Complete amino acid sequences of the three collagen-like regions present in subcomponent C1q of the first component of human complement.

Possible interactions between polymerized (F-) actin and insulin-storage granules from rat islets of Langerhans were examined in vitro by comparing the sedimentation of the granules in the presence of various actin concentrations. Actin in the concentration range 0.1--0.5 mg/ml produced a retardation in granule-sedimentation rates consistent with binding of the granules to the actin filaments. The interaction was increased by addition of ATP (2mM), but was decreased by CaCl2 (0.1 mM). Binding of granules to actin was unaffected by cyclic AMP or by preincubation of the granules with phospholipase C. Specificity of the interaction was confirmed by the use of depolymerized (G-) actin and of myosin to provide a solution of comparable viscosity; neither of these caused any alteration of granule sedimentation. Possible implications of this interaction of insulin-storage granules with actin for the mechanism of insulin secretion are briefly discussed.     

Quick-freeze,deep-etch visualization of exocytosis in anterior pituitary secretory cells: localization and possible roles of actin and annexin II

The exocytotic process in the anterior pituitary secretory cells was studied using quick-freeze deep-etch electron microscopy, fluorescein-isothiocyanate-phalloidin staining, heavy meromyosin decoration, and immuno-electron microscopy. The subcortical actin filaments are distributed unevenly in the peripheral cytoplasm. Few secretory granules are seen beneath the plasma membrane in the region where the peripheral cytoplasm is occupied by numerous subcortical actin filaments. On the contrary, in the region free of the subcortical actin filaments, many secretory granules lie in contact with the plasma membrane. Thus, the subcortical actin filaments may control the approach of the secretory granules to the plasma membrane in these cells. The granule and plasma membranes that lie in close proximity are linked by intervening strands. Unfused portions of both membranes remain linked by these strands during membrane fusion and opening. These strands may be involved in membrane contact, fusion and opening during exocytosis. Annexin II (calpactin I) has been demonstrated immunocytochemically to be localized at the contact sites between the granule and plasma membranes, and is therefore a possible component of the intervening strands. Membrane fusion starts within focal regions of both membranes less than 50 nm in diameter. The plasma membrane shows inward depressions toward the underlying granules immediately before fusion. The disappearance of intramembranous particles from the exocytotic site of the membrane has not been observed.     

Actomyosin interactions with insulin-storage granules in vitro.

Interactions between actomyosin and insulin storage granules isolated from rat islets of Langerhans have been examined in a simple system in vitro, which allows comparison of the sedimentation of the granules in the presence of absence of actomyosin in various conditions. Actomyosin altered granule-sedimentation rates in a manner consistent with the binding of the granules of actomyosin filaments. This interaction was enhanced by addition of ATP (1.5 mM) but unaltered by addition of CaCl2, by calmodulin or by calmodulin in the presence of 10 microM-CaCl2. Addition of EGTA (0.1 mM), cyclic AMP (10 microM) of cytochalasin B (10 microgram/ml) were also without effects in these conditions. Pre-incubation of granules with phospholipase c did not affect granule-actomyosin interaction. Ultrastructural studies showed close contacts between the membranes of the granules and actomyosin filaments. The results indicate the possibility that actomyosin might provide the motile force for granule translocation during the insulin secretory process.     

Ultrastructural and immunocytochemical studies on the cytoskeleton in the anterior pituitary of rats,with special regard to the relationship between actin filaments and secretory granules

Summary As previously reported, in anterior pituitary cells of the rat, secretory granules are linked with adjacent granules, cytoorganelles, microtubules, and plasma membrane by thin filaments, 4–10 nm in diameter. The quick-freeze, deep-etching method revealed that some of the filaments linking adjacent secretory granules show 5 nm-spaced striations on their surface which are known to be characteristic of actin. Immunocytochemistry showed that actin is localized in the cytoplasm beneath the plasma membrane, and around or between secretory granules. The heavy meromyosin decoration method demonstrated that actin filaments are mainly located in the cytoplasm beneath the plasma membrane, while some actin filaments are connected with the limiting membrane of the secretory granules. The actin filaments associated with the secretory granules are considered to be involved in the intracellular transport of the granules, while those localized in the peripheral cytoplasmic matrix might control the approach of the secretory granules to the plasma membrane and their release.This study was supported in part by grants from the Research Fund of the Ministry of Education, Science, and Culture, Japan     

Binding of microtubules to pituitary secretory granules and secretory granule membranes

Microtubules assembled in vitro were bound to purified porcine pituitary secretory granules and to isolated granule membranes. The interaction between microtubules and whole secretory granules was demonstrated by alteration in the sedimentation properties of the microtubules. Incubation of secretory granules with microtubules resulted in pelleting of microtubules which increased as a function of the number of granules added. Binding was quantitated by measurement of the tubulin remaining in the supernate after centrifugation. The interaction of secretory granules and microtubules was inhibited by nucleoside triphosphates and augmented by adenosine 5'-monophosphate and adenosine. When depolymerized protein from microtubules was incubated with secretory granules, the granules did not appear to bind the soluble tubulin dimer present in these preparations. However, the high molecular weight protein associated with microtubules was adsorbed by secretory granules during the binding process. Incubation of isolated secretory granule membranes with microtubules followed by centrifugation to density equilibrium in a discontinuous sucrose density gradient caused pelleting of the membranes, which otherwise banded higher in the gradient. The visible alteration in membrane sedimentation was confirmed by measurements of the membrane-associated magnesium-ATPase activity and by a shift in radioactivity in iodinated membrane preparations. Our data suggest a role for microtubules in the intracellular movement of secretory granules; this movement is perhaps brought about by dynein-like cross bridges which link the tubulin backbone and granule surface.     

Association of actin with chromaffin granule membranes and the effect of cytochalasin B on the polarity of actin filament elongation

Membranes of chromaffin granules isolated from bovine adrenal medulla are shown to bind dihydrocytochalasin B with high affinity. These membranes also bound [3H]actin in a time- and Mg2+-dependent manner and electron microscopy showed the presence of membrane-attached actin filaments following addition of exogenous actin. Binding of [3H]actin was partially inhibited by cytochalasin B. Electron microscopic analysis of heavy meromyosin-decorated, membrane-attached filaments showed terminally (end-on) attached filaments with both possible polarities (i.e., filaments with arrowheads pointing both towards and away from the membranes). Treatment of samples with cytochalasin B preferentially inhibited growth of filaments with their 'barbed' ends pointing away from membranes. These results are discussed with respect to the role of actin in secretory granule function and the mechanism of cytochalasin action.     

Involvement of the Rab27 binding protein Slac2c/MyRIP in insulin exocytosis

Rab27a is a GTPase associated with insulin-containing secretory granules of pancreatic beta-cells. Selective reduction of Rab27a expression by RNA interference did not alter granule distribution and basal secretion but impaired exocytosis triggered by insulin secretagogues. Screening for potential effectors of the GTPase revealed that the Rab27a-binding protein Slac2c/MyRIP is associated with secretory granules of beta-cells. Attenuation of Slac2c/MyRIP expression by RNA interference did not modify basal secretion but severely impaired hormone release in response to secretagogues. Although beta-cells express Myosin-Va, a potential partner of Slac2c/MyRIP, no functional link between the two proteins could be demonstrated. In fact, overexpression of the Myosin-Va binding domain of Slac2c/MyRIP did not affect granule localization and hormone exocytosis. In contrast, overexpression of the actin-binding domain of Slac2c/MyRIP led to a potent inhibition of exocytosis without detectable alteration in granule distribution. This effect was prevented by point mutations that abolish actin binding. Taken together our data suggest that Rab27a and Slac2c/MyRIP are part of a complex mediating the interaction of secretory granules with cortical actin cytoskeleton and participate to the regulation of the final steps of insulin exocytosis.     

Role of actin in regulated exocytosis and compensatory membrane retrieval: insights from an old acquaintance

This review summarizes new insights into the role of the actin cytoskeleton in exocytosis and compensatory membrane retrieval from mammalian regulated secretory cells. Data from our lab and others now indicate that the actin cytoskeleton is involved in exocytosis both as a negative regulator of membrane fusion under resting conditions and as a facilitator of movement of secretory granules to their site of fusion with the apical plasmalemma. Coating of docked secretory granules with actin filaments correlates with the dissociation of secretory-granule-associated rab3D, pointing out a novel role for rab proteins in modulating the actin cytoskeleton during regulated exocytosis. Compensatory membrane retrieval following regulated exocytosis is also critically dependent on the actin cytoskeleton both in initiating the formation of clathrin-coated retrieval vesicles and subsequent trafficking back into the cell. We propose that insertion of secretory granule membrane into the plasmalemma initiates a trigger for membrane retrieval, possibly by exposing sites where proteins involved in compensatory membrane retrieval are assembled. The results summarized in this review were derived primarily from investigations on the pancreatic acinar cell, an old friend who is providing modern wisdom not attainable in other simpler systems.     

Actomyosin II contractility expels von Willebrand factor from Weibel-Palade bodies during exocytosis

The study of actin in regulated exocytosis has a long history with many different results in numerous systems. A major limitation on identifying precise mechanisms has been the paucity of experimental systems in which actin function has been directly assessed alongside granule content release at distinct steps of exocytosis of a single secretory organelle with sufficient spatiotemporal resolution. Using dual-color confocal microscopy and correlative electron microscopy in human endothelial cells, we visually distinguished two sequential steps of secretagogue-stimulated exocytosis: fusion of individual secretory granules (Weibel-Palade bodies [WPBs]) and subsequent expulsion of von Willebrand factor (VWF) content. Based on our observations, we conclude that for fusion, WPBs are released from cellular sites of actin anchorage. However, once fused, a dynamic ring of actin filaments and myosin II forms around the granule, and actomyosin II contractility squeezes VWF content out into the extracellular environment. This study therefore demonstrates how discrete actin cytoskeleton functions within a single cellular system explain actin filament-based prevention and promotion of specific exocytic steps during regulated secretion.     

Changes in mobility of chromaffin granules in actin network with its assembly and Ca(2+)-dependent disassembly by gelsolin.

As a final stage of cell signal transduction, secretory cells release hormones by exocytosis. Before secretory granules contact with the cell membrane for fusion, an actin-network barrier must dissociate as a prelude. To elucidate dynamical behaviors of secretory granules in actin networks, in vitro assembly and disassembly processes of actin networks were examined by means of dynamic light-scattering spectroscopy. We studied actin polymerization in the presence of chromaffin granules isolated from bovine adrenal medullas and found that the entanglement of actin filaments rapidly formed cages that confined granules in them. We also studied the effect of gelsolin, one of actin-severing proteins, on the network of actin filaments preformed in the presence of chromaffin granules. It turned out that the cages that confined granules rapidly disappeared when gelsolin was added in the presence of free Ca2+ ions. A semiquantitative analysis of dynamic light-scattering spectra permitted us to estimate the changes in the mobility (or the translational diffusion coefficient) of chromaffin granules in the actin network with its assembly and Ca(2+)-dependent disassembly by gelsolin. Based on the present results and some pieces of evidence in the literature, a model is proposed for biophysical situations before, during, and after an exocytotic event.     

Localization and organization of actin in melanophores

Melanophores of the angelfish, Pterophyllum scalare, were studied in an attempt to demonstrate the existence of actin in these cells although microfilaments had previously not been found. By use of a variety of procedures, including immunofluorescence microscopy of intact and detergent-extracted cells, transmission electron microscopy, high voltage electron microscopy of whole-mount preparations, and labeling with heavy meromyosin-subfragment 1, the presence of a loose cortical mesh of actin filaments is demonstrated. In addition, a more parallel array of filaments is detected in microspike- and microvillus-like surface projections. There seem to be no changes in the arrangement of these filaments as a function of the state of pigment distribution. No actin filaments could be found in association with pigment granules or microtubules in more central cell portions. For reasons presently unknown, the preservation of the cortical filament network in lysed cell preparations depends strongly on the presence of an intact microtubular system. The involvement of this subplasmalemmal actin filament network in pigment granule transport remains unclear.     

Secretion in dissociated human pulmonary mast cells. Evidence for solubilization of granule contents before discharge

Mast cells were enzymatically dissociated from human lung fragments that had been sensitized with serum from human allergic to ragweed and were enriched by isopyknic and velocity gradient sedimentation. Electron microscope examination showed that the mast cells were well preserved at the end of the dissociation and isolation and that the majority of their secretory granules contained crystalline structures. These structures exhibited three patterns--scrolls, gratings, and lattices--which all could be found in the same granule. The period of crystalline structures was found to be bimodal, with maxima at 150 and 75 A. Both periods were observed in gratings that had been tilted and in scrolls that had been cut obliquely, indicating that the various gross patterns are composed of the same basic substructure. After the mast cells were stimulated by rabbit anti-human IgE to release histamine, the contents of the granule were transformed from a crystalline to an amorphous state, and only granules with amorphous contents were seen discharging from the cell. Clusters of intermediate filaments were present around the granules with amorphous contents, both deep in the cytoplasm and discharging at the cell surface. Discharge occurred both by fusion of granule membranes with the plasma membrane and by fusion of granule membranes with other granule membranes that ultimately were continuous with the plasma membrane. After discharge, the granule residue was fibrillar. Cells challenged with anti-human IgE in calcium-free medium neither released histamine nor demonstrated morphologic changes in their granules. We conclude that the crystalline state represents a storage form for materials that are solubilized before fusion of the granule membrane with the plasma membrane and discharge.     

Unfertilized sea urchin eggs contain a discrete cortical shell of actin that is subdivided into two organizational states

Changes in the distribution and organizational state of actin in the cortex of echinoderm eggs are believed to be important events following fertilization. To examine the initial distribution and form of actin in unfertilized eggs, we have adapted immunogold-labeling procedures for use with eggs of Strongylocentrotus purpuratus. Using these procedures, as well as fluorescence microscopy, we have revealed a discrete 1-micron-thick concentrated shell of actin in the unfertilized egg cortex. This actin is located in the short surface projections of unfertilized eggs and around the cortical granules in a manner that suggests it is associated with the cortical granule surface. The actin in the short surface projections appears to be organized into filaments. However, most if not all of the actin surrounding the cortical granules is organized in a form that does not bind phalloidin, even though it is accessible to actin antibody. The lack of phalloidin binding is consistent with either the presence of nonfilamentous actin associated with the cortical granules or the masking of actin-filament phalloidin-binding sites by some cellular actin-binding component. In addition to the concentrated shell of actin found in the cortex, actin was also found to be concentrated in the nuclei of unfertilized eggs.     

Isolation and partial characterization of two populations of secretory granules from rat parotid glands

Summary A method is described for the isolation of two populations of secretory granules from rat parotid glands utilizing differences in their sedimentation characteristics. The granule preparations were analyzed for homogeneity by electron microscopy and chemical analyses. The soluble contents of both types of granules were obtained by hypotonic lysis, and the proteins compared by SDS-PAGE and ion exchange-gel filtration chromatography. Both populations of secretory granules appear to have the same protein composition as that of the parotid saliva. The secretory granules with the smaller apparent buoyant density became labelled with radioactive leucine earlier than the heavier granules when a pulse of this amino acid was supplied to a gland slice system. The lighter granules appear to represent an earlier stage in maturation.     

β2-Syntrophin is a Cdk5 substrate that restrains the motility of insulin secretory granules

The molecular basis for the interaction of insulin granules with the cortical cytoskeleton of pancreatic β-cells remains unknown. We have proposed that binding of the granule protein ICA512 to the PDZ domain of β2-syntrophin anchors granules to actin filaments and that the phosphorylation/dephosphorylation of β2-syntrophin regulates this association. Here we tested this hypothesis by analyzing INS-1 cells expressing GFP-β2-syntrophin through the combined use of biochemical approaches, imaging studies by confocal and total internal reflection fluorescence microscopy as well as electron microscopy. Our results support the notion that β2-syntrophin restrains the mobility of cortical granules in insulinoma INS-1 cells, thereby reducing insulin secretion and increasing insulin stores in resting cells, while increasing insulin release upon stimulation. Using mass spectrometry, in vitro phosphorylation assays and β2-syntrophin phosphomutants we found that phosphorylation of β2-syntrophin on S75 near the PDZ domain decreases its binding to ICA512 and correlates with increased granule motility, while phosphorylation of S90 has opposite effects. We further show that Cdk5, which regulates insulin secretion, phosphorylates S75. These findings provide mechanistic insight into how stimulation displaces insulin granules from cortical actin, thus promoting their motility and exocytosis.     

Actin filament content and organization in unstimulated platelets

The extent of actin polymerization in unstimulated, discoid platelets was measured by DNase I inhibition assay in Triton X-100 lysates of platelets washed at 37 degrees C by gel filtration, or in Triton X-100 lysates of platelets washed at ambient temperatures by centrifugation in the presence of prostacyclin. About 40% of the actin in the discoid platelets obtained by either method existed as filaments. These filaments could be visualized by electron microscopy of thin sections. Similar results were obtained when the actin filament content of discoid platelets was measured by sedimentation of filaments from Triton X-100 lysates at high g forces (145,000 g for 45 min). However, few of these filaments sedimented at the lower g forces often used to isolate networks of actin filaments from cell extracts. These results indicate that actin filaments in discoid cells are not highly crosslinked. Platelets isolated by centrifugation in the absence of prostacyclin were not discoid, but were instead irregular with one or more pseudopodia. These platelets also contained approximately 40-50% of their actin in a filamentous form; many of these filaments sedimented at low g forces, however, indicating that they were organized into networks. The discoid shape of these centrifuged platelets could be restored by incubating them for 1-3 h at 37 degrees C, which resulted in the reversal of filament organization. High g forces were then required for the sedimentation of the actin. Approximately 80-90% of the actin in platelets washed at 4 degrees C was filamentous; this high actin filament content could be attributed to actin polymerization during the preparation of the platelets at low temperatures. These studies show that platelet activation involves mechanisms for the structural reorganization of existing filaments, in addition to those previously described for mediating actin polymerization.     

Aggregation and dispersity of isolated chromaffin granules studied by intensity fluctuation spectroscopy

The aggregation and dispersity of isolated bovine adrenal secretory vesicles (chromaffin granules) were studied by intensity fluctuation spectroscopy. The degree of dispersity and the Z-average translational diffusion coefficients were calculated from the autocorrelation functions of the intensity fluctuations in lase light scattered from the granules in solution. Granules purified by sedimentation through 0.3 M sucrose/Ficoll/²H₂O showed greater dispersity than granules purified by sedimentation through 1.6 M sucrose. By monitoring the scattered light intensity and the diffusion coefficients of the granules, many of the difficulties encountered in the interpretation of absorbance measurements were avoided. Measurements over a range of granule concentrations in sucrose solutions (10 mM HEPES, pH 7.0), indicated that aggregation of the granules occurred at concentrations above 150 μg protein/ml. At low granule concentrations (15–30 μg protein/ml) Ca²⁺-induced aggregation was detected at a threshold of 2–10 mM calcium.     

Light and electron microscopic observation on the cervical epithelium of the rabbit. I

The light and electron microscopy of the cervical epithelium of ovulatory, estrous, and long-term ovariectomized rabbits have been studied to determine what structural changes occur under different hormonal conditions. The percentage of nonciliated secretory cells is 49.6 in ovulatory, 43.6 in estrous, and 23.7 in long-term ovariectomized rabbits, and of ciliated cells is 50.2 in ovulatory, 56.2 in estrous, and 76.3 in long-term ovariectomized animals. The values for the ovulatory and estrous rabbits are significantly different at the P less than 0.05 level from those of the ovariectomized animals. In all 3 groups the general ultrastructure of the normal ciliated cells is similar. Interestingly, the Golgi complex is very prominent in all. Glycogen bodies occur frequently only in ciliated cells of ovariectomized and occasionally of estrous animals. Abnormalities in ciliation are quite common in the ovariectomized rabbits. The structure of the nonciliated secretory cells varies appreciably within and between the 3 groups. In these cells from well-developed epithelia of certain ovulatory and estrous animals, the apical cytoplasm contains secretory granules of at least three types. In addition, very irregularly shaped, dense, perinuclear granules occur, which may be another type of secretory granule or lysosomes. As compared to ciliated cells, the secretory cells have less prominent Golgi complexes, more abundant bundles of intermediate filaments, a more extensive glycocalyx on their apical surface, and more heterochromatic nuclei. In comparison to the cells of well-developed epithelia, the nonciliated cells of some other ovulatory and estrous rabbits are less well differentiated with fewer or no secretory granules and less well developed organelles. In the nonciliated cells of the long-term ovariectomized rabbits, there are no secretory or dense perinuclear granules. There is a decrease in the number of organelles that are involved in secretion, in the size of the cells, and in the amount of nuclear euchromatin.     

Localization of three types of the inositol 1,4,5-trisphosphate receptor/Ca(2+) channel in the secretory granules and coupling with the Ca(2+) storage proteins chromogranins A and B

Although the role of secretory granules as the inositol 1,4,5-trisphosphate (IP(3))-sensitive intracellular Ca(2+) store and the presence of the IP(3) receptor (IP(3)R)/Ca(2+) channel on the secretory granule membrane have been established, the identity of the IP(3)R types present in the secretory granules is not known. We have therefore investigated the presence of different types of IP(3)R in the secretory granules of bovine adrenal medullary chromaffin cells using immunogold electron microscopy and found the existence of all three types of IP(3)R in the secretory granules. To determine whether these IP(3)Rs interact with CGA and CGB, each IP(3)R isoform was co-transfected with CGA or CGB into NIH3T3 or COS-7 cells, and the expressed IP(3)R isoform and CGA or CGB were co-immunoprecipitated. From these studies it was shown that all three types of IP(3)R form complexes with CGA and CGB in the cells. To further confirm whether the IP(3)R isoforms and CGA and CGB form a complex in the secretory granules the potential interaction between all three isoforms of IP(3)R and CGA and CGB was tested by co-immunoprecipitation experiments of the mixture of secretory granule lysates and the granule membrane proteins. The three isoforms of IP(3)R were shown to form complexes with CGA and CGB, indicating the complex formation between the three isoforms of IP(3)R and CGA and CGB in the secretory granules. Moreover, the pH-dependent Ca(2+) binding property of CGB was also studied using purified recombinant CGB, and it was shown that CGB bound 93 mol of Ca(2+)/mol with a dissociation constant (K(d)) of 1.5 mm at pH 5.5 but virtually no Ca(2+) at pH 7.5. The high capacity, low affinity Ca(2+)-binding property of CGB at pH 5.5 is comparable with that of CGA and is in line with its role as a Ca(2+) storage protein in the secretory granules.     

Control of local actin assembly by membrane fusion-dependent compartment mixing

Local actin assembly is associated with sites of exocytosis in processes ranging from phagocytosis to compensatory endocytosis. Here, we examine whether the trigger for actin-coat assembly around exocytosing Xenopus egg cortical granules is 'compartment mixing'--the union of the contents of the plasma membrane with that of the secretory granule membrane. Consistent with this model, compartment mixing occurs on cortical granule-plasma membrane fusion and is required for actin assembly. Compartment mixing triggers actin assembly, at least in part, through diacylglycerol (DAG), which incorporates into the cortical granule membranes from the plasma membrane after cortical granule-plasma membrane fusion. DAG, in turn, directs long-term recruitment of protein kinase Cbeta (PKCbeta) to exocytosing cortical granules, where it is required for activation of Cdc42 localized on the cortical granules. The results demonstrate that mixing of two membrane compartments can direct local actin assembly and indicate that this process is harnessed during Xenopus egg cortical granule exocytosis to drive compensatory endocytosis.