RESUMEN
Exocytosis of secretory granules (SGs) requires their delivery to the actin-rich cell cortex followed by their attachment to the plasma membrane (PM). How these reactions are executed and coordinated is still unclear. Myrip, which is also known as Slac-2c, binds to the SG-associated GTPase Rab27 and is thought to promote the delivery of SGs to the PM by recruiting the molecular motor myosin Va. Myrip also interacts with actin and the exocyst complex, suggesting that it may exert multiple roles in the secretory process. By combining total internal reflection fluorescence microscopy, single-particle tracking, a photoconversion-based assay, and mathematical modeling, we show that, in human enterochromaffin cells, Myrip (1) inhibits a class of SG motion characterized by fast and directed movement, suggesting that it facilitates the dissociation of SGs from microtubules; (2) enhances their motion toward the PM and the probability of SG attachment to the PM; and (3) increases the characteristic time of immobilization at the PM, indicating that it is a component of the molecular machinery that tether SGs to the PM. Remarkably, while the first two effects of Myrip depend on its ability to recruit myosin Va on SGs, the third is myosin Va independent but relies on the C-terminal domain of Myrip. We conclude that Myrip couples the retention of SGs in the cell cortex, their transport to the PM, and their attachment to the PM, and thus promotes secretion. These three steps of the secretory process are thus intimately coordinated.
Asunto(s)
Actinas/metabolismo , Membrana Celular/metabolismo , Exocitosis/fisiología , Vesículas Secretoras/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Adhesión Celular/fisiología , Línea Celular Tumoral , Membrana Celular/ultraestructura , Células Enterocromafines/metabolismo , Células Enterocromafines/ultraestructura , Humanos , Unión Proteica/fisiología , Vesículas Secretoras/ultraestructuraRESUMEN
Factors affecting the accuracy of molecular dynamics (MD) simulations are investigated by comparing generalized order parameters for backbone NH vectors of the B3 immunoglobulin-binding domain of streptococcal protein G (GB3) derived from simulations with values obtained from NMR spin relaxation (Yao L, Grishaev A, Cornilescu G, Bax A, J Am Chem Soc 2010;132:4295-4309.). Choices for many parameters of the simulations, such as buffer volume, water model, or salt concentration, have only minor influences on the resulting order parameters. In contrast, seemingly minor conformational differences in starting structures, such as orientations of sidechain hydroxyl groups, resulting from applying different protonation algorithms to the same structure, have major effects on backbone dynamics. Some, but not all, of these effects are mitigated by increased sampling in simulations. Most discrepancies between simulated and experimental results occur for residues located at the ends of secondary structures and involve large amplitude nanosecond timescale transitions between distinct conformational substates. These transitions result in autocorrelation functions for bond vector reorientation that do not converge when calculated over individual simulation blocks, typically of length similar to the overall rotational diffusion time. A test for convergence before averaging the order parameters from different blocks results in better agreement between order parameters calculated from different sets of simulations and with NMR-derived order parameters. Thus, MD-derived order parameters are more strongly affected by transitions between conformational substates than by fluctuations within individual substates themselves, while conformational differences in the starting structures affect the frequency and scale of such transitions.
Asunto(s)
Proteínas Bacterianas/química , Biología Computacional/métodos , Simulación de Dinámica Molecular , Glicina/química , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Mutación , Estructura Secundaria de Proteína , Reproducibilidad de los Resultados , Streptococcus/química , Factores de Tiempo , Agua/químicaRESUMEN
The purpose of our study is to develop and evaluate a method for radiopaque 3-D printing (R3P) of soft tissue computed tomography (CT) phantoms with office laser printers. Five laser printers from different vendors are tested for toner CT attenuation. A liver phantom is created by printing CT images of a patient liver on office paper. One thousand eight hundred sixty paper sheets are printed with three repeated prints per page, resulting in a stack of 18.6 cm. The phantom is examined with 12 tube current settings. Images are reconstructed using filtered back projection (FBP) and iterative reconstruction [adaptive iterative dose reduction 3D (AIDR 3D)]. Seven radiologists rated image quality of all acquisitions. Toner attenuation of all investigated printers increased linearly with the print template grayscale. The liver phantom reproduced anatomic detail and attenuation values of the patient ( mean ± SD HU difference 12.68 ± 7.74 ). Image quality scores increased with dose but did not vary significantly above a threshold dose for AIDR 3D. Overall, AIDR 3D reconstructed images are rated superior to FBP reconstructions ( p < 0.001 ). In conclusion, R3P with standard office laser printers can generate soft tissue CT phantoms without hardware manipulations but with limited flexibility regarding attenuation properties of the printed toner material.
RESUMEN
Transcription factors bind to their binding sites over a wide range of affinities, yet how differences in affinity are encoded in DNA sequences is not well understood. Here, we report X-ray crystal structures of four heterodimers of the Hox protein AbdominalB bound with its cofactor Extradenticle to four target DNA molecules that differ in affinity by up to â¼20-fold. Remarkably, despite large differences in affinity, the overall structures are very similar in all four complexes. In contrast, the predicted shapes of the DNA binding sites (i.e., the intrinsic DNA shape) in the absence of bound protein are strikingly different from each other and correlate with affinity: binding sites that must change conformations upon protein binding have lower affinities than binding sites that have more optimal conformations prior to binding. Together, these observations suggest that intrinsic differences in DNA shape provide a robust mechanism for modulating affinity without affecting other protein-DNA interactions.
Asunto(s)
Sitios de Unión/genética , ADN/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Datos de Secuencia MolecularRESUMEN
Thermodynamic stability is a central requirement for protein function, and one goal of protein engineering is improvement of stability, particularly for applications in biotechnology. Herein, molecular dynamics simulations are used to predict in vitro thermostability of members of the bacterial ribonuclease HI (RNase H) family of endonucleases. The temperature dependence of the generalized order parameter, S, for four RNase H homologues, from psychrotrophic, mesophilic, and thermophilic organisms, is highly correlated with experimentally determined melting temperatures and with calculated free energies of folding at the midpoint temperature of the simulations. This study provides an approach for in silico mutational screens to improve thermostability of biologically and industrially relevant enzymes.