Keith J. Mickolajczyk

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

Significance We use high spatiotemporal resolution single-molecule microscopy to directly visualize the structural transitions underlying each step of the molecular motor kinesin-1 at physiological stepping rates. Our results identify a one-head–bound intermediate in the stepping cycle that is initiated by ATP binding and is terminated by ATP hydrolysis. These results supersede previous functional studies because they identify the transitions that must occur to produce a step as opposed to transitions that may occur if the motor is studied under controlled conditions. We thus show that kinesin utilizes a two-step powerstroke mechanism to walk at maximum velocity. The single-molecule methods developed here are broadly applicable for resolving protein conformational changes as small as 2 nm with millisecond temporal resolution. To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the one-head–bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.

The Kinesin-5 Chemomechanical Cycle is Dominated by a Two-heads-bound State.

Single-molecule microscopy and stopped-flow kinetics assays were carried out to understand the microtubule polymerase activity of kinesin-5 (Eg5). Four lines of evidence argue that the motor primarily resides in a two-heads-bound (2HB) state. First, upon microtubule binding, dimeric Eg5 releases both bound ADPs. Second, microtubule dissociation in saturating ADP is 20-fold slower for the dimer than for the monomer. Third, ATP-triggered mant-ADP release is 5-fold faster than the stepping rate. Fourth, ATP binding is relatively fast when the motor is locked in a 2HB state. Shortening the neck-linker does not facilitate rear-head detachment, suggesting a minimal role for rear-head-gating. This 2HB state may enable Eg5 to stabilize incoming tubulin at the growing microtubule plus-end. The finding that slowly hydrolyzable ATP analogs trigger slower nucleotide release than ATP suggests that ATP hydrolysis in the bound head precedes stepping by the tethered head, leading to a mechanochemical cycle in which processivity is determined by the race between unbinding of the bound head and attachment of the tethered head.

Kinesin Processivity Is Determined by a Kinetic Race from a Vulnerable One-Head-Bound State

Kinesin processivity, defined as the average number of steps that occur per interaction with a microtubule, is an important biophysical determinant of the motors intracellular capabilities. Despite its fundamental importance to the diversity of tasks that kinesins carry out in cells, no existing quantitative model fully explains how structural differences between kinesins alter kinetic rates in the ATPase cycle to produce functional changes in processivity. Here we use high-resolution single-molecule microscopy to directly observe the stepping behavior of kinesin-1 and -2 family motors with different length neck-linker domains. We characterize a one-head-bound posthydrolysis vulnerable state where a kinetic race occurs between attachment of the tethered head to its next binding site and detachment of the bound head from the microtubule. We find that greater processivity is correlated with faster attachment of the tethered head from this vulnerable state. In compliment, we show that slowing detachment from this vulnerable state by strengthening motor-microtubule electrostatic interactions also increases processivity. Furthermore, we provide evidence that attachment of the tethered head is irreversible, suggesting a first passage model for exit from the vulnerable state. Overall, our results provide a kinetic framework for explaining kinesin processivity and for mapping structural differences to functional differences in diverse kinesin isoforms.

The Axonal Transport Motor Kinesin‐2 Navigates Microtubule Obstacles via Protofilament Switching

Axonal transport involves kinesin motors trafficking cargo along microtubules that are rich in microtubule‐associated proteins (MAPs). Much attention has focused on the behavior of kinesin‐1 in the presence of MAPs, which has overshadowed understanding the contribution of other kinesins such as kinesin‐2 in axonal transport. We have previously shown that, unlike kinesin‐1, kinesin‐2 in vitro motility is insensitive to the neuronal MAP Tau. However, the mechanism by which kinesin‐2 efficiently navigates Tau on the microtubule surface is unknown. We hypothesized that mammalian kinesin‐2 side‐steps to adjacent protofilaments to maneuver around MAPs. To test this, we used single‐molecule imaging to track the characteristic run length and protofilament switching behavior of kinesin‐1 and kinesin‐2 motors in the absence and presence of 2 different microtubule obstacles. Under all conditions tested, kinesin‐2 switched protofilaments more frequently than kinesin‐1. Using computational modeling that recapitulates run length and switching frequencies in the presence of varying roadblock densities, we conclude that kinesin‐2 switches protofilaments to navigate around microtubule obstacles. Elucidating the kinesin‐2 mechanism of navigation on the crowded microtubule surface provides a refined view of its contribution in facilitating axonal transport.

Nicotinamide is an endogenous agonist for a C. elegans TRPV OSM-9 and OCR-4 channel

TRPV ion channels are directly activated by sensory stimuli and participate in thermo-, mechano- and chemo-sensation. They are also hypothesized to respond to endogenous agonists that would modulate sensory responses. Here, we show that the nicotinamide (NAM) form of vitamin B3 is an agonist of a Caenorhabditis elegans TRPV channel. Using heterologous expression in Xenopus oocytes, we demonstrate that NAM is a soluble agonist for a channel consisting of the well-studied OSM-9 TRPV subunit and relatively uncharacterized OCR-4 TRPV subunit as well as the orthologous Drosophila Nan-Iav TRPV channel, and we examine stoichiometry of subunit assembly. Finally, we show that behaviours mediated by these C. elegans and Drosophila channels are responsive to NAM, suggesting conservation of activity of this soluble endogenous metabolite on TRPV activity. Our results in combination with the role of NAM in NAD+ metabolism suggest an intriguing link between metabolic regulation and TRPV channel activity.

Interferometric Scattering Microscopy for the Study of Molecular Motors

Our understanding of molecular motor function has been greatly improved by the development of imaging modalities, which enable real-time observation of their motion at the single-molecule level. Here, we describe the use of a new method, interferometric scattering microscopy, for the investigation of motor protein dynamics by attaching and tracking the motion of metallic nanoparticle labels as small as 20nm diameter. Using myosin-5, kinesin-1, and dynein as examples, we describe the basic assays, labeling strategies, and principles of data analysis. Our approach is relevant not only for motor protein dynamics but also provides a general tool for single-particle tracking with high spatiotemporal precision, which overcomes the limitations of single-molecule fluorescence methods.

Crystal structure of Zen4 in the apo state reveals a missing conformation of kinesin

Kinesins hydrolyse ATP to transport intracellular cargoes along microtubules. Kinesin neck linker (NL) functions as the central mechano-chemical coupling element by changing its conformation through the ATPase cycle. Here we report the crystal structure of kinesin-6 Zen4 in a nucleotide-free, apo state, with the NL initial segment (NIS) adopting a backward-docked conformation and the preceding α6 helix partially melted. Single-molecule fluorescence resonance energy transfer (smFRET) analyses indicate the NIS of kinesin-1 undergoes similar conformational changes under tension in the two-head bound (2HB) state, whereas it is largely disordered without tension. The backward-docked structure of NIS is essential for motility of the motor. Our findings reveal a key missing conformation of kinesins, which provides the structural basis of the stable 2HB state and offers a tension-based rationale for an optimal NL length to ensure processivity of the motor.

Direct observation of individual tubulin dimers binding to growing microtubules

The biochemical basis of microtubule growth has remained elusive for over thirty years despite being fundamental for both cell division and associated chemotherapy strategies. Here, we combine interferometric scattering microscopy with recombinant tubulin to monitor individual tubulins binding to and dissociating from growing microtubule tips. We make the first direct, single-molecule measurements of tubulin on- and off-rates. We detect two populations of transient dwell times, and determine via binding-interface mutants that they are separated by the formation of inter-protofilament bonds. Applying a computational model, we find that slow association kinetics with strong interactions along protofilaments best recapitulate our data, and furthermore predict plus-end tapering. Overall, we provide the most direct and complete quantification of how microtubules grow to date. SIGNIFICANCE Microtubule polymerization dynamics are fundamental to cell migration and cell division, where they are targets for chemotherapy drugs. Despite significant progress, the precise structural and biochemical events occurring at growing microtubule tips are not well defined, and better understanding is necessary for discriminating mechanisms of microtubule dynamics regulation in cells. Here, we visualize individual tubulin subunits reversibly and irreversibly interacting with dynamic microtubule tips, and thereby directly measure tubulin on- and off-rates. By analyzing plus-tip residence times of wild-type and mutant tubulin, we characterize the relative contributions of longitudinal (along protofilaments) and lateral (between protofilaments) bond energies to microtubule growth. This work provides insights into microtubule tip structure and potential modes of microtubule dynamics regulation.

The Orphan Kinesin PAKRP2 Achieves Processive Motility Via Noncanonical Stepping

PAKRP2 is an orphan kinesin in Arabidopsis thaliana that is thought to transport vesicles along phragmoplast microtubules for cell plate formation. Here, using single-molecule fluorescence microscopy, we show that PAKRP2 exhibits processive plus-end-directed motility on single microtubules as individual homodimers despite having an exceptionally long (32 residues) neck linker. Furthermore, using high-resolution nanoparticle tracking to visualize motor stepping dynamics, we find that PAKRP2 achieves processivity via a noncanonical stepping mechanism that includes small step sizes and frequent lateral steps to adjacent protofilaments. We propose that the small steps sizes are due to a transient intermediate step that involves a prolonged diffusional search of the tethered head due to its long neck linker. Despite this different stepping behavior, ATP is tightly coupled to each 8-nm step. Collectively, this study reveals PAKRP2 as the first orphan kinesin to demonstrate processive motility and broadens our understanding of the diverse kinesin stepping mechanisms.

The S6 gate in regulatory Kv6 subunits restricts heteromeric K+ channel stoichiometry

&NA; The Shaker‐like family of voltage‐gated K+ channels comprises four functionally independent gene subfamilies, Shaker (Kv1), Shab (Kv2), Shaw (Kv3), and Shal (Kv4), each of which regulates distinct aspects of neuronal excitability. Subfamily‐specific assembly of tetrameric channels is mediated by the N‐terminal T1 domain and segregates Kv1‐4, allowing multiple channel types to function independently in the same cell. Typical Shaker‐like Kv subunits can form functional channels as homotetramers, but a group of mammalian Kv2‐related genes (Kv5.1, Kv6s, Kv8s, and Kv9s) encodes subunits that have a “silent” or “regulatory” phenotype characterized by T1 self‐incompatibility. These channels are unable to form homotetramers, but instead heteromerize with Kv2.1 or Kv2.2 to diversify the functional properties of these delayed rectifiers. While T1 self‐incompatibility predicts that these heterotetramers could contain up to two regulatory (R) subunits, experiments show a predominance of 3:1R stoichiometry in which heteromeric channels contain a single regulatory subunit. Substitution of the self‐compatible Kv2.1 T1 domain into the regulatory subunit Kv6.4 does not alter the stoichiometry of Kv2.1:Kv6.4 heteromers. Here, to identify other channel structures that might be responsible for favoring the 3:1R stoichiometry, we compare the sequences of mammalian regulatory subunits to independently evolved regulatory subunits from cnidarians. The most widespread feature of regulatory subunits is the presence of atypical substitutions in the highly conserved consensus sequence of the intracellular S6 activation gate of the pore. We show that two amino acid substitutions in the S6 gate of the regulatory subunit Kv6.4 restrict the functional stoichiometry of Kv2.1:Kv6.4 to 3:1R by limiting the formation and function of 2:2R heteromers. We propose a two‐step model for the evolution of the asymmetric 3:1R stoichiometry, which begins with evolution of self‐incompatibility to establish the regulatory phenotype, followed by drift of the activation gate consensus sequence under relaxed selection to limit stoichiometry to 3:1R. &NA; Atypical substitutions in the S6 activation gate sequence distinguish “regulatory” Kv subunits, which cannot homotetramerize due to T1 self‐incompatibility. Pisupati et al. show that such substitutions in Kv6 work together with self‐incompatibility to restrict Kv2:Kv6 heteromeric stoichiometry to 3:1.