No abstract

Secretion of inflammatory mediators prestored in mast cells secretory granules (SGs) enhances immune responses such as in allergy and host defense. However, the mechanisms underlying the biogenesis of the SGs remain largely unresolved. By combining high-resolution live cell imaging and quantitative morphometric analyses, we show that the small GTPase Rab5 controls the SG size and cargo composition by a VAMP8-dependent fusion mechanism. Knockdown of the endogenous Rab5, or expression of constitutively negative mutants, significantly reduces the size of SGs and increases their number. Conversely, expression of constitutively active Rab5 mutants induces few, but giant, SGs. Both the small and giant SGs maintain their exocytosis competence. Finally, we show that Rab5-mediated fusion between Golgi-derived SGs and early endosomes precedes the maturation of the SGs, as reflected by the recruitment of Rab27B, and allows the incorporation of cargo, such as CD63, that traffics through endosomes. Collectively, our results assign Rab5 a key role in mediating mast cell SG fusion during biogenesis, thereby controlling the amount and composition of the SGs content and maintaining the communication between new and pre-existing SGs.

No abstract

Carbohydrates of the zona pellucida (ZP) in mammals are believed to have a role in sperm-egg interaction. We have characterized the biochemical nature and distribution of the carbohydrate residues of rat ZP at the light (LM) and electron microscope (EM) levels, using lectins as probes. Immature female rats were induced to superovulate and cumulus-oocyte complexes were isolated from the oviduct, fixed with glutaraldehyde, and embedded in araldite for LM and LR-Gold for EM histochemistry. For examination of follicular oocytes, rat ovaries were fixed with glutaraldehyde and embedded in paraffin. The araldite or paraffin sections were deresined or deparaffinized, respectively, labeled with biotin-tagged lectins as probes, and avidin-biotin-peroxidase complex as visualant. For EM examination, thin LR-Gold sections were labeled with RCA-I colloidal gold complex (RCA/G) and stained with uranyl acetate. LM analyses indicate that in ovulated oocytes the ZP intensely binds peanut agglutinin (PNA); succinylated wheat germ agglutinin, (S-WGA), Griffonia simplisifolia agglutinin-I (GS-I) and soybean agglutinin (SBA), and to a lesser extent, lectins from Ricinus communis (RCA-I), Concanavaia ensiformis (Con A), Ulex europoeus (UEA-I), and wheat germ agglutinin (WGA). The neighboring cumulus cells are considerably less reactive and exhibit membrane staining only with Con A, WGA, and PNA. EM analysis of RCA/G binding revealed intensive binding to the inner layer region of the ZP and moderate binding to cytoplasmic vesicles of the cumulus cells. The ZP of follicular oocytes exhibits a different lectin binding pattern, expressed in staining strongly with PNA and S-WGA, and in a tendency of the lectin receptors to occur in the outer portion of the ZP.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract

The delivery of newly-formed secretory content to the granule inventory occurs through direct fusion of recently formed granules and mature granules. The introduction of knockout mice allowed us to investigate the characteristics of the delivery process and to determine the core protein machinery required for granule growth. The SNARE machinery mediates membrane fusion and is essential for the granule lifecycle. In the current work, we use VAMP8 knockout mice to show that the SNARE machinery plays a critical role in the process of granule homotypic fusion. Consistent with this, the mutated mouse pancreatic acinar secretory granules are significantly smaller compared to the control group, demonstrating few granule profiles that might be the result of homotypic fusion.

Secretory granule formation in pancreatic acinar cells is known to involve massive membrane flow. In previous studies we have undertaken morphometry of the regranulation mechanism in these cells and in mast cells as a model for cellular membrane movement. In our current work, electron micrographs of pancreatic acinar cells from ICR mice were taken at several time points after extensive degranulation induced by pilocarpine injection in order to investigate the volume changes of rough endoplasmic reticulum (RER), nucleus, mitochondria and autophagosomes. At 2-4 h after stimulation, when the pancreatic cells demonstrated a complete loss of granules, this was accompanied by an increased proportion of autophagosomal activity. This change primarily reflected a greatly increased proportion of profiles retaining autophagic vacuoles containing recognisable cytoplasmic structures such as mitochondria, granule profiles and fragments of RER. The mitochondrial structures reached a significant maximal size 4 h following injection (before degranulation 0.178p0.028 µm$ ; at 4 h peak value, 0.535p0.109 µm$). Nucleus size showed an early volume increase approaching a maximum value 2 h following degranulation. The regranulation span was thus divided into 3 stages. The first was the membrane remodelling stage (0-2 h). During this period the volume of the RER and secretory granules was greatly decreased. At the intermediate stage (2-4 h) a significant increase of the synthesis zone was observed within the nucleus. The volume of the mitochondria was increasing. At the last step, the major finding was a significant granule accumulation in parallel with an active Golgi zone.

The effect of furosemide, a blocker of the Na+/K+/Cl- cotransporter, on hypothermic preservation of rat hearts was studied with use of the Langendorff perfusion system and electron microscopy. Furosemide significantly improved the mechanical recovery and the coronary flow of the hearts preserved for 8 hours in St. Thomas' Hospital cardioplegic solution at a temperature of 4 degrees C. Furosemide at the concentration of 100 mumol/L was found to have an optimal effect, whereas at high concentrations (1000 mumol/L) it was found to have toxic effects. In addition, furosemide reduces the time elapsed between the end of the preservation time and the resumption of myocardial contractions. Ultrastructural evaluations were done in which the presence of swollen mitochondria was chosen as a criterion of hypothermic ischemic damage to the myocardium. Morphometric analysis indicated that the mitochondrial volume of hearts stored for 8 hours in St. Thomas' Hospital cardioplegic solution increased by 72% as compared with the mitochondrial volume of hearts that were not exposed to the hypothermic ischemic conditions (control group). The addition of 100 mumol/L furosemide to the cardioplegic solution resulted in a significant reduction of mitochondrial swelling during the period of 8 hours' storage, which amounted only to 28% as compared with the figure for the control group. The reduction of mitochondrial swelling by furosemide and the improved mechanical and coronary flow recoveries are thought to be related to the blocking of the sarcolemmal Na+/K+/Cl- cotransporter and consequently the reduction of the Na+ influx during hypothermic ischemic storage.