Stephen R. Norris

Direct evidence for BBSome-associated intraflagellar transport reveals distinct properties of native mammalian cilia

Cilia dysfunction underlies a class of human diseases with variable penetrance in different organ systems. Across eukaryotes, intraflagellar transport (IFT) facilitates cilia biogenesis and cargo trafficking, but our understanding of mammalian IFT is insufficient. Here we perform live analysis of cilia ultrastructure, composition and cargo transport in native mammalian tissue using olfactory sensory neurons. Proximal and distal axonemes of these neurons show no bias towards IFT kinesin-2 choice, and Kif17 homodimer is dispensable for distal segment IFT. We identify Bardet–Biedl syndrome proteins (BBSome) as bona fide constituents of IFT in olfactory sensory neurons, and show that they exist in 1:1 stoichiometry with IFT particles. Conversely, subpopulations of peripheral membrane proteins, as well as transmembrane olfactory signalling pathway components, are capable of IFT but with significantly less frequency and/or duration. Our results yield a model for IFT and cargo trafficking in native mammalian cilia and may explain the penetrance of specific ciliopathy phenotypes in olfactory neurons.

Dimerization of mammalian kinesin-3 motors results in superprocessive motion.

Significance The kinesin-3 family is one of the largest among the kinesin superfamily and its members play important roles in a variety of cellular functions ranging from intracellular transport to mitosis. Defects in kinesin-3 transport have been implicated in a variety of neurodegenerative, developmental, and cancer diseases, yet the molecular mechanisms of kinesin-3 regulation and cargo transport are largely unknown. We show that kinesin-3 motors undergo cargo-mediated dimerization to transport cellular cargoes. We also show that dimerization results in kinesin-3 motors that are fast and superprocessive. 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. 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.

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

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.

Kinesin-5 inhibitor resistance is driven by kinesin-12

The kinesin-5 Eg5 is essential for mitotic progression, and the lethal effects of Eg5 inhibitors make these inhibitors attractive candidates for chemotherapy drugs. Sturgill et al. show that kinesin-12 and a nonmotile Eg5 mutant form an alternative spindle assembly pathway that provides resistance to Eg5 inhibitors.

Influence of Fluorescent Tag on the Motility Properties of Kinesin-1 in Single-Molecule Assays

Molecular motors such as kinesin and dynein use the energy derived from ATP hydrolysis to walk processively along microtubule tracks and transport various cargoes inside the cell. Recent advancements in fluorescent protein (FP) research enable motors to be fluorescently labeled such that single molecules can be visualized inside cells in multiple colors. The performance of these fluorescent tags can vary depending on their spectral properties and a natural tendency for oligomerization. Here we present a survey of different fluorescent tags fused to kinesin-1 and studied by single-molecule motility assays of mammalian cell lysates. We tested eight different FP tags and found that seven of them display sufficient fluorescence intensity and photostability to visualize motility events. Although none of the FP tags interfere with the enzymatic properties of the motor, four of the tags (EGFP, monomeric EGFP, tagRFPt, and mApple) cause aberrantly long motor run lengths. This behavior is unlikely to be due to electrostatic interactions and is probably caused by tag-dependent oligomerization events that appear to be facilitated by fusion to the dimeric kinesin-1. We also compared the single-molecule performance of various fluorescent SNAP and HALO ligands. We found that although both green and red SNAP ligands provide sufficient fluorescent signal, only the tetramethyl rhodamine (TMR) HALO ligand provides sufficient signal for detection in these assays. This study will serve as a valuable reference for choosing fluorescent labels for single-molecule motility assays.

Two mechanisms coordinate the recruitment of the chromosomal passenger complex to the plane of cell division

Proper positioning of the chromosomal passenger complex (CPC) at the cell division plane is required for cytokinesis. We show here that CPC targeting to the equatorial cortex depends on both the kinesin MKlp2 and a direct interaction with actin. These recruitment mechanisms converge to promote successful cleavage furrow ingression.

Microtubule minus-end aster organization is driven by processive HSET-tubulin clusters

Higher-order structures of the microtubule (MT) cytoskeleton are comprised of two architectures: bundles and asters. Although both architectures are critical for cellular function, the molecular pathways that drive aster formation are poorly understood. Here, we study aster formation by human minus-end-directed kinesin-14 (HSET/KIFC1). We show that HSET is incapable of forming asters from preformed, nongrowing MTs, but rapidly forms MT asters in the presence of soluble (non-MT) tubulin. HSET binds soluble (non-MT) tubulin via its N-terminal tail domain to form heterogeneous HSET-tubulin clusters containing multiple motors. Cluster formation induces motor processivity and rescues the formation of asters from nongrowing MTs. We then show that excess soluble (non-MT) tubulin stimulates aster formation in HeLa cells overexpressing HSET during mitosis. We propose a model where HSET can toggle between MT bundle and aster formation in a manner governed by the availability of soluble (non-MT) tubulin.Microtubules (MT) form higher-order structures such as asters, but the molecular pathway underlying aster formation remains unclear. Here authors demonstrate that the kinesin-14, HSET, clusters with soluble (nonMT) tubulin via its N-terminal tail domain and thereby promotes MT aster formation.

Cell Division: Centrosomes Have Separation Anxiety

Prior to mitosis, duplicated centrosomes are tethered together, which is thought to prevent mitotic defects. A new study establishes the role of tetrameric Kif25, a microtubule minus-end-directed kinesin-14 motor, in preventing premature centrosome separation through a microtubule-dependent pathway.