Dynamin recruitment and membrane scission at the neck of a clathrin-coated pit

Dynamin, the GTPase required for clathrin-mediated endocytosis, is recruited to clathrin-coated pits in two sequential phases. The first is associated with coated pit maturation; the second, with fission of the membrane neck of a coated pit. Using gene-edited cells that express dynamin2-EGFP instead of dynamin2 and live-cell TIRF imaging with single-molecule EGFP sensitivity and high temporal resolution, we detected the arrival of dynamin at coated pits and defined dynamin dimers as the preferred assembly unit. We also used live-cell spinning-disk confocal microscopy calibrated by single-molecule EGFP detection to determine the number of dynamins recruited to the coated pits. A large fraction of budding coated pits recruit between 26 and 40 dynamins (between 1 and 1.5 helical turns of a dynamin collar) during the recruitment phase associated with neck fission; 26 are enough for coated vesicle release in cells partially depleted of dynamin by RNA interference. We discuss how these results restrict models for the mechanism of dynamin-mediated membrane scission

Vesicle coat proteins: Finding the missing link

The discovery of an ancient protein complex reveals the evolutionary relationships between the proteins that help to form vesicles

Regulation of ligand-independent notch signal through intracellular trafficking

Notch signaling is an evolutionarily conserved mechanism that defines a key cell fate control mechanism in metazoans. Notch signaling relies on the surface interaction between the Notch receptor and membrane bound ligands in an apposing cell. In our recent study, we uncover a non-canonical receptor activation path that relies on a ligand-independent, intracellular activation of the receptor as it travels through the endosomal compartments. We found that Notch receptor, targeted for degradation lysosomal degradation through multivesicular bodies (MVBs) is “diverted” toward activation upon mono-ubiquitination through a synergy between the ubiquitin ligase Deltex, the non-visual β-arrestin Kurtz and the ESCRT-III component Shrub. This activation path is not universal but appears to depend on the cellular context

J Phys Act Health

BackgroundSince 1995, an 8-day Physical Activity and Public Health Course for Researchers has been offered yearly in the United States.MethodsIn 2013, an evaluation quantified time that fellows spent in different course offerings, surveyed fellows on course impact, documented grant funding, and identified fellow participation on leading physical activity-related journals.ResultsThe number of fellows that attended the course ranged from 20\u201335/year. Fellows who participated in the web survey (n=322) agreed that the course: met their expectations (99%), had a positive impact on the physical activity research or practice work they did (98%), and helped increase their professional networking in the field (93%). Following the course, 73% of fellows had further contact with course faculty and 71% had further contact with other fellows. From the National Institutes of Health, 117 grants were awarded to 82 fellows (21% of eligible fellows). Out of 14 journals reviewed, 11 had at least one fellow on their staff as editor, associate editor, or editorial board member.ConclusionThe Physical Activity and Public Health Course for Researchers helps address a training need by providing instruction and building capacity in the US and abroad for conducting research on physical activity and public health.20142016-08-01T00:00:00ZU48 DP000059/DP/NCCDPHP CDC HHS/United StatesU48/DP000059/DP/NCCDPHP CDC HHS/United States25271475PMC4949596695

Live-Cell Visualization of Pre-mRNA Splicing with Single-Molecule Sensitivity

SummaryRemoval of introns from pre-messenger RNAs (pre-mRNAs) via splicing provides a versatile means of genetic regulation that is often disrupted in human diseases. To decipher how splicing occurs in real time, we directly examined with single-molecule sensitivity the kinetics of intron excision from pre-mRNA in the nucleus of living human cells. By using two different RNA labeling methods, MS2 and λN, we show that β-globin introns are transcribed and excised in 20–30 s. Furthermore, we show that replacing the weak polypyrimidine (Py) tract in mouse immunoglobulin μ (IgM) pre-mRNA by a U-rich Py decreases the intron lifetime, thus providing direct evidence that splice-site strength influences splicing kinetics. We also found that RNA polymerase II transcribes at elongation rates ranging between 3 and 6 kb min−1 and that transcription can be rate limiting for splicing. These results have important implications for a mechanistic understanding of cotranscriptional splicing regulation in the live-cell context

The Small Molecule Dispergo Tubulates the Endoplasmic Reticulum and Inhibits Export

The mammalian endoplasmic reticulum (ER) is an organelle that maintains a complex, compartmentalized organization of interconnected cisternae and tubules while supporting a continuous flow of newly synthesized proteins and lipids to the Golgi apparatus. Using a phenotypic screen, we identify a small molecule, dispergo, that induces reversible loss of the ER cisternae and extensive ER tubulation, including formation of ER patches comprising densely packed tubules. Dispergo also prevents export from the ER to the Golgi apparatus, and this traffic block results in breakdown of the Golgi apparatus, primarily due to maintenance of the constitutive retrograde transport of its components to the ER. The effects of dispergo are reversible, since its removal allows recovery of the ER cisternae at the expense of the densely packed tubular ER patches. This recovery occurs together with reactivation of ER-to-Golgi traffic and regeneration of a functional Golgi with correct morphology. Because dispergo is the first small molecule that reversibly tubulates the ER and inhibits its export function, it will be useful in studying these complex processes.Chemistry and Chemical Biolog

Structural Correlates of Rotavirus Cell Entry

Cell entry by non-enveloped viruses requires translocation into the cytosol of a macromolecular complex—for double-strand RNA viruses, a complete subviral particle. We have used live-cell fluorescence imaging to follow rotavirus entry and penetration into the cytosol of its ∼700 Å inner capsid particle (“double-layered particle”, DLP). We label with distinct fluorescent tags the DLP and each of the two outer-layer proteins and track the fates of each species as the particles bind and enter BSC-1 cells. Virions attach to their glycolipid receptors in the host cell membrane and rapidly become inaccessible to externally added agents; most particles that release their DLP into the cytosol have done so by ∼10 minutes, as detected by rapid diffusional motion of the DLP away from residual outer-layer proteins. Electron microscopy shows images of particles at various stages of engulfment into tightly fitting membrane invaginations, consistent with the interpretation that rotavirus particles drive their own uptake. Electron cryotomography of membrane-bound virions also shows closely wrapped membrane. Combined with high resolution structural information about the viral components, these observations suggest a molecular model for membrane disruption and DLP penetration