Quantitative microscopy of mole rat eosinophil granule morphology.

Mole rat bone marrow cells and peritoneal eosinophils are used to study granule morphological maturation by quantitative microscopy. The bulk eosinophil granule content is pre-stored in unique granular structures known as crystalloid or secondary granules. Mole rat eosinophil granules exhibit the basic structure of an electron-dense crystalloid core surrounded by a lighter, homogeneous matrix. Morphometric analysis demonstrated that bone marrow-derived eosinophil sphere-like granules display a periodic, multimodal granule volume distribution. In contrast, peritoneal eosinophils display cigar-shaped granules, whose crystalloid cores are more variable in size and shape as compared to bone marrow eosinophil granules. Using a morphometric approach, we deduced that the basic granule volume quantum is similar in both cases, suggesting that the sphere-like young eosinophil granules turn into dense ellipsoidal ones by intragranular processes in which both volume and membrane surface are conserved. Crystalloid granule mediators are known to be widely associated with allergic inflammatory events, which may damage the host tissue following secretion to the extracellular environment. Based on mathematical modeling, we suggest that this deviation from sphere-like to ellipsoidal shape reflects an adaptive response of the mole rat to its unique solitary life.

Rab5 is a novel regulator of mast cell secretory granules: impact on size, cargo, and exocytosis.

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.

VAMP8/endobrevin is a critical factor for the homotypic granule growth in pancreatic acinar cells.

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.

Combining an EGFR directed tyrosine kinase inhibitor with autophagy-inducing drugs: a beneficial strategy to combat non-small cell lung cancer.

The potential therapeutic value of combinatorial regimens based on an EGF receptor tyrosine kinase inhibitor (TKI) and autophagy inducing drugs was evaluated by comparing their molecular impacts on H1299 and A549 non-small cell lung cancer (NSCLC) cells, which overexpress wild type EGF receptor, but are either deficient or have wild type p53 alleles, respectively. We show that H1299 cells display a considerably lower sensitivity to erlotinib treatment, which can be restored by combining erlotinib with rapamycin or with imatinib, though to a lesser extent. Cytotoxicity was associated with increased autophagy and hyperpolarization of the mitochondrial membrane potential. Therefore, combining an EGF receptor directed TKI with an autophagy-inducing drug, preferably, rapamycin, might be beneficial in treating poor responding NSCLC patients.

Localization of anionic constituents in mast cell granules of brachymorphic (bm/bm) mice by using avidin-conjugated colloidal gold.

We used the egg avidin gold complex as a polycationic probe for the localization of negatively charged sites in the secretory granules of mouse mast cells. We compared the binding of this reagent to mast cell granules in wild-type mice and in congenic brachymorphic mice in which mast cell secretory granules contained undersulfated proteoglycans. We localized anionic sites by post-embedding labeling of thin sections of mouse skin and tongue tissues fixed in Karnovsky's fixative and OsO(4) and embedded in Araldite. Transmission electron microscopy revealed that the mast cell granules of bm/bm mice had a lower optical density than those of wild-type mice (P<0.001) and a lower avidin gold binding density (by approximately 50%, P<0.001). The latter result provided additional evidence that the contents of mast cell granules in bm/bm mice were less highly sulfated than in those of wild-type mice. In both wild-type and bm/bm mast cells, the distribution of granule equivalent volumes was multimodal, but the unit granule volume was approximately 19% lower in bm/bm cells than in wild-type cells (P<0.05). Thus, bm/bm mast cells develop secretory granules that differ from those of wild-type mice in exhibiting a lower optical density and slightly smaller unit granules, however the processes that contribute to granule maturation and granule-granule fusion in mast cells are operative in bm/bm cells.

Human oocyte cryopreservation and the fate of cortical granules.

OBJECTIVE: To examine the effect of the commonly used oocyte cryopreservation protocol on the cortical granules (CGs) of human immature germinal vesicle (GV) and mature metaphase II (MII) oocytes. DESIGN: Laboratory study. SETTING: IVF unit. INTERVENTION(S): Unfertilized, intracytoplasmic sperm injected (ICSI) oocytes, and immature oocytes were cryopreserved using a slow freezing-rapid thawing program with 1,2-propanediol (PROH) as a cryoprotectant. MAIN OUTCOME MEASURE(S): Cortical granule exocytosis (CGE) was assessed by either confocal microscopy or transmission electron microscopy (TEM). RESULT(S): The survival rates of frozen-thawed oocytes (mature and immature) were significantly lower compared with zygotes. Both mature and immature oocytes exhibited increased fluorescence after cryopreservation, indicating the occurrence of CGE. Mere exposure of oocytes to cryoprotectants induced CGE of 70% the value of control zygotes. The TEM revealed a drastic reduction in the amount of CGs at the cortex of frozen-thawed GV and MII oocytes, as well as appearance of vesicles in the ooplasm. CONCLUSION(S): The commonly used PROH freezing protocol for human oocytes resulted in extensive CGE. This finding explains why ICSI is needed to achieve fertilization of frozen-thawed human oocytes.