run length, from the image. Our experiments and analyses show that kinesin-II motility parameters are far less affected by (self) crowding than OSM-3. Our observations are supported by numerical calculations based on the TASEP-LK model (Totally Asymmetric Simple Exclusion Process-Langmuir Kinetics). From a comparison of data and modelling of OSM-3 and kinesin-II, a general picture emerges of the collective dynamics of the kinesin motors driving IFT in C. elegans chemosensory cilia and the way the motors deal with crowding.

Molecular motor proteins fulfill the critical function of transporting organelles and other building blocks along the biopolymer network of the cell’s cytoskeleton, but crowding effects are believed to crucially affect this motor-driven transport due to motor interactions. Physical transport models, like the paradigmatic, totally asymmetric simple exclusion process (TASEP), have been used to predict these crowding effects based on simple exclusion interactions, but verifying them in experiments remains challenging. Here, we introduce a correlation imaging technique to precisely measure the motor density, velocity, and run length along filaments under crowding conditions, enabling us to elucidate the physical nature of crowding and test TASEP model predictions. Using the kinesin motor proteins kinesin-1 and OSM-3, we identify crowding effects in qualitative agreement with TASEP predictions, and we achieve excellent quantitative agreement by extending the model with motor-specific interaction ranges and crowding-dependent detachment probabilities. These results confirm the applicability of basic nonequilibrium models to the intracellular transport and highlight motor-specific strategies to deal with crowding.

Neuronal cells are highly crowded with various types of vesicles, organelles, proteins and cytoskeletal filaments densely packed inside the cytoplasm. One would expect crowding to pose challenges to motor-protein mediated transport, but in spite of the very crowded interior of the cell, motors appear to be able to deliver their cargo efficiently, overcoming traffic jams and roadblocks. The motors could be a possible source for crowding by creating traffic jams, limiting the number of motor-binding sites thus blocking the forward motion of motors. We use in vitro motility assays to take Total Internal Reflection Microscopy sequences of images of motors moving along microtubules. Here, we have developed an advanced analysis technique to obtain quantitative motor parameters from the image sequences in an automated way, using correlation of the fluorescence intensities. This new analysis method speeds up analysis and requires less data than the single-particle tracking approach. Correlating intensities allows analysis of data obtained under conditions inaccessible for single-particle tracking, including those performed at high motor densities that can provide insight into traffic-jam like interactions between motors. Apart from Kinesin-1 motors, this advanced analysis technique is used to understand the behavior of two kinesin-2 family motors in a crowded environment. These two kinesin-2 family motors, heterotrimeric kinesin-II and homodimeric OSM-3, cooperate to drive intraflagellar transport (IFT) in the sensory cilia of C. elegans. Our results based on the new analysis approach provide novel insights into the way kinesin motor proteins handle traffic-jam situations

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.