Cytoplasmic dynein and kinesin are both microtubule-based molecular motors but are structurally and evolutionarily unrelated. Under standard conditions, both move with comparable unloaded velocities toward either the microtubule minus (dynein) or plus (most kinesins) end. This similarity is important because it is often implicitly incorporated into models that examine the balance of cargo fluxes in cells and into models of the bidirectional motility of individual cargos. We examined whether this similarity is a robust feature, and specifically whether it persists across the biologically relevant temperature range. The velocity of mammalian cytoplasmic dynein, but not of mammalian kinesin-1, exhibited a break from simple Arrhenius behavior below 15°C-just above the restrictive temperature of mammalian fast axonal transport. In contrast, the velocity of yeast cytoplasmic dynein showed a break from Arrhenius behavior at a lower temperature (∼8°C). Our studies implicate cytoplasmic dynein as a more thermally tunable motor and therefore a potential thermal regulator of microtubule-based transport. Our theoretical analysis further suggests that motor velocity changes can lead to qualitative changes in individual cargo motion and hence net intracellular cargo fluxes. We propose that temperature can potentially be used as a noninvasive probe of intracellular transport.

Intracellular transport of cargos along microtubules is often complicated by the topology of the underlying filament network. The fundamental building blocks for this complex arrangement are filament intersections. The navigation of cargos across microtubule intersections remains poorly understood. Here, we demonstrate that kinesin-driven cargos are engaged in a tug-of-war at microtubule intersections. Tug-of-war events result in long pauses that can last from a few seconds to several minutes. We demonstrate that the extent of the tug-of-war and the duration of pauses change with the number of motors on the cargo and can be regulated by ionic strength. We also show that dwell times at intersections depend on the angle between crossing microtubules. Our data suggest that local microtubule geometry can regulate microtubule-based transport.

Developing abilities to assemble nanoscale structures is a major scientific and engineering challenge. We report a technique which allows precise positioning and manipulation of individual rigid filaments, enabling construction of custom-designed 3D filament networks. This approach uses holographic optical trapping (HOT) for nano-positioning and microtubules (MTs) as network building blocks. MTs are desirable engineering components due to their high aspect ratio, rigidity, and their ability to serve as substrate for directed nano-transport, reflecting their roles in the eukaryotic cytoskeleton. The 3D architecture of MT cytoskeleton is a significant component of its function, however experimental tools to study the roles of this geometric complexity in a controlled environment have been lacking. We demonstrate the broad capabilities of our system by building a self-supporting 3D MT-based nanostructure and by conducting a MT-based transport experiment on a dynamically adjustable 3D MT intersection. Our methodology not only will advance studies of cytoskeletal networks (and associated processes such as MT-based transport) but will also likely find use in engineering nanostructures and devices.

In Figure 2, the first part of the caption should read: Temperature dependence of kinesin and mammalian cytoplasmic dynein processivity and stall force. (A and B) Kinesin processivity at 5 C (A) is 497.7 5 0.09 nm/s, which is significantly lower than the 841.5 5 0.2 nm/s obtained at room temperature (B).

We report a minimal microtubule-based motile system displaying signatures of unconventional diffusion.