Rab5 is critical for SNAP23 regulated granule-granule fusion during compound exocytosis.

Compound exocytosis is considered the most massive mode of exocytosis, during which the membranes of secretory granules (SGs) fuse with each other to form a channel through which the entire contents of their granules is released. The underlying mechanisms of compound exocytosis remain largely unresolved. Here we show that the small GTPase Rab5, a known regulator of endocytosis, is pivotal for compound exocytosis in mast cells. Silencing of Rab5 shifts receptor-triggered secretion from a compound to a full exocytosis mode, in which SGs individually fuse with the plasma membrane. Moreover, we show that Rab5 is essential for FcεRI-triggered association of the SNARE protein SNAP23 with the SGs. Direct evidence is provided for SNAP23 involvement in homotypic SG fusion that occurs in the activated cells. Finally, we show that this fusion event is prevented by inhibition of the IKKß2 kinase, however, neither a phosphorylation-deficient nor a phosphomimetic mutant of SNAP23 can mediate homotypic SG fusion in triggered cells. Taken together our findings identify Rab5 as a heretofore-unrecognized regulator of compound exocytosis that is essential for SNAP23-mediated granule-granule fusion. Our results also implicate phosphorylation cycles in controlling SNAP23 SNARE function in homotypic SG fusion.

Different activation signals induce distinct mast cell degranulation strategies.

Mast cells (MCs) influence intercellular communication during inflammation by secreting cytoplasmic granules that contain diverse mediators. Here, we have demonstrated that MCs decode different activation stimuli into spatially and temporally distinct patterns of granule secretion. Certain signals, including substance P, the complement anaphylatoxins C3a and C5a, and endothelin 1, induced human MCs rapidly to secrete small and relatively spherical granule structures, a pattern consistent with the secretion of individual granules. Conversely, activating MCs with anti-IgE increased the time partition between signaling and secretion, which was associated with a period of sustained elevation of intracellular calcium and formation of larger and more heterogeneously shaped granule structures that underwent prolonged exteriorization. Pharmacological inhibition of IKK-ß during IgE-dependent stimulation strongly reduced the time partition between signaling and secretion, inhibited SNAP23/STX4 complex formation, and switched the degranulation pattern into one that resembled degranulation induced by substance P. IgE-dependent and substance P-dependent activation in vivo also induced different patterns of mouse MC degranulation that were associated with distinct local and systemic pathophysiological responses. These findings show that cytoplasmic granule secretion from MCs that occurs in response to different activating stimuli can exhibit distinct dynamics and features that are associated with distinct patterns of MC-dependent inflammation.

Function Suggests Nano-Structure: Quantitative Structural Support for SNARE-Mediated Pore Formation.

Granule secretory content is released in either basal or calcium-activated complete exocytosis mode. A vital element in these processes is the establishment of a fusion pore between the granule membrane and the plasma membrane, initiated by the formation of a circular rosette docking arrangement of SNARE protein complexes. The controversially disputed number of SNARE complexes needed for granule priming leading to the formation of the fusion pore, is granule-size dependent and varies between secretion modes. Resorting to a statistical mechanics approach that views SNARE complexes and Ca(2+) ions as interacting particles, we have developed a relationship that links secretion rate to SNARE rosette size, Ca(2+) concentration and Ca(2+) ion cooperativity. Data are presented and discussed which suggest this SNARE-dependent generalization of existing narrow-range biophysical models that correlate secretion rate with Ca(2+) concentration and maximal Ca(2+) ion cooperativity. Evidence from dozens of examples in the literature advocate for this relation, which holds through the entire biological range. The coalescence of so many areas of diverse research methodologies has greatly augmented our understanding of so many different sequences of granule life cycle. Accordingly, these new tools may become valuable in a variety of electrophysiological experiments.

The econobiology of pancreatic acinar cells granule inventory and the stealthy nano-machine behind it.

The pancreatic gland secretes most of the enzymes and many other macromolecules needed for food digestion in the gastrointestinal tract. These molecules play an important role in digestion, host defense and lubrication. The secretion of pancreatic proteins ensures the availability of the correct mix of proteins when needed. This review describes model systems available for the study of the econobiology of secretory granule content. The secretory pancreatic molecules are stored in large dense-core secretory granules that may undergo either constitutive or evoked secretion, and constitute the granule inventory of the cell. It is proposed that the Golgi complex functions as a distribution center for secretory proteins in pancreatic acinar cells, packing the newly formed secretory molecules into maturing secretory granules, also known functionally as condensing vacuoles. Mathematical modelling brings forward a process underlying granule inventory maintenance at various physiological states of condensation and aggregation by homotypic fusion. These models suggest unique but simple mechanisms accountable for inventory buildup and size, as well as for the distribution of secretory molecules into different secretory pathways in pancreatic acinar cells.

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.

The stealthy nano-machine behind mast cell granule size distribution.

The classical model of mast cell secretory granule formation suggests that newly synthesized secretory mediators, transported from the rough endoplasmic reticulum to the Golgi complex, undergo post-transitional modification and are packaged for secretion by condensation within membrane-bound granules of unit size. These unit granules may fuse with other granules to form larger granules that reside in the cytoplasm until secreted. A novel stochastic model for mast cell granule growth and elimination (G&E) as well as inventory management is presented. Resorting to a statistical mechanics approach in which SNAP (Soluble NSF Attachment Protein) REceptor (SNARE) components are viewed as interacting particles, the G&E model provides a simple 'nano-machine' of SNARE self-aggregation that can perform granule growth and secretion. Granule stock is maintained as a buffer to meet uncertainty in demand by the extracellular environment and to serve as source of supply during the lead time to produce granules of adaptive content. Experimental work, mathematical calculations, statistical modeling and a rationale for the emergence of nearly last-in, first out inventory management, are discussed.

Characterization of mast cell secretory granules and their cell biology.

Exocytosis and secretion of secretory granule (SG) contained inflammatory mediators is the primary mechanism by which mast cells exert their protective immune responses in host defense, as well as their pathological functions in allergic reactions and anaphylaxis. Despite their central role in mast cell function, the molecular mechanisms underlying the biogenesis and secretion of mast cell SGs remain largely unresolved. Early studies have established the lysosomal nature of the mast cell SGs and implicated SG homotypic fusion as an important step occurring during both their biogenesis and compound secretion. However, the molecular mechanisms that account for key features of this process largely remain to be defined. A novel high-resolution imaging based methodology allowed us to screen Rab GTPases for their phenotypic and functional impact and identify Rab networks that regulate mast cell secretion. This screen has identified Rab5 as a novel regulator of homotypic fusion of the mast cell SGs that thereby regulates their size and cargo composition.

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.

Statistical analysis of the quantal basis of secretory granule formation.

The size distribution of vesicles exocytosed from secretory cells displays quantal nature, vesicle volume is periodic multi-modal, suggesting that these heterogeneous vesicles are aggregate sums of a variable number of homogeneous basic granules. Whether heterogeneity is a lumping-together artifact of the measurement or an inherent intra-cell feature of the vesicles is an unresolved question. Recent empirical evidence will be provided for the quantal nature of intra-cell vesicle volume, supporting the controversial paradigm of homotypic fusion: basic cytoplasmic granules fuse with each other to create heterogeneously sized vesicles. An EM-algorithm-based method is presented for the conversion of multi-modal to quantal data that provides as by-product estimates of means and variances of basic granule packaging.

Quantal Basis of Secretory Granule Biogenesis and Inventory Maintenance: the Surreptitious Nano-machine Behind It.

Proteins are molecular machines with the capacity to perform diverse physical work as response to signals from the environment. Proteins may be found as monomers or polymers, two states that represent an important subset of protein interactions and generate considerable functional diversity, leading to regulatory mechanisms closely akin to decision-making in service systems. Polymerization is not unique to proteins. Other cell compartments (e.g. secretory granules) or tissue states (e.g. miniature end plate potential) are associated with polymerization of some sort, leading to information transport. This data-processing mechanism has similarities with (and led us to the investigation of) granule homotypic polymerization kinetics. Using information theory, we demonstrate the role played by the heterogeneity induced by polymerization: granule size distribution and the stealthy machine behind granule life cycle increase system entropy, which modulates the source/receiver potential that affects communication between the cell and its environment. The granule inventory management by the same nano-machine is discussed.

Function suggests nano-structure: towards a unified theory for secretion rate, a statistical mechanics approach.

The inventory of secretory granules along the plasma membrane can be viewed as maintained in two restricted compartments. The release-ready pool represents docked granules available for an initial stage of fast, immediate secretion, followed by a second stage of granule set-aside secretion pool, with significantly slower rate. Transmission electron microscopy ultra-structural investigations correlated with electrophysiological techniques and mathematical modelling have allowed the categorization of these secretory vesicle compartments, in which vesicles can be in various states of secretory competence. Using the above-mentioned approaches, the kinetics of single vesicle exocytosis can be worked out. The ultra-fast kinetics, explored in this study, represents the immediately available release-ready pool, in which granules bound to the plasma membrane are exocytosed upon Ca(2+) influx at the SNARE rosette at the base of porosomes. Formalizing Dodge and Rahamimoff findings on the effect of calcium concentration and incorporating the effect of SNARE transient rosette size, we postulate that secretion rate (rate), the number (X) of intracellular calcium ions available for fusion, calcium capacity (0 ≤ M ≤ 5) and the fusion nano-machine size (as measured by the SNARE rosette size K) satisfy the parsimonious M-K relation rate ≈ C × [Ca(2+)](min(X,M))e(-K/2).

Function suggests nano-structure: electrophysiology supports that granule membranes play dice.

Cellular communication depends on membrane fusion mechanisms. SNARE proteins play a fundamental role in all intracellular fusion reactions associated with the life cycle of secretory vesicles, such as vesicle-vesicle and vesicle plasma membrane fusion at the porosome base in the cell plasma membrane. We present growth and elimination (G&E), a birth and death model for the investigation of granule growth, its evoked and spontaneous secretion and their information content. Using a statistical mechanics approach in which SNARE components are viewed as interacting particles, the G&E model provides a simple 'nano-machine' of SNARE self-aggregation behind granule growth and secretion. Results from experimental work, mathematical calculations and statistical modelling suggest that for vesicle growth a minimal aggregation of three SNAREs is required, while for the evoked secretion one SNARE is enough. Furthermore, the required number of SNARE aggregates (which varies between cell types and is nearly proportional to the square root of the mean granule diameter) affects and is statistically identifiable from the size distributions of spontaneous and evoked secreted granules. The new statistical mechanics approach to granule fusion is bound to have a significant changing effect on the investigation of the pathophysiology of secretory mechanisms and methodologies for the investigation of secretion.

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.

Quantal basis of vesicle growth and information content, a unified approach.

Secretory vesicles express a periodic multimodal size distribution. The successive modes are integral multiples of the smallest mode (G(1)). The vesicle content ranges from macromolecules (proteins, mucopolysaccharides and hormones) to low molecular weight molecules (neurotransmitters). A steady-state model has been developed to emulate a mechanism for the introduction of vesicles of monomer size, which grow by a unit addition mechanism, G(1)+G(n)-->G(n+1) which, at a later stage are eliminated from the system. We describe a model of growth and elimination transition rates which adequately illustrates the distributions of vesicle population size at steady-state and upon elimination. Consequently, prediction of normal behavior and pathological perturbations is feasible. Careful analysis of spontaneous secretion, as compared to short burst-induced secretion, suggests that the basic character-code for reliable communication should be within a range of only 8-10 vesicles' burst which may serve as a yes/no message.

Regulation of secretory granule size by the precise generation and fusion of unit granules.

Morphometric evidence derived from studies of mast cells, pancreatic acinar cells and other cell types supports a model in which the post-Golgi processes that generate mature secretory granules can be resolved into three steps: (1) fusion of small, Golgi-derived progranules to produce immature secretory granules which have a highly constrained volume; (2) transformation of such immature granules into mature secretory granules, a process often associated with a reduction in the maturing granule's volume, as well as changes in the appearance of its content and (3) fusion of secretory granules of the smallest size, termed 'unit granules', forming granules whose volumes are multiples of the unit granule's volume. Mutations which perturb this process can cause significant pathology. For example, Chediak-Higashi syndrome / lysosomal trafficking regulator (CHS)/(Lyst) mutations result in giant secretory granules in a number of cell types in human beings with the Chediak-Higashi syndrome and in 'beige' (Lyst(bg)/Lyst(bg)) mice. Analysis of the secretory granules of mast cells and pancreatic acinar cells in Lyst-deficient beige mice suggests that beige mouse secretory granules retain the ability to fuse randomly with other secretory granules no matter what the size of the fusion partners. By contrast, in normal mice, the pattern of granule-granule fusion occurs exclusively by the addition of unit granules, either to each other or to larger granules. The normal pattern of fusion is termed unit addition and the fusion evident in cells with CHS/Lyst mutations is called random addition. The proposed model of secretory granule formation has several implications. For example, in neurosecretory cells, the secretion of small amounts of cargo in granules constrained to a very narrow size increases the precision of the information conveyed by secretion. By contrast, in pancreatic acinar cells and mast cells, large granules composed of multiple unit granules permit the cells to store large amounts of material without requiring the amount of membrane necessary to package the same amount of cargo into small granules. In addition, the formation of mature secretory granules that are multimers of unit granules provides a mechanism for mixing in large granules the contents of unit granules which differ in their content of cargo.

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.

Imaging of zymogen granules in fully wet cells: evidence for restricted mechanism of granule growth.

The introduction of wet SEM imaging technology permits electron microscopy of wet samples. Samples are placed in sealed specimen capsules and are insulated from the vacuum in the SEM chamber by an impermeable, electron-transparent membrane. The complete insulation of the sample from the vacuum allows direct imaging of fully hydrated, whole-mount tissue. In the current work, we demonstrate direct inspection of thick pancreatic tissue slices (above 400 mum). In the case of scanning of the pancreatic surface, the boundaries of intracellular features are seen directly. Thus no unfolding is required to ascertain the actual particle size distribution based on the sizes of the sections. This method enabled us to investigate the true granule size distribution and confirm early studies of improved conformity to a Poisson-like distribution, suggesting that the homotypic granule growth results from a mechanism, which favors the addition of a single unit granule to mature granules.

Regional and developmental variations of blood vessel morphometry in the chick embryo chorioallantoic membrane.

Avian eggs contain all the necessary materials for embryonic development except for oxygen, which diffuses in from the environment via pores in the hard, calcified eggshell to the chorioallantoic membrane (CAM), the respiratory organ, which is rich in blood vessels. An air cell is formed at the blunt pole of the egg between the two membranes of the eggshell and enlarges during incubation due to water vapor loss. In this study of the CAM of chicken eggs, we compared blood vessel numerical density [N(A(v))], area fraction of blood vessels [A(A(v))], CAM thickness (D(CAM)), total length of blood vessels (L) and surface area of the CAM attached to the eggshell (CAMre) with those under the air cell (CAMac) during incubation. We found that N(A(v)), A(A(v)), D(CAM) and L of the CAM increase with embryonic age and development. The N(A(v)), A(A(v)) and L under the air cell were higher in relation to the rest of the CAM at all ages tested, while the D(CAM) under the air cell was always lower than around the rest of the egg. Since the eggshell over the air cell has a relatively greater porosity, and the respiratory gas exchange ratio there is higher than at other areas of the egg, there is a correlation between all the above morphometric data and the eggshell porosity. This suggests optimization of embryonic gas exchange in the chicken egg. We would like to propose that, during natural incubation, an increased gas diffusion under the air cell, together with increased blood vessel numerical density, may compensate for covering of the central part of the eggshell by the incubating parent.

Quantitative microscopy of human DNA topoisomerase II-alpha expression in transitional cell carcinoma of the bladder.

OBJECTIVE: To compare the nuclear size of various grades of transitional cell carcinoma (TCC) stained immunohistochemically with the nuclear enzyme topoisomerase II-alpha (topo II-alpha) in bladder urothelial neoplasms. STUDY DESIGN: Histologic sections from 53 consecutive papillary bladder neoplasms were stained immunohistochemically for topo II-alpha expression. There were 18 (33.9%) urothelial neoplasms of low malignant potential (UNLMP), 18 (33.9%) low grade urothelial carcinoma (LGUCa), and 17 (32%) with high grade urothelial carcinoma (HGUCa). The histologic slides were photographed at 400 x magnification and then projected on a screen, and the area with stained nuclei was measured. RESULTS: The cells and nuclei in HGUCa were significantly larger than in LGUCa (P < .05) and UNLMP (P < .01). CONCLUSION: Calculation of the area fraction of nuclei in TCC of the bladder stained with topo II-alpha is an additional method of establishing the grade of these tumors.