The assembly and maintenance of all cilia and flagella require intraflagellar transport (IFT) along the axoneme. IFT has been implicated in sensory and motile ciliary functions, but the mechanisms of this relationship remain unclear. Here, we used Chlamydomonas flagellar surface motility (FSM) as a model to test whether IFT provides force for gliding of cells across solid surfaces. We show that IFT trains are coupled to flagellar membrane glycoproteins (FMGs) in a Ca2+-dependent manner. IFT trains transiently pause through surface adhesion of their FMG cargos, and dynein-1b motors pull the cell towards the distal tip of the axoneme. Each train is transported by at least four motors, with only one type of motor active at a time. Our results demonstrate the mechanism of Chlamydomonas gliding motility and suggest that IFT plays a major role in adhesion-induced ciliary signaling pathways.DOI: http://dx.doi.org/10.7554/eLife.00744.001

Cytoplasmic dynein is a dimeric motor that transports intracellular cargoes towards the minus-end of microtubules (MTs). In contrast to other processive motors, stepping of the dynein motor domains (heads) is not precisely coordinated. Therefore, the mechanism of dynein processivity remains unclear. Here, by engineering the mechanical and catalytic properties of the motor, we show that dynein processivity minimally requires a single active head and a second inert MT binding domain. Processivity arises from a high ratio of MT-bound to unbound time, and not from interhead communication. Additionally, nucleotide-dependent microtubule release is gated by tension on the linker domain. Intramolecular tension sensing is observed in dynein’s stepping motion at high interhead separations. We developed a quantitative model for the stepping characteristics of dynein and its response to chemical and mechanical perturbation.

The rotary motor F 1 -ATPase from the thermophilic Bacillus PS3 (TF 1 ) is one of the best-studied of all molecular machines. F 1 -ATPase is the part of the enzyme F 1 F O -ATP synthase that is responsible for generating most of the ATP in living cells. Single-molecule experiments have provided a detailed understanding of how ATP hydrolysis and synthesis are coupled to internal rotation within the motor. In this work, we present evidence that mesophilic F 1 -ATPase from Escherichia coli (EF 1 ) is governed by the same mechanism as TF 1 under laboratory conditions. Using optical microscopy to measure rotation of a variety of marker particles attached to the γ-subunit of single surface-bound EF 1 molecules, we characterized the ATP-binding, catalytic and inhibited states of EF 1 . We also show that the ATP-binding and catalytic states are separated by 35±3°. At room temperature, chemical processes occur faster in EF 1 than in TF 1 , and we present a methodology to compensate for artefacts that occur when the enzymatic rates are comparable to the experimental temporal resolution. Furthermore, we show that the molecule-to-molecule variation observed at high ATP concentration in our single-molecule assays can be accounted for by variation in the orientation of the rotating markers.

We report statistical time-series analysis tools providing improvements in the rapid, precision extraction of discrete state dynamics from time traces of experimental observations of molecular machines. By building physical knowledge and statistical innovations into analysis tools, we provide techniques for estimating discrete state transitions buried in highly correlated molecular noise. We demonstrate the effectiveness of our approach on simulated and real examples of steplike rotation of the bacterial flagellar motor and the F1-ATPase enzyme. We show that our method can clearly identify molecular steps, periodicities and cascaded processes that are too weak for existing algorithms to detect, and can do so much faster than existing algorithms. Our techniques represent a step in the direction toward automated analysis of high-sample-rate, molecular-machine dynamics. Modular, open-source software that implements these techniques is provided.