Neck linker docking is critical for Kinesin-1 force generation in cells but at a cost to motor speed and processivity.

Kinesin force generation involves ATP-induced docking of the neck linker (NL) along the motor core. However, the roles of the proposed steps of NL docking, cover-neck bundle (CNB) and asparagine latch (N-latch) formation, during force generation are unclear. Furthermore, the necessity of NL docking for transport of membrane-bound cargo in cells has not been tested. We generated kinesin-1 motors impaired in CNB and/or N-latch formation based on molecular dynamics simulations. The mutant motors displayed reduced force output and inability to stall in optical trap assays but exhibited increased speeds, run lengths, and landing rates under unloaded conditions. NL docking thus enhances force production but at a cost to speed and processivity. In cells, teams of mutant motors were hindered in their ability to drive transport of Golgi elements (high-load cargo) but not peroxisomes (low-load cargo). These results demonstrate that the NL serves as a mechanical element for kinesin-1 transport under physiological conditions.

Intracellular cargo transport by single-headed kinesin motors.

Kinesin motor proteins that drive intracellular transport share an overall architecture of two motor domain-containing subunits that dimerize through a coiled-coil stalk. Dimerization allows kinesins to be processive motors, taking many steps along the microtubule track before detaching. However, whether dimerization is required for intracellular transport remains unknown. Here, we address this issue using a combination of in vitro and cellular assays to directly compare dimeric motors across the kinesin-1, -2, and -3 families to their minimal monomeric forms. Surprisingly, we find that monomeric motors are able to work in teams to drive peroxisome dispersion in cells. However, peroxisome transport requires minimal force output, and we find that most monomeric motors are unable to disperse the Golgi complex, a high-load cargo. Strikingly, monomeric versions of the kinesin-2 family motors KIF3A and KIF3B are able to drive Golgi dispersion in cells, and teams of monomeric KIF3B motors can generate over 8 pN of force in an optical trap. We find that intracellular transport and force output by monomeric motors, but not dimeric motors, are significantly decreased by the addition of longer and more flexible motor-to-cargo linkers. Together, these results suggest that dimerization of kinesin motors is not required for intracellular transport; however, it enables motor-to-motor coordination and high force generation regardless of motor-to-cargo distance. Dimerization of kinesin motors is thus critical for cellular events that require an ability to generate or withstand high forces.

A method for tethering single viral particles for virus-cell interaction studies with optical tweezers.

Direct optical trapping of single viral particles allows characterization of individual particles in suspension with single-molecule sensitivity. Alternative to direct optical trapping of particles, individual particles may be tethered specifically in suspension for manipulation by optical tweezers indirectly, which could be useful for studies of virus-cell interactions. One specific example is the interactions between cell surface receptors and the envelope glycoproteins (Env) on the surface of human immunodeficiency virus type 1 (HIV-1). Env binds to cellular receptors and undergoes a series of conformational changes, culminating in fusion of the viral and cellular membranes that mediates viral entry into cells. In addition to being required for cellular infection, Env is also the sole target for neutralizing antibodies. Thus, significant research has focused on elucidating the structure of Env and the mechanism of HIV-1 entry. However, current methods are unable to resolve the dynamics and stoichiometry of Env binding to cellular receptors during the entry process. Fluorescence and electron microscopy have visualized Env clusters in the viral membrane, but the extent to which these clusters actually bind to cellular receptors, and the mechanism of cluster formation, remain unclear. We describe the development of an optical tweezers technique that can potentially address these questions by delivering a single HIV-1 virion to a live cell with minimal perturbation to the system. Our method can be used to quantitatively probe the physical interactions between Env and cellular receptors in their native environment, which may reveal critical parameters in HIV-1 entry. Furthermore, our method can be used to investigate other protein-protein interactions in the context of live cells, such as the recognition of particulate antigens by B cells, thus offering insight into fundamental features of protein-mediated receptor activation.

A method for multiprotein assembly in cells reveals independent action of kinesins in complex.

Teams of processive molecular motors are critical for intracellular transport and organization, yet coordination between motors remains poorly understood. Here, we develop a system using protein components to generate assemblies of defined spacing and composition inside cells. This system is applicable to studying macromolecular complexes in the context of cell signaling, motility, and intracellular trafficking. We use the system to study the emergent behavior of kinesin motors in teams. We find that two kinesin motors in complex act independently (do not help or hinder each other) and can alternate their activities. For complexes containing a slow kinesin-1 and fast kinesin-3 motor, the slow motor dominates motility in vitro but the fast motor can dominate on certain subpopulations of microtubules in cells. Both motors showed dynamic interactions with the complex, suggesting that motor-cargo linkages are sensitive to forces applied by the motors. We conclude that kinesin motors in complex act independently in a manner regulated by the microtubule track.