Actin and dynamin2 dynamics and interplay during clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) involves the recruitment of numerous proteins to sites on the plasma membrane with prescribed timing to mediate specific stages of the process. However, how choreographed recruitment and function of specific proteins during CME is achieved remains unclear. Using genome editing to express fluorescent fusion proteins at native levels and live-cell imaging with single-molecule sensitivity, we explored dynamin2 stoichiometry, dynamics, and functional interdependency with actin. Our quantitative analyses revealed heterogeneity in the timing of the early phase of CME, with transient recruitment of 2-4 molecules of dynamin2. In contrast, considerable regularity characterized the final 20 s of CME, during which ∼26 molecules of dynamin2, sufficient to make one ring around the vesicle neck, were typically recruited. Actin assembly generally preceded dynamin2 recruitment during the late phases of CME, and promoted dynamin recruitment. Collectively, our results demonstrate precise temporal and quantitative regulation of the dynamin2 recruitment influenced by actin polymerization.

Targeted integration in rat and mouse embryos with zinc-finger nucleases.

Gene targeting is indispensible for reverse genetics and the generation of animal models of disease. The mouse has become the most commonly used animal model system owing to the success of embryonic stem cell-based targeting technology, whereas other mammalian species lack convenient tools for genome modification. Recently, microinjection of engineered zinc-finger nucleases (ZFNs) in embryos was used to generate gene knockouts in the rat and the mouse by introducing nonhomologous end joining (NHEJ)-mediated deletions or insertions at the target site. Here we use ZFN technology in embryos to introduce sequence-specific modifications (knock-ins) by means of homologous recombination in Sprague Dawley and Long-Evans hooded rats and FVB mice. This approach enables precise genome engineering to generate modifications such as point mutations, accurate insertions and deletions, and conditional knockouts and knock-ins. The same strategy can potentially be applied to many other species for which genetic engineering tools are needed.

Identification and characterization of endogenous Langerin ligands in murine extracellular matrix.

Langerin is a C-type lectin that is expressed by Langerhans cells (LC) and related immune cells, and believed to play an important role in antigen recognition and uptake. To determine if Langerin has endogenous ligands, we generated S protein binding, bacterial recombinant, mouse soluble Langerin, and utilized it as a probe. Recombinant soluble Langerin did not bind to lymph node or spleen cells, or keratinocytes as assessed via flow cytometry. However, Langerin did bind to surfaces of primary skin fibroblasts and NIH3T3 cells. "Ligand blotting" of fibroblast membrane-enriched fractions with Langerin revealed reproducible binding to 140 and 240 kDa proteins resolved in reduced denaturing gels. Characterization of these proteins using mass spectrometry suggested types I and III procollagen and fibronectin as candidate ligands. Langerin bound to type I procollagen that was immunoprecipitated from fibroblast lysates, but did not bind to fibronectin that was immunoprecipitated from fibroblast-conditioned media or mouse plasma fibronectin. These results indicate that Langerin selectively interacts with at least one ligand in extracellular matrix (type I procollagen). Langerin may have an unanticipated role in cell-matrix interactions that modulate LC development, localization, or function.