Intracellular transport is interspersed with frequent reversals in direction due to the presence of opposing kinesin and dynein motors on organelles that are carried as cargo. The cause and the mechanism of reversals are unknown, but are a key to understanding how cargos are delivered in a regulated manner to specific cellular locations. Unlike established single-motor biophysical assays, this problem requires understanding of the cooperative behavior of multiple interacting motors. Here we present measurements inside live Dictyostelium cells, in a cell extract and with purified motors to quantify such an ensemble function of motors. We show through precise motion analysis that reversals during endosome motion are caused by a tug-of-war between kinesin and dynein. Further, we use a combination of optical trap-based force measurements and Monte Carlo simulations to make the surprising discovery that endosome transport uses many (approximately four to eight) weak and detachment-prone dyneins in a tug-of-war against a single strong and tenacious kinesin. We elucidate how this clever choice of dissimilar motors and motor teams achieves net transport together with endosome fission, both of which are important in controlling the balance of endocytic sorting. To the best of our knowledge, this is a unique demonstration that dynein and kinesin function differently at the molecular level inside cells and of how this difference is used in a specific cellular process, namely endosome biogenesis. Our work may provide a platform to understand intracellular transport of a variety of organelles in terms of measurable quantities.asymmetric motor competition ͉ coordination of motors ͉ molecular motor dynein kinesin ͉ regulation of bidirectional motion ͉

The kinesin-3 family is one of the largest among the kinesin superfamily and its members play important roles in a wide range of cellular transport activities, yet the molecular mechanisms of kinesin-3 regulation and cargo transport are largely unknown. We performed a comprehensive analysis of mammalian kinesin-3 motors from three different subfamilies (KIF1, KIF13, and KIF16). Using Forster resonance energy transfer microscopy in live cells, we show for the first time to our knowledge that KIF16B motors undergo cargo-mediated dimerization. The molecular mechanisms that regulate the monomer-to-dimer transition center around the neck coil (NC) segment and its ability to undergo intramolecular interactions in the monomer state versus intermolecular interactions in the dimer state. Regulation of NC dimerization is unique to the kinesin-3 family and in the case of KIF13A and KIF13B requires the release of a proline-induced kink between the NC and subsequent coiled-coil 1 segments. We show that dimerization of kinesin-3 motors results in superprocessive motion, with average run lengths of ∼10 μm, and that this property is intrinsic to the dimeric kinesin-3 motor domain. This finding opens up studies on the mechanistic basis of motor processivity. Such high processivity has not been observed for any other motor protein and suggests that kinesin-3 motors are evolutionarily adapted to serve as the marathon runners of the cellular world.microtubule | molecular motor | helical plot | intracellular transport

Long-distance transport in eukaryotic cells is driven by molecular motors that move along microtubule tracks. Molecular motors of the kinesin superfamily contain a kinesin motor domain attached to family-specific sequences for cargo binding, regulation, and oligomerization. The biochemical and biophysical properties of the kinesin motor domain have been widely studied, yet little is known about how kinesin motors work in the complex cellular environment. We discuss recent studies on the three major families involved in intracellular transport (kinesin-1, kinesin-2, and kinesin-3) that have begun to bridge the gap in knowledge between the in vitro and in vivo behaviors of kinesin motors. These studies have increased our understanding of how kinesin subunits assemble to produce a functional motor, how kinesin motors are affected by biochemical cues and obstacles present on cellular microtubules, and how multiple motors on a cargo surface can work collectively for increased force production and travel distance.

The kinesin-3 family–specific, positively charged insert, the K-loop, in loop 12 of the motor domain plays a critical role in cargo transport by enhancing the initial interaction of cargo-bound dimeric motors with the microtubule. The replacement of the K-loop, however, does not abolish the superprocessive motion of this class of kinesin motors.

A new system for generating cellular protein assemblies of defined spacing and composition reveals that kinesin motors located near each other function independently rather than cooperatively and are influenced primarily by the characteristics of the microtubule track on which they are moving.