Virupakshi Soppina

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.

Luminal Localization of α-tubulin K40 Acetylation by Cryo-EM Analysis of Fab-Labeled Microtubules

The αβ-tubulin subunits of microtubules can undergo a variety of evolutionarily-conserved post-translational modifications (PTMs) that provide functional specialization to subsets of cellular microtubules. Acetylation of α-tubulin residue Lysine-40 (K40) has been correlated with increased microtubule stability, intracellular transport, and ciliary assembly, yet a mechanistic understanding of how acetylation influences these events is lacking. Using the anti-acetylated tubulin antibody 6-11B-1 and electron cryo-microscopy, we demonstrate that the K40 acetylation site is located inside the microtubule lumen and thus cannot directly influence events on the microtubule surface, including kinesin-1 binding. Surprisingly, the monoclonal 6-11B-1 antibody recognizes both acetylated and deacetylated microtubules. These results suggest that acetylation induces structural changes in the K40-containing loop that could have important functional consequences on microtubule stability, bending, and subunit interactions. This work has important implications for acetylation and deacetylation reaction mechanisms as well as for interpreting experiments based on 6-11B-1 labeling.

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.

The family-specific K-loop influences the microtubule on-rate but not the superprocessivity of kinesin-3 motors

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.

Mapping the Processivity Determinants of the Kinesin-3 Motor Domain

Kinesin superfamily members play important roles in many diverse cellular processes, including cell motility, cell division, intracellular transport, and regulation of the microtubule cytoskeleton. How the properties of the family-defining motor domain of distinct kinesins are tailored to their different cellular roles remains largely unknown. Here, we employed molecular-dynamics simulations coupled with energetic calculations to infer the family-specific interactions of kinesin-1 and kinesin-3 motor domains with microtubules in different nucleotide states. We then used experimental mutagenesis and single-molecule motility assays to further assess the predicted residue-wise determinants of distinct kinesin-microtubule binding properties. Collectively, our results identify residues in the L8, L11, and α6 regions that contribute to family-specific microtubule interactions and whose mutation affects motor-microtubule complex stability and processive motility (the ability of an individual motor to take multiple steps along its microtubule filament). In particular, substitutions of prominent kinesin-3 residues with those found in kinesin-1, namely, R167S/H171D, K266D, and R346M, were found to decrease kinesin-3 processivity 10-fold and thus approach kinesin-1 levels.

Acetylation of Alpha Tubulin Lysine-40 Alone is not Sufficient for Changes in Kinesin-1 Motility

Kinesin-1 is a processive, plus end-directed motor that is responsible for major microtubule-based intracellular transport. It has been previously shown through in vivo studies that kinesin-1 preferentially translocates along certain subsets of microtubules, which are marked with specific posttranslational modifications (PTMs). We hypothesize that PTMs of tubulin directly influence the interaction of kinesin-1 with microtubules. In order to investigate the role of acetylation of α-tubulin at Lysine 40 (K40) in this context, we examined the binding affinity of kinesin in solution to acetylated and deacetylated microtubules in the presence of AMPPNP. We further characterized the single molecule motility properties of kinesin-1 on acetylated and deacetylated microtubules using Total Internal Reflection Fluorescence (TIRF) microscopy. To generate acetylated and deacetyalated microtubules, purified bovine tubulin was treated with the enzymes MEC-17 and SIRT2, respectively. Kinesin-1 motors were either expressed in COS cells or purified from bacterial cells. We found that kinesin-1 shows similar binding affinity, velocity and run length on acetylated and deacetylated microtubules as measured in these in vitro assays. Our results suggest that an alteration in the state of acetylation of K40 on α-tubulin in the microtubules does not result in changes in the catalytic cycle and strong or weak- binding states of the motor. We conclude that kinesin-1 cannot directly recognize the presence of an acetyl group on K40 of α-tubulin and hence this modification alone is not sufficient to explain the preferential motility of kinesin-1 observed in vivo. Rather, K40 acetylation appears to mark a subset of microtubules with other structural or biochemical alterations that are recognized as trafficking cues by kinesin-1.