Altered chemomechanical coupling causes impaired motility of the kinesin-4 motors KIF27 and KIF7.

Kinesin-4 motors play important roles in cell division, microtubule organization, and signaling. Understanding how motors perform their functions requires an understanding of their mechanochemical and motility properties. We demonstrate that KIF27 can influence microtubule dynamics, suggesting a conserved function in microtubule organization across the kinesin-4 family. However, kinesin-4 motors display dramatically different motility characteristics: KIF4 and KIF21 motors are fast and processive, KIF7 and its Drosophila melanogaster homologue Costal2 (Cos2) are immotile, and KIF27 is slow and processive. Neither KIF7 nor KIF27 can cooperate for fast processive transport when working in teams. The mechanistic basis of immotile KIF7 behavior arises from an inability to release adenosine diphosphate in response to microtubule binding, whereas slow processive KIF27 behavior arises from a slow adenosine triphosphatase rate and a high affinity for both adenosine triphosphate and microtubules. We suggest that evolutionarily selected sequence differences enable immotile KIF7 and Cos2 motors to function not as transporters but as microtubule-based tethers of signaling complexes

Two binding partners cooperate to activate the molecular motor Kinesin-1

The regulation of molecular motors is an important cellular problem, as motility in the absence of cargo results in futile adenosine triphosphate hydrolysis. When not transporting cargo, the microtubule (MT)-based motor Kinesin-1 is kept inactive as a result of a folded conformation that allows autoinhibition of the N-terminal motor by the C-terminal tail. The simplest model of Kinesin-1 activation posits that cargo binding to nonmotor regions relieves autoinhibition. In this study, we show that binding of the c-Jun N-terminal kinase–interacting protein 1 (JIP1) cargo protein is not sufficient to activate Kinesin-1. Because two regions of the Kinesin-1 tail are required for autoinhibition, we searched for a second molecule that contributes to activation of the motor. We identified fasciculation and elongation protein ζ1 (FEZ1) as a binding partner of kinesin heavy chain. We show that binding of JIP1 and FEZ1 to Kinesin-1 is sufficient to activate the motor for MT binding and motility. These results provide the first demonstration of the activation of a MT-based motor by cellular binding partners

Autoinhibition of the kinesin-2 motor KIF17 via dual intramolecular mechanisms

Kinesin-2 motor KIF17 autoinhibition is visualized in vivo; in the absence of cargo, this homodimer’s C-terminal tail blocks microtubule binding, and a coiled-coil segment blocks motility

Dual Chromatin and Cytoskeletal Remodeling by SETD2

Posttranslational modifications (PTMs) of tubulin specify microtubules for specialized cellular functions and comprise what is termed a "tubulin code." PTMs of histones comprise an analogous "histone code," although the "readers, writers, and erasers" of the cytoskeleton and epigenome have heretofore been distinct. We show that methylation is a PTM of dynamic microtubules and that the histone methyltransferase SET-domain-containing 2 (SETD2), which is responsible for H3 lysine 36 trimethylation (H3K36me3) of histones, also methylates α-tubulin at lysine 40, the same lysine that is marked by acetylation on microtubules. Methylation of microtubules occurs during mitosis and cytokinesis and can be ablated by SETD2 deletion, which causes mitotic spindle and cytokinesis defects, micronuclei, and polyploidy. These data now identify SETD2 as a dual-function methyltransferase for both chromatin and the cytoskeleton and show a requirement for methylation in maintenance of genomic stability and the integrity of both the tubulin and histone codes

Mammalian Kinesin-3 Motors Are Dimeric In Vivo and Move by Processive Motility upon Release of Autoinhibition

Kinesin-3 motors drive the transport of synaptic vesicles and other membrane-bound organelles in neuronal cells. In the absence of cargo, kinesin motors are kept inactive to prevent motility and ATP hydrolysis. Current models state that the Kinesin-3 motor KIF1A is monomeric in the inactive state and that activation results from concentration-driven dimerization on the cargo membrane. To test this model, we have examined the activity and dimerization state of KIF1A. Unexpectedly, we found that both native and expressed proteins are dimeric in the inactive state. Thus, KIF1A motors are not activated by cargo-induced dimerization. Rather, we show that KIF1A motors are autoinhibited by two distinct inhibitory mechanisms, suggesting a simple model for activation of dimeric KIF1A motors by cargo binding. Successive truncations result in monomeric and dimeric motors that can undergo one-dimensional diffusion along the microtubule lattice. However, only dimeric motors undergo ATP-dependent processive motility. Thus, KIF1A may be uniquely suited to use both diffuse and processive motility to drive long-distance transport in neuronal cells

NPHP proteins are binding partners of nucleoporins at the base of the primary cilium.

Cilia are microtubule-based organelles that protrude from the surface of eukaryotic cells to generate motility and to sense and respond to environmental cues. In order to carry out these functions, the complement of proteins in the cilium must be specific for the organelle. Regulation of protein entry into primary cilia has been shown to utilize mechanisms and components of nuclear gating, including nucleoporins of the nuclear pore complex (NPC). We show that nucleoporins also localize to the base of motile cilia on the surface of trachea epithelial cells. How nucleoporins are anchored at the cilium base has been unclear as transmembrane nucleoporins, which anchor nucleoporins at the nuclear envelope, have not been found to localize at the cilium. Here we use the directed yeast two-hybrid assay to identify direct interactions between nucleoporins and nephronophthisis proteins (NPHPs) which localize to the cilium base and contribute to cilium assembly and identity. We validate NPHP-nucleoporin interactions in mammalian cells using the knocksideways assay and demonstrate that the interactions occur at the base of the primary cilium using bimolecular fluorescence complementation. We propose that NPHP proteins anchor nucleoporins at the base of primary cilia to regulate protein entry into the organelle

Recycling of Kinesin-1 Motors by Diffusion after Transport

<div><p>Kinesin motors drive the long-distance anterograde transport of cellular components along microtubule tracks. Kinesin-dependent transport plays a critical role in neurogenesis and neuronal function due to the large distance separating the soma and nerve terminal. The fate of kinesin motors after delivery of their cargoes is unknown but has been postulated to involve degradation at the nerve terminal, recycling via retrograde motors, and/or recycling via diffusion. We set out to test these models concerning the fate of kinesin-1 motors after completion of transport in neuronal cells. We find that kinesin-1 motors are neither degraded nor returned by retrograde motors. By combining mathematical modeling and experimental analysis, we propose a model in which the distribution and recycling of kinesin-1 motors fits a “loose bucket brigade” where individual motors alter between periods of active transport and free diffusion within neuronal processes. These results suggest that individual kinesin-1 motors are utilized for multiple rounds of transport.</p></div

Diffusion of KHC-mCit in neurites.

<p>Differentiated CAD cells expressing (A–C) the control mCit-GST-NUS fusion protein or (D–F) mCit-KHC were imaged by confocal microscopy. Low-expressing cells were chosen for imaging but the brightness of the image was digitally enhanced to aid in visualization. A 30–40 µm stretch in the middle of the neurite (white boxed region) was photobleached and then the fluorescence recovery was monitored over time. (A,D) Representative pre-bleach images. Kymographs of fluorescence recovery in the bleached region (right panels) were generated by drawing a line along the bleached region of the neurite (white box) and plotting the line from each time point down the y-axis. (B,E) Graphs of fluorescence recovery in the bleached regions of the representative cells in (A,D). The fluorescence recovery data (black circles) were fit by an analytical solution (gray lines). (C,F) The average fluorescence recovery for multiple cells expressing (C) mCit-GST-Nus or (F) mCit-KHC. <i>n</i> = 8 cells each.</p

The distribution of kinesin-1 fits with a Loose Bucket Brigade.

<p>(A–D) Models for kinesin-1 transport and recycling in neuronal processes. In the Diligent Worker model (A), active kinesin-1 motors (green) transport vesicular cargoes to the plus ends of the microtubules (solid green arrow). The motors disengage from the cargo and track (black arrow), assume the inactive autoinhibited conformation (red), and diffuse in the neurite (dotted red arrows). In the Loose Bucket Brigade (B), active kinesin-1 motors (green) transport vesicular cargoes to the plus ends of the microtubules (solid green arrow) but stochastically fall off the cargo (orange arrows). These motors diffuse within the neurite (dotted red arrows) and can reattach to cargoes (green arrows) and again contribute to the transport of vesicles to the plus ends of microtubules. The Hybrid 1 model (C) assumes that kinesin-1 motors can stochastically fall off the cargo (orange arrows) but cannot reattach while diffusing in the neurite (like the Sloppy Bucket Brigade during active transport and the Diligent Worker during recycling). The Hybrid 2 model (D) assumes that kinesin-1 motors do not fall off during transport but can reattach to cargoes while diffusing in the neurite (like the Diligent Worker during active transport and the Sloppy Bucket Brigade during recycling). For each model, computer simulations were used to determine the predicted distribution of kinesin-1 motors in neuronal processes (bottom panels). (E, F) Experimental determination of kinesin-1 distribution in neuronal processes. (E) Differentiated CAD cells or (F) primary hippocampal neurons expressing EGFP (a marker of cell volume) were stained with an antibody to KHC. The fluorescence intensity of KHC (red lines) and EGFP (green lines) was plotted along the length of the neurite or axon (right panels). The fluorescence intensity of KHC represents the spatial distribution of the total kinesin concentration (a sum of concentrations of bound and freely diffusing kinesin-1). (G) The increasing portion of the kinesin-1 profile (red line) in the neuronal process of the CAD cell in (E) was plotted point-by-point as a ratio of the local fluorescence intensities of KHC and EGFP. The distribution fits with an exponential curve (blue line) and is consistent with the Loose Bucket Brigade model.</p