Roles of motor on-rate and cargo mobility in intracellular transport

Abstract

Molecular motors like kinesin are critical for cellular organization and biological function including in neurons. There is detailed understanding of how they move and how factors such as applied force and the presence of microtubule-associated proteins can alter this single-motor travel. In order to walk, the cargo-motor complex must first attach to a microtubule. This attachment process is less studied. Here, we use a combination of single-molecule bead experiments, modeling, and simulation to examine how cargos with kinesin-1 bind to microtubules. In experiment, we find that increasing cargo size and environment viscosity both significantly slow cargo binding time. We use modeling and simulation to examine how the single motor on rate translates to the on rate of the cargo. Combining experiment and modeling allows us to estimate the single motor on rate as 100 s-1. This is a much higher value than previous estimates. We attribute the difference between our measurements and previous estimates to two factors: first, we are directly measuring initial motor attachment (as opposed to re-binding of a second motor) and second, the theoretical framework allows us to account for missed events (i.e. binding events not detected by the experiments due to their short duration). This indicates that the mobility of the cargo itself, determined by its size and interaction with the cytoplasmic environment, play a previously underestimated role in determining intracellular transport kinetics.

Diffusion of kinesin motors on cargo can enhance binding and run lengths during intracellular transport

“…It is clear that in some situations kinesin binds quickly ( Bovyn et al. , 2020 ), while in others it binds slowly ( Jiang et al.…”

“…We reiterate another subtlety, which is that there are distinct biophysical notions of attachment ( Bovyn et al. , 2020 ): The time for a cargo, on first approach to a MT (e.g., from the organelle that generated it), the time to rebind after pulling out of a trap, the concentration-dependent local attachment rates of the motor head to the MT lattice, and the local attachment rate of a second motor to the MT on a multimotor cargo.…”

“…In Bovyn et al. (2020) , we measure the time it takes for beads with single kinesin motors to bind to the MT after detaching in an optical trap.…”

“…One key step that has been less studied so far is initiation of transport, specifically, how long it takes to attach a cargo to the MT in the first place. In vitro, we recently reported that the time for a cargo to reattach to the MT after detaching in an optical trap is on the order of 1 s at viscosities 1–10× water ( Bovyn et al. , 2020 ) and depends strongly on the size of the cargo, indicating that cargo rotation plays an significant role.…”

“…For cytoplasmic viscosity estimates ( Figure 1A , gray line), for a cargo of 500–1000 nm in diameter, we estimate the time to be 10 2 –10 3 s. (For details on the calculation see Materials and Methods . Note that, because the times for motor repositioning are so slow, the choice of local motor attachment rate value k on ≥ 5 s − 1 ( Bovyn et al. , 2020 ) does not significantly affect total cargo attachment time.)…”