Submaximal Responses in Calcium-triggered Exocytosis Are Explained by Differences in the Calcium Sensitivity of Individual Secretory Vesicles

A graded response to calcium is the defining feature of calcium-regulated exocytosis. That is, there exist calcium concentrations that elicit submaximal exocytotic responses in which only a fraction of the available population of secretory vesicles fuse. The role of calcium-dependent inactivation in defining the calcium sensitivity of sea urchin egg secretory vesicle exocytosis in vitro was examined. The cessation of fusion in the continued presence of calcium was not due to calcium-dependent inactivation. Rather, the calcium sensitivity of individual vesicles within a population of exocytotic vesicles is heterogeneous. Any specific calcium concentration above threshold triggered subpopulations of vesicles to fuse and the size of the subpopulations was dependent upon the magnitude of the calcium stimulus. The existence of multiple, stable subpopulations of vesicles is consistent with a fusion process that requires the action of an even greater number of calcium ions than the numbers suggested by models based on the assumption of a homogeneous vesicle population.     

Calcium Transport in the Green Alga Mesotaenium caldariorum: Preliminary Characterization and Subcellular Distribution

The subcellular localization and biochemical characterization of calcium transport were studied in the unicellular green alga Mesotaenium caldariorum. Membrane fractions prepared by osmotic lysis of Mesotaenium protoplasts exhibit high rates of ATP-dependent calcium uptake. Sucrose gradient centrifugation separates two pools of activity, which display specific activities for calcium transport as high as 15 nanomoles Ca²⁺ per minute per milligram of protein. Marker enzyme analysis shows that this dual distribution of calcium transport activity is similar to that of vanadate-insensitive ATPase and pyrophosphatase, activities considered to be associated with the tonoplast. Plasma membranes, endoplasmic reticulum vesicles, mitochondrial membranes, and thylakoids band at higher densities than either calcium transport fraction. Both pools of ATP-dependent calcium uptake contain two components which are not separable on sucrose gradients but can be distinguished on the basis of inhibitor sensitivity. One component is inhibited by nigericin or trimethyltin chloride (I₅₀ values of 3 nanomolar and 4 micromolar, respectively), while the other component is vanadate sensitive (I₅₀ of 25 micromolar). These results suggest that direct Ca²⁺ transport and Ca²⁺/H⁺ antiport activities are present in both sucrose gradient fractions.     

Calcium requirements for exocytosis do not delimit the releasable neuropeptide pool

Recently, it was proposed that secretory vesicles have widely varying Ca(2+) thresholds for exocytosis. This model can explain adaptation of secretory responses and predicts that incomplete release is a consequence of insufficient Ca(2+). However, membrane capacitance-based measurements have not supported varying Ca(2+) thresholds. Here, Green Fluorescent Protein (GFP) imaging is used to test whether a Ca(2+) limitation determines the size of the releasable neuropeptide pool in differentiated PC12 cells. We show that depolarization-evoked release correlates with failure to sustain fully elevated [Ca(2+)](i). However, this is coincidental because release remains incomplete when [Ca(2+)](i) is maintained at a relatively high level by application of an ionophore or by dialysis with a buffered Ca(2+) solution. Furthermore, in contradiction with the existence of high threshold vesicles, stimulating maximal release with moderate [Ca(2+)](i) prevents secretory responses to large increases in [Ca(2+)](i) induced by photolysis of the caged dimethoxynitrophenyl-EGTA-4 (DMNPE-4). Thus, optical measurements show that limited capacity for neuropeptide release in response to depolarization is not caused by an insufficient duration of [Ca(2+)](i) elevation or by variation among vesicles in Ca(2+) sensitivity for exocytosis.     

Exocytosis reconstituted from the sea urchin egg is unaffected by calcium pretreatment of granules and plasma membrane

Micromolar calcium ion concentrations stimulate exocytosis in a reconstituted system made by recombining in the plasma membrane and cortical secretory granules of the sea urchin egg. The isolated cortical granules are unaffected by calcium concentrations up to 1 mM, nor do granule aggregates undergo any mutual fusion at this concentration. Both isolated plasma membrane and cortical granules can be pretreated with 1 mM Ca before reconstitution without affecting the subsequent exocytosis of the reconstituted system in response to micromolar calcium concentrations. On reconstitution, aggregated cortical granules will fuse with one another in response to micromolar calcium provided that one of their number is in contact with the plasma membrane. If exocytosis involves the generation of lipid fusogens, then these results suggest that the calcium-stimulated production of a fusogen can occur only when contiguity exists between cortical granules and plasma membrane. They also suggest that a substance involved in exocytosis can diffuse and cause piggy-back fusion of secretory granules that are in contact with the plasma membrane. Our results are also consistent with a scheme in which calcium ions cause a reversible, allosteric activation of an exocytotic protein.     

Calcium/sodium exchange in purified secretory vesicles from bovine neurohypophyses

Purified secretory vesicles isolated from bovine neurohypophyses take up Na+ under the same circumstances where an efflux of Ca2+ takes place, suggesting a Na+/Ca2+ exchange. Potassium cannot substitute for Na+ in this process. Also, a Ca2+/Ca2+ exchange can occur. Inhibiting the latter process by Mg2+ allowed to estimate an apparent KM of 0.7 microM free Ca2+ and a maximal uptake of 1.5 nmol X mg protein-1 X min-1 Ca2+ in exchange for Na+. The vesicles did not contain plasma membrane marker (Na+/K+ ATPase) as shown by distribution analyses on the density gradients on which they were purified. Similarly, distribution studies also showed that no other ATPase activity could be detected in the purified vesicle fraction. It is concluded that a Na+/Ca2+ exchange is operating across the secretory vesicle membrane and that it is not directly dependent on ATP hydrolysis.     

Fusion between disk membranes and plasma membrane of bovine photoreceptor cells is calcium dependent.

Disk membranes and plasma membrane vesicles were prepared from bovine retinal rod outer segments (ROS). The plasma membrane vesicles were labeled with the fluorescent probe octadecylrhodamine B chloride (R18) to a level at which the R18 fluorescence was self-quenched. At pH 7.4 and 37 degrees C and in the presence of micromolar calcium, an increase in R18 fluorescence with time was observed when R18-labeled plasma membrane vesicles were introduced to a suspension of disks. This result was interpreted as fusion between the disk membranes and the plasma membranes, the fluorescence dequenching resulting from dilution of the R18 into the unlabeled membranes as a result of lipid mixing during membrane fusion. While the disk membranes exposed exclusively their cytoplasmic surface, plasma membrane vesicles were found with both possible orientations. These vesicles were fractionated into subpopulations with homogeneous orientation. Plasma membrane vesicles that were oriented with the cytoplasmic surface exposed were able to fuse with the disk membranes in a Ca(2+)-dependent manner. Fusion was not detected between disk membranes and plasma membrane vesicles oriented such that the cytoplasmic surface was on the interior of the vesicles. ROS plasma membrane-disk membrane fusion was stimulated by calcium, inhibited by EGTA, and unaffected by magnesium. Rod photoreceptor cells of vertebrate retinas undergo diurnal shedding of disk membranes containing the photopigment rhodopsin. Membrane fusion is required for the shedding process.     

Synaptic vesicle ceramide kinase. A calcium-stimulated lipid kinase that co-purifies with brain synaptic vesicles

Much current work on the mechanism of neurosecretion has focused on proteins specific to neural secretory vesicles (synaptic vesicles). We report a calcium-stimulated lipid kinase that co-purifies with rat brain synaptic vesicles. This enzyme activity is found only in membrane fractions that contain synaptic vesicle markers. Based on identification of the lipid product as ceramide 1-phosphate and on the finding that ceramide kinase activity co-purifies with synaptic vesicles, the enzyme is proposed to be a ceramide kinase. Kinase activity is stimulated by micromolar concentrations of calcium. Calcium increases the apparent Vmax of the reaction with little effect on the Km for ceramide. The vesicular localization of this enzyme, the requirement for ATP, and the stimulation of enzyme activity by micromolar calcium suggest that ceramide phosphorylation may be associated with neurotransmitter release.     

High calcium concentrations shift the mode of exocytosis to the kiss-and-run mechanism

Exocytosis, the fusion of secretory vesicles with the plasma membrane to allow release of the contents of the vesicles into the extracellular environment, and endocytosis, the internalization of these vesicles to allow another round of secretion, are coupled. It is, however, uncertain whether exocytosis and endocytosis are tightly coupled, such that secretory vesicles fuse only transiently with the plasma membrane before being internalized (the 'kiss-and-run' mechanism), or whether endocytosis occurs by an independent process following complete incorporation of the secretory vesicle into the plasma membrane. Here we investigate the fate of single secretory vesicles after fusion with the plasma membrane by measuring capacitance changes and transmitter release in rat chromaffin cells using the cell-attached patch-amperometry technique. We show that raised concentrations of extracellular calcium ions shift the preferred mode of exocytosis to the kiss-and-run mechanism in a calcium-concentration-dependent manner. We propose that, during secretion of neurotransmitters at synapses, the mode of exocytosis is modulated by calcium to attain optimal conditions for coupled exocytosis and endocytosis according to synaptic activity.     

Calcium dependencies of regulated exocytosis in different endocrine cells

Exocytotic machinery in neuronal and endocrine tissues is sensitive to changes in intracellular Ca(2+) concentration. Endocrine cell models, that are most frequently used to study the mechanisms of regulated exocytosis, are pancreatic beta cells, adrenal chromaffin cells and pituitary cells. To reliably study the Ca(2+) sensitivity in endocrine cells, accurate and fast determination of Ca(2+) dependence in each tested cell is required. With slow photo-release it is possible to induce ramp-like increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) that leads to a robust exocytotic activity. Slow increases in the [Ca(2+)](i) revealed exocytotic phases with different Ca(2+) sensitivities that have been largely masked in step-like flash photo-release experiments. Strikingly, in the cells of the three described model endocrine tissues (beta, chromaffin and melanotroph cells), distinct Ca(2+) sensitivity 'classes' of secretory vesicles have been observed: a highly Ca(2+)-sensitive, a medium Ca(2+)-sensitive and a low Ca(2+)-sensitive kinetic phase of secretory vesicle exocytosis. We discuss that a physiological modulation of a cellular activity, e.g. by activating cAMP/PKA transduction pathway, can switch the secretory vesicles between Ca(2+) sensitivity classes. This significantly alters late steps in the secretory release of hormones even without utilization of an additional Ca(2+) sensor protein.     

A Fine Structural Analysis of Auxin-induced Elongation of Cucumber Hypocotyls, and the Effects of Calcium Antagonists and Ionophores

The fine structure of epidermal cells, particularly in relationto dictyosomes, has been examined in different regions of dark-growncucumber hypocotyls and in response to auxin treatment, usingboth dot overlay and image analysis techniques. The most noticeablechange in cell structure along the hypocotyls is the increasein vacuolar volume. The volume fraction occupied by dictyosomesand secretory vesicles also increased, whereas that for mitochondriaremained relatively constant. During auxin treatment, the volumefraction for dictyosomes showed an increase after 30 min followedby a fall, whereas that occupied by secretory vesicles fellsteadily over 90 min. The number of cisternae per dictyosomeshowed some increase after 2 h of auxin treatment, althoughthe increase in dictyosomal material with cell expansion waslargely accounted for by an increase in the number of dictyosomes. Auxin-stimulated elongation growth of the hypocotyls was inhibitedby a range of calcium antagonists, chelators and ionophores.The most marked inhibitions were observed with calcium chloride,the chelator chlortetracycline and the ionophores verapamil,nigericin and monensin. Linear transducer experiments showedthat these compounds generally caused an immediate reductionin the rate of growth. Fine structural observations carriedout on epidermal cells showed the most obvious effects withmonensin and nigericin which caused dictyosomes and secretoryvesicles to swell. EGTA and LaCl₃ caused secretory vesiclesto accumulate around dictyosomes, while the ionophore A23187had little effect. The results suggest that the concentration of Ca²⁺ in the cytoplasmmay be critical for cell elongation. Compounds which chelateCa²⁺ appear to be more effective inhibitors of growth in theinitial acid-induced phase, whereas those which affect ionicgradients are more disruptive in the second phase.Copyright1993, 1999 Academic Press Calcium, Cucumis sativus hypocotyle, dictyosomes, elongation growth, indoleacetic acid, stereology     

Calcium gradients and the Golgi

Changes in intracellular free calcium regulate many intracellular processes. With respect to the secretory pathway and the Golgi apparatus, changes in calcium concentration occurring either in the adjacent cytosol or within the lumen of the Golgi act to regulate Golgi function. Conversely, the Golgi sequesters calcium to shape cytosolic calcium signals as well as initiate them by releasing calcium via inositol-1,4,5-triphosphate (IP(3)) receptors, located on Golgi membranes. Local calcium transients juxtaposed to the Golgi (arising from release by the Golgi or other organelles) can activate calcium dependent signalling molecules located on or around the Golgi. This review focuses on the reciprocal relationship between the cell biology of the Golgi apparatus and intracellular calcium homeostasis.     

Ca++-induced fusion of fragmented sarcoplasmic reticulum with artificial planar bilayers

Summary Addition of fragmented sarcoplasmic reticulum (SR) vesicles to the aqueous phase of a black lipid membrane (BLM) causes a large increase in BLM conductance within 10 min. The conductance increase is absolutely dependent on three conditions: The presence of at least 0.5mm Ca⁺⁺, an acidic phospholipid such as phosphatidylserine or diphosphatidylglycerol in the BLM phospholipid mixture, and an osmotic gradient across the SR vesicle membrane, with the internal osmolarity greater than the external. These requirements are identical to conditions under which the fusion of phospholipid vesicles occurs.When the early part of the time course of conductance rise is examined at high sensitivity, the conductance is seen to increase in discrete steps. The probability of a step increases with the concentration of Ca⁺⁺ in the medium, with the fraction of acidic phospholipid in the BLM, and with the size of the osmotic gradient across the SR vesicle membrane. On the other hand, the average conductance change per step is independent of the above parameters, but varies with the type and concentration of ions present in the aqueous phase. For a given ion, the mean specific conductance per step is independent of the ion's concentration between 10 and 100mm.The probability distribution of the step-conductances agrees well with the distribution of SR vesicle surface areas, both before and after sonication of the vesicles.The evidence indicates that SR vesicles fuse with the BLM, thereby inserting SR membrane conductance pathways into it. Each discrete conductance jump appears to be the result of the fusion of a single SR vesicle with the BLM. This technique may serve as a general method for inserting membrane vesicles into an electrically accessible system.     

Calcium oxalate and calcium phosphate capacities of cardiac sarcoplasmic reticulum

Both oxalate-supported and phosphate-supported calcium uptake by canine cardiac sarcoplasmic reticulum initially increase linearly with time but fall to a steady-state level within 20 min. The departure from linearity could be due to a decrease in influx or to an increase in efflux of calcium. Because Ca2+-ATPase activity is linear, a decrease in the influx of calcium is an unlikely cause of the non-linear calcium uptake curves. A possible cause of an increase in calcium efflux is rupture of the vesicles. This hypothesis was tested by investigating the amount of calcium which could be released upon addition of 5 mM EGTA. The amount of rapidly releasable calcium was zero until a threshold calcium uptake of about 4-6 mumol calcium oxalate or calcium phosphate per mg was reached. After that point the rapidly releasable calcium continued to increase with calcium oxalate to reach more than 23 mumol/mg, but stayed constant at about 0.7 mumol/mg for calcium phosphate. The rapidly releasable calcium was attributed to calcium oxalate or calcium phosphate crystals externalized by vesicle rupture. The differences in the amounts of rapidly releasable calcium were attributed to different kinetics of calcium phosphate and calcium oxalate dissolution. Addition of ryanodine caused a marked increase in the threshold for rapidly releasable calcium oxalate. Transmission electron micrographs showed that vesicles can become filled with calcium oxalate crystals, but the vesicles were heterogeneous with respect to their size and their sensitivity to ryanodine. These observations support the hypothesis that calcium oxalate and calcium phosphate capacities are limited by vesicle rupture and that ryanodine increases the capacity by closing a calcium channel in a subpopulation of vesicles that otherwise would not accumulate calcium.     

Calcium regulation by lens plasma membrane vesicles

The role of the plasma membrane in the regulation of lens fiber cell cytosolic Ca2+ concentration has been examined using a vesicular preparation derived from calf lenses. Calcium accumulation by these vesicles was ATP dependent, and was releasable by the ionophore A23187, indicating that calcium was transported into a vesicular space. Calcium accumulation was stimulated by Ca2+ (K1/2 = 0.08 microM Ca2+) potassium (maximally at 50 mM K+), and cAMP-dependent protein kinase; it was inhibited by both vanadate (IC50 = 5 microM) and the calmodulin inhibitor R24571 (IC50 = 5 microM), indicating that this pump was plasma-membrane derived and likely calmodulin dependent. Valinomycin, in the presence of K+, stimulated calcium uptake, suggesting that the calcium pump either countertransports K+, or is regulated in an electrogenic fashion. Inhibition of calcium uptake by selenite and p-chloromercuribenzoate demonstrates the presence of an essential -SH group(s) in this enzyme. Calcium release from calcium-filled lens vesicles was enhanced by Na+, demonstrating that these vesicles also contain a Na:Ca exchange carrier. p-Chloromercuribenzoate and p-chloromercuribenzoate sulfonic acid also promoted calcium release from calcium-filled vesicles, suggesting that this release, like calcium uptake, is in part mediated by a cysteine-containing protein. We conclude that lens fiber cell cytosolic Ca2+ concentration could be regulated by a number of plasma membrane processes. The sensitivity of both calcium uptake and release to -SH reagents has implications in lens cataract formation, where oxidation of lens proteins has been proposed to account for the elevated cytosolic Ca2+ in this condition.     

Pancreatic beta cell heterogeneity in glucose-induced insulin secretion.

Rat pancreatic beta cells differ in their individual sensitivity to glucose-inducible metabolic changes. The present study examines whether beta cells with a higher metabolic threshold require higher glucose levels for stimulation of their secretory activity. Purified beta cells were distributed according to their metabolic redox state at 7.5 mM glucose; the metabolically responsive (high responsive) and unresponsive (low responsive) subpopulations of comparable size and viability were reaggregated in the presence of [3H]tyrosine and then perfused at 2.8 mM glucose with 10-min pulses of increasing glucose concentration. Glucose elicited first-phase insulin release in both high and low responsive subpopulations from, respectively, 4.2 and 8.3 mM on. The amplitude of both secretory responses increased dose dependently, the rates in the high responsive subpopulation being 2-fold higher than in the low responsive one. At all stimulating glucose levels, fractional release of 3H-labeled insulin was 3- to 4-fold higher than that of immunoreactive insulin. Preferential release of newly formed insulin was already maximally stimulated at 4.2 mM glucose in the high responsive subpopulation, whereas it increased dose-dependently in the low responsive one. These results indicate the existence of intercellular differences in the secretory activity of glucose-exposed beta cells, both in terms of glucose sensitivity and of amplitude. This heterogeneity in beta cell secretory responsiveness parallels that which has been previously described for the cellular metabolic and biosynthetic functions. It is concluded that glucose dose-dependently recruits beta cells into both biosynthetic and secretory activities. Co-existence of inactive and activated cells can explain preferential release of newly synthesized over preformed hormone during glucose stimulation.     

Secretory vesicle formation in the secretory cavity of glandular trichomes of Cannabis sativa L. (Cannabaceae)

The disc cell wall facing the secretory cavity in lipophilic glands of Cannabis was studied for origin and distribution of hyaline areas, secretory vesicles, fibrillar matrix and particulate material. Secretions evident as light areas in the disc cell cytoplasm pass through modified regions in the plasma membrane and appear as hyaline areas in the cell wall. Hyaline areas, surrounded with a filamentous outline, accumulate near the wall surface facing the secretory cavity where they fuse to form enlarged hyaline areas. Fibrillar matrix is related to and may originate from the dense outer layer of the plasma membrane. This matrix becomes distributed throughout the wall material and contributes in part to the composition of the surface feature of secretory vesicles. Thickening of the cell wall is associated with secretions from the disc cells that facilitates movement of hyaline areas, fibrillar matrix and other possible secretions through the wall to form secretory vesicles and intervesicular materials in the secretory cavity. The outer wall of disc cells in aggregate forms the basilar wall surface of the secretory cavity which facilitates the organization of secretory vesicles that fill the secretory cavity.     

Calcium snapshots in the stigma and style of medlar (Lycium barbarum L.) during pollen germination and pollen tube growth suggest active calcium oscilations

Abstract

The stigma (tip of the pistil) of medlar is wet and covered with stigmatic exudate at anthesis. The exudate contains many vesicles with abundant calcium precipitates. After deposition on the stigma, the pollen grain undergoes hydration, displaying signs of calcium ion (Ca²⁺) transfer from the exudate vesicles into the pollen grains. Calcium precipitates in the pollen cytoplasm are concentrated into small vacuoles that fuse to form large vacuoles, which provide turgor pressure to push the cytoplasm to the apical region of the growing pollen tube. Many calcium precipitates are present in the stylar transmitting tract, which displays a calcium gradient: fewer precipitates are localised in the distal (upper) transmitting tissue below the stigma, and more precipitates are present in the transmitting tract at the style base. The emporal and spatial distribution of calcium in the stigma and style of medlar suggests that it satisfies the demand for calcium in vivo and played some functional significance.     

Calcium activation of mougeotia potassium channels

Phytochrome mediates chloroplast movement in the alga Mougeotia, possibly via changes in cytosolic calcium. It is known to regulate a calcium-activated potassium channel in the algal plasma membrane. As part of a characterization of the potassium channel, we examined the properties of calcium activation. The calcium ionophore A23187 activates the channel at external [Ca(2+)] as low as 20 micromolar. However, external [Ca(2+)] is not required for activation of the channel by photoactivated phytochrome. Furthermore, when an inhibitor of calcium release from internal stores, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, hydrochloride (TMB-8), is present, red light no longer stimulates channel activity. We conclude that phytochrome activates the plasma membrane potassium channel by releasing calcium from intracellular calcium vesicles; the elevated cytosolic calcium then stimulates channel activity by an unknown mechanism. In the presence of TMB-8, red light does induce chloroplast rotation; thus, potassium channel activation may not be coupled to chloroplast rotation.     

Calcium reduces the sodium permeability of luminal membrane vesicles from toad bladder. Studies using a fast-reaction apparatus

Regulation of the sodium permeability of the luminal membrane is the major mechanism by which the net rate of sodium transport across tight epithelia is varied. Previous evidence has suggested that the permeability of the luminal membrane might be regulated by changes in intracellular sodium or calcium activities. To test this directly, we isolated a fraction of the plasma membrane from the toad urinary bladder, which contains a fast, amiloride-sensitive sodium flux with characteristics similar to those of the native luminal membrane. Using a flow-quench apparatus to measure the initial rate of sodium efflux from these vesicles in the millisecond time range, we have demonstrated that the isotope exchange permeability of these vesicles is very sensitive to calcium. Calcium reduces the sodium permeability, and the half-maximal inhibitory concentration is 0.5 microM, well within the range of calcium activity found in cells. Also, the permeability of the luminal membrane vesicles is little affected by the ambient sodium concentration. These results, when taken together with studies on whole tissue, suggest that cell calcium may be an important regulator of transepithelial sodium transport by its effect on luminal sodium permeability. The effect of cell sodium on permeability may be mediated by calcium rather than by sodium itself.     

Co-localization of vesicles and P/Q Ca2+-channels explains the preferential distribution of exocytotic active zones in neurites emitted by bovine chromaffin cells.

We have taken advantage of the differences between the preferential localization of secretion in the terminals of neurite-emitting bovine chromaffin cells in contrast with the random distribution secretion in spherical cells to study the possible molecular factors determining such localization by using immunofluorescence and confocal microscopy techniques. By analyzing the distribution of dopamine beta-hydroxylase present in the membrane of chromaffin granules, we found that vesicles migrate and accumulate in dense packages in the terminals of neurite processes. Neither members of the fusion core complex such as SNAP-25, nor nicotinic receptors are preferentially located in the terminals as would be expected from elements defining sites of release, thereby suggesting the presence of additional factors. Interestingly, we observed a preferential distribution of the P/Q subtype of Ca2+ channels in these neurite terminals and co-localization with vesicles present in these structures, in sharp contrast with the overall distribution of the L subtype channels. Using the same immunofluorescence techniques we were unable to detect N-type calcium channels. In addition, omega-agatoxin IVA was able to block 70% of the exocytotic release occurring into the neurites, whereas L-type blockers had a weak effect. Taken together our results strongly indicate that the co-localization of vesicles and clusters of P/Q Ca2+ channels may explain the precise localization of exocytotic sites in the terminals of neurite-emitting chromaffin cells, whereas the distribution of secretory sites in round cells may arise from the random presence of these factors as indicated by their partial co-localization.