RESUMEN
For a long time, traditional purification and extraction methods for the native Torpedo californica nicotinic acetylcholine receptor in lipid-like detergent complex (nAChR-DC) have compromised its purity, functionality and X-ray structural studies possibility. The dataset presented in this article provide a characterization of the Torpedo californica nAChR-DC purified using a sequential purification processes developed in our laboratory [1]. This purification takes in consideration all of the physicochemical and functional requirements stablished by several researchers for the past three decades for the nAChR. These requirements were addressed in order to preserve the stability and functionality of nAChR-DC while ensuring the highest degree of protein purity. We focused on the effect of cholesteryl hemisuccinate (CHS) supplementation on nAChR conformational changes during the purification process. Data from the size exclusion chromatography of the nAChR-DC supplemented with CHS in concentrations ranging from 0.01 mM, 0.1 mM, 0.2 mM and 0.5 mM consistently demonstrated that 0.5 mM CHS affects receptor stability via disassemble of the pentameric oligomer. However, 0.2 mM CHS produced negligible nAChR-DC subunit disruption. The purified nAChR-DC has been characterized by circular dichroism (CD) and fluorescence recovery after photobleaching (FRAP), in order to assess its stability. The CD data was recorded in the wavelength range of 190-250 nm, showed that CHS induce a âº-helix to ß-sheet transition of the nAChR-DC. The nAChR-LFC-16 delipidation with Methyl-ß-Cyclodextrin decreased the percentage of α-helix and increased the ß-sheet antiparallel secondary structure and levels the percentage of turns to that of the nAChR-DC without CHS treatment. Additionally, the stability of the nAChR-DC supplemented with CHS and incorporated into lipid cubic phase (LCP) was monitored for a period of 30 days by means of FRAP. The LCP-FRAP data allowed to establish possible optimal crystallization conditions for the development of crystals from purified nAChR-conjugated to α-Bungarotoxin, Alexa Fluor ™ 488 (α-BTX) in order to obtain a high-resolution atomic structure by X-ray diffraction.
RESUMEN
The fungal cell wall building processes are the ultimate determinants of hyphal shape. In Neurospora crassa the main cell wall components, ß-1,3-glucan and chitin, are synthesized by enzymes conveyed by specialized vesicles to the hyphal tip. These vesicles follow different secretory routes, which are delicately coordinated by cargo-specific Rab GTPases until their accumulation at the Spitzenkörper. From there, the exocyst mediates the docking of secretory vesicles to the plasma membrane, where they ultimately get fused. Although significant progress has been done on the cellular mechanisms that carry cell wall synthesizing enzymes from the endoplasmic reticulum to hyphal tips, a lot of information is still missing. Here, the current knowledge on N. crassa cell wall composition and biosynthesis is presented with an emphasis on the underlying molecular and cellular secretory processes.
RESUMEN
Diaphragmatic myoblasts (DMs) are precursors of type-1 muscle cells displaying high exhaustion threshold on account that they contract and relax 20 times/min over a lifespan, making them potentially useful in cardiac regeneration strategies. Besides, it has been shown that biomaterials for stem cell delivery improve cell retention and viability in the target organ. In the present study, we aimed at developing a novel approach based on the use of poly (L-lactic acid) (PLLA) scaffolds seeded with DMs overexpressing connexin-43 (cx43), a gap junction protein that promotes inter-cell connectivity. DMs isolated from ovine diaphragm biopsies were characterized by immunohistochemistry and ability to differentiate into myotubes (MTs) and transduced with a lentiviral vector encoding cx43. After confirming cx43 expression (RT-qPCR and Western blot) and its effect on inter-cell connectivity (fluorescence recovery after photobleaching), DMs were grown on fiber-aligned or random PLLA scaffolds. DMs were successfully isolated and characterized. Cx43 mRNA and protein were overexpressed and favored inter-cell connectivity. Alignment of the scaffold fibers not only aligned but also elongated the cells, increasing the contact surface between them. This novel approach is feasible and combines the advantages of bioresorbable scaffolds as delivery method and a cell type that on account of its features may be suitable for cardiac regeneration. Future studies on animal models of myocardial infarction are needed to establish its usefulness on scar reduction and cardiac function.
RESUMEN
In our previous study we examined the functionality and stability of nicotinic acetylcholine receptor (nAChR)-detergent complexes (nAChR-DCs) from affinity-purified Torpedo californica (Tc) using fluorescence recovery after photobleaching (FRAP) in Lipidic Cubic Phase (LCP) and planar lipid bilayer (PLB) recordings for phospholipid and cholesterol like detergents. In the present study we enhanced the functional characterization of nAChR-DCs by recording macroscopic ion channel currents in Xenopus oocytes using the two electrode voltage clamp (TEVC). The use of TEVC allows for the recording of macroscopic currents elicited by agonist activation of nAChR-DCs that assemble in the oocyte plasma membrane. Furthermore, we examined the stability of nAChR-DCs, which is obligatory for the nAChR crystallization, using a 30 day FRAP assay in LCP for each detergent. The present results indicate a marked difference in the fractional fluorescence recovery (ΔFFR) within the same detergent family during the 30 day period assayed. Within the cholesterol analog family, sodium cholate and CHAPSO displayed a minimum ΔFFR and a mobile fraction (MF) over 80%. In contrast, CHAPS and BigCHAP showed a marked decay in both the mobile fraction and diffusion coefficient. nAChR-DCs containing phospholipid analog detergents with an alkylphosphocholine (FC) and lysofoscholine (LFC) of 16 carbon chains (FC-16, LFC-16) were more effective in maintaining a mobile fraction of over 80% compared to their counterparts with shorter acyl chain (C12, C14). The significant differences in macroscopic current amplitudes, activation and desensitization rates among the different nAChR-DCs evaluated in the present study allow to dissect which detergent preserves both, agonist activation and ion channel function. Functionality assays using TEVC demonstrated that LFC16, LFC14, and cholate were the most effective detergents in preserving macroscopic ion channel function, however, the nAChR-cholate complex display a significant delay in the ACh-induce channel activation. In summary, these results suggest that the physical properties of the lipid analog detergents (headgroup and acyl chain length) are the most effective in maintaining both the stability and functionality of the nAChR in the detergent solubilized complex.