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 onehead-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.kinesin | iSCAT | microscopy | structural kinetics | structure-function K inesin-1 is a motor protein that steps processively toward microtubule plus-ends, tracking single protofilaments and hydrolyzing one ATP molecule per step (1-6).Step sizes corresponding to the tubulin dimer spacing of 8.2 nm are observed when the molecule is labeled by its C-terminal tail (7-10) and to a two-dimer spacing of 16.4 nm when a single motor domain is labeled (4,11,12), consistent with the motor walking in a handover-hand fashion. Kinesin has served as an important model system for advancing single-molecule techniques (7-10) and is clinically relevant for its role in neurodegenerative diseases (13), making dissection of its step a popular ongoing target of study.Despite decades of work, many essential components of the mechanochemical cycle remain disputed, including (i) how much time kinesin-1 spends in a one-head-bound (1HB) state when stepping at physiological ATP concentrations, (ii) whether the motor waits for ATP in a 1HB or two-heads-bound (2HB) state, and (iii) whether ATP hydrolysis occurs before or after tethered head attachment (4,11,(14)(15)(16)(17)(18)(19)(20). These questions are important because they are fundamental to the mechanism by which kinesins harness nucleotide-dependent structural changes to generate mechanical force in a manner optimized for their specific cellular tasks. Addressing these questions requires characterizing a transient 1HB state in the stepping cycle in which the unattached head is located between successive binding sites on the microtubule. This 1HB intermediate is associated with the force-generating powerstroke of the motor and underlies the detachment pathway that limits motor processivity. Optical trapping (7,19,21,22) and sin...

Fluorescence photobleaching is used to estimate the number of cellulose synthase subunits in Arabidopsis cellulose synthesis complexes. To overcome challenges with counting many (>10) fluorophores from photobleaching traces, a general set of statistical procedures is developed for objectively detecting steps and accurately estimating high copy numbers.

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. The high spatiotemporal resolution of this method enabled real-time recording of structural changes in the protein as it walked at~100 steps per second. 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 the structural transitions to and from this one-headbound intermediate 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, meaning that there is one rate-limited step in each the one-and two-heads bound states. 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, and show that the mechanism underlying stepping is a two-step process. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.

Pancreatic islets secrete multiple hormones, including insulin, that are required to maintain euglycemia while meeting the energy demand of the body during everyday activities. We are interested in understanding the interplay between molecular mechanisms that precisely regulate these secretions. One specific paracrine modulator, dopamine, functions as a negative regulator of insulin secretion in the context of the pancreatic islet. It is secreted by the insulinproducing b-cells, activates an autocrine negative feedback that decreases the frequency of glucose-stimulated [Ca 2þ ] i oscillations, and in turn, inhibits insulin secretion. The G-protein coupled dopamine receptors are present in islet cells, but it is not clear how activation of the these receptors results in the observed changes in [Ca 2þ ] i in intact pancreatic islet cells. We are using an mTurquoise-Based cAMP biosensor with an improved dynamic range (Klarenbeek, J.B., et al., PLoS One, 2011), along with organic and genetically encoded Ca 2þ -indicator dyes. Labeled cells are studied by live imaging using perfused pancreatic islet. Spectral unmixing is used to extract the fluorescence emissions. This experimental setup allows us to monitor the effect of specific dopamine receptor agonists and antagonists on the two main cellular second messengers. Also, islets from mice with a genetic target mutation of the DRD3 (D3-KO) are used to measure how the deletion of the dopaminergic feedback changes the second messenger dynamics. With the same approach we are measuring the effects of the overexpression of dopamine receptor D3 in wild-type and D3-KO islets. The information from these experiments will help elucidate the mechanism of dopamine signaling in the pancreatic islet.

Member of kinesin-7 family CENP-E is a kinetochore-associated plus-end-directed motor, which is important for faithful chromosome segregation in mitosis. CENP-E assists chromosome transport to the spindle midzone, where the microtubule plus ends are located. Here we report that once reaching the dynamic microtubule ends in vitro, CENP-E converts from a lateral transporter into a microtubule tip-tracker, stably associating with the tips of both assembling and disassembling microtubules. We show that the binding between kinetochores and dynamic microtubule ends is destabilized in live cells when CENP-E function is perturbed via an inhibition or RNAi depletion, implying 326a Monday,