MINFLUX reveals dynein stepping in live neurons

Dynein is the primary molecular motor responsible for retrograde intracellular transport of a variety of cargoes, performing successive nanometer-sized steps within milliseconds. Due to the limited spatiotemporal precision of established methods for molecular tracking, current knowledge of dynein stepping is essentially limited to slowed-down measurements in vitro. Here, we use MINFLUX fluorophore localization to directly track CRISPR/Cas9-tagged endogenous dynein with nanometer/millisecond precision in living primary neurons. We show that endogenous dynein primarily takes 8 nm steps, including frequent sideways steps but few backward steps. Strikingly, the majority of direction reversals between retrograde and anterograde movement occurred on the time scale of single steps (16 ms), suggesting a rapid regulatory reversal mechanism. Tug-of-war-like behavior during pauses or reversals was unexpectedly rare. By analyzing the dwell time between steps, we concluded that a single rate-limiting process underlies the dynein stepping mechanism, likely arising from just one adenosine 5′-triphosphate hydrolysis event being required during each step. Our study underscores the power of MINFLUX localization to elucidate the spatiotemporal changes underlying protein function in living cells

Membrane protein sequestering by ionic protein–lipid interactions.

Neuronal exocytosis is catalyzed by the SNARE protein syntaxin-1A(1). Syntaxin-1A is clustered in the plasma membrane at sites where synaptic vesicles undergo exocytosis(2,3). However, how syntaxin-1A is sequestered is unknown. Here, we show that syntaxin clustering is mediated by electrostatic interactions with the strongly anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2). We found with super-resolution STED microscopy on the plasma membrane of PC12 cells that PIP2 is the dominant inner-leaflet lipid in ~73 nm-sized microdomains. This high accumulation of PIP2 was required for syntaxin-1A sequestering, as destruction of PIP2 by the phosphatase synaptojanin-1 reduced syntaxin-1A clustering. Furthermore, co-reconstitution of PIP2 and the C-terminal part of syntaxin-1A in artificial giant unilamellar vesicles resulted in segregation of PIP2 and syntaxin-1A into distinct domains even when cholesterol was absent. Our results demonstrate that electrostatic protein-lipid interactions can result in the formation of microdomains independent of cholesterol or lipid phases

The Dynamic Striated Metropolis-Hastings Sampler for High-Dimensional Models

Having efficient and accurate samplers for simulating the posterior distribution is crucial for Bayesian analysis. We develop a generic posterior simulator called the "dynamic striated Metropolis-Hastings (DSMH)" sampler. Grounded in the Metropolis-Hastings algorithm, it draws its strengths from both the equienergy sampler and the sequential Monte Carlo sampler by avoiding the weaknesses of the straight Metropolis-Hastings algorithm as well as those of importance sampling. In particular, the DSMH sampler possesses the capacity to cope with incredibly irregular distributions that are full of winding ridges and multiple peaks and has the flexibility to take full advantage of parallelism on either desktop computers or clusters. The high-dimensional application studied in this paper provides a natural platform to put to the test generic samplers such as the DSMH sampler

The Interplay between Financial Conditions and Monetary Policy Shocks

We study the interplay between monetary policy and financial conditions shocks. Such shocks have a significant and similar impact on the real economy, though with different degrees of persistence. The systematic fed funds rate response to a financial shock contributes to bringing the economy back towards trend, but a zero lower bound on policy rates can prevent this from happening, with a significant cost in terms of output and investment. In a retrospective analysis of the U.S. economy over the past 20 years, we decompose the realization of economic variables into the contributions of financial, monetary policy, and other shocks

Does Banque de France control inflation and unemployment?

We re-estimate statistical properties and predictive power of a set of Phillips curves, which are expressed as linear and lagged relationships between the rates of inflation, unemployment, and change in labour force. For France, several relationships were estimated eight years ago. The change rate of labour force was used as a driving force of inflation and unemployment within the Phillips curve framework. The set of nested models starts with a simplistic version without autoregressive terms and one lagged term of explanatory variable. The lag is determined empirically together with all coefficients. The model is estimated using the Boundary Element Method (BEM) with the least squares method applied to the integral solutions of the differential equations. All models include one structural break might be associated with revisions to definitions and measurement procedures in the 1980s and 1990s as well as with the change in monetary policy in 1994-1995. For the GDP deflator, our original model provided a root mean squared forecast error (RMSFE) of 1.0% per year at a four-year horizon for the period between 1971 and 2004. The rate of CPI inflation is predicted with RMSFE=1.5% per year. For the naive (no change) forecast, RMSFE at the same time horizon is 2.95% and 3.3% per year, respectively. Our model outperforms the naive one by a factor of 2 to 3. The relationships for inflation were successfully tested for cointegration. We have formally estimated several vector error correction (VEC) models for two measures of inflation. At a four year horizon, the estimated VECMs provide significant statistical improvements on the results obtained by the BEM: RMSFE=0.8% per year for the GDP deflator and ~1.2% per year for CPI. For a two year horizon, the VECMs improve RMSFEs by a factor of 2, with the smallest RMSFE=0.5% per year for the GDP deflator.Comment: 25 pages, 12 figure

"CAN Stop" - Implementation and evaluation of a secondary group prevention for adolescent and young adult cannabis users in various contexts - study protocol

<p>Abstract</p> <p>Background</p> <p>Current research shows that overall numbers for cannabis use among adolescents and young adults dropped in recent years. However, this trend is much less pronounced in continuous cannabis use. With regard to the heightened risk for detrimental health- and development-related outcomes, adolescents and young adults with continuous cannabis use need special attention. The health services structure for adolescents and young adults with substance related problems in Germany, is multifaceted, because different communal, medical and judicial agencies are involved. This results in a rather decentralized organizational structure of the help system. This and further system-inherent characteristics make the threshold for young cannabis users rather high. Because of this, there is a need to establish evidence-based low-threshold help options for young cannabis users, which can be easily disseminated. Therefore, a training programme for young cannabis users (age 14-21) was developed in the "CAN Stop" project. Within the project, we seek to implement and evaluate the training programme within different institutions of the help system. The evaluation is sensitive to the different help systems and their specific prerequisites. Moreover, within this study, we also test the practicability of a training provision through laypersons.</p> <p>Methods/Design</p> <p>The CAN Stop study is a four-armed randomized wait-list controlled trial. The four arms are needed for the different help system settings, in which the CAN Stop training programme is evaluated: (a) the drug addiction aid and youth welfare system, (b) the out-patient medical system, (c) the in-patient medical system and (d) prisons for juvenile offenders. Data are collected at three points, before and after the training or a treatment as usual, and six months after the end of either intervention.</p> <p>Discussion</p> <p>The CAN Stop study is expected to provide an evidence-based programme for young cannabis users seeking to reduce or quit their cannabis use. Moreover, we seek to gain knowledge about the programme's utility within different settings of the German help system for young cannabis users and information about the settings' specific clientele. The study protocol is discussed with regard to potential difficulties within the different settings.</p> <p>Trial registration</p> <p>ISRCTN: <a href="http://www.controlled-trials.com/ISRCTN57036983">ISRCTN57036983</a></p