Membrane recycling after exocytosis: An ultrastructural study of cultured chromaffin cells

When exocytosis of granule contents is induced by nicotine stimulation, glycoprotein III (a chromaffin granule membrane constituent) is exposed on the surface of cultured chromaffin cells, where it may be labeled with an immunocytochemical tracer. The subsequent fate of this glycoprotein after endocytosis was followed at the ultrastructural level using immunogold methods and was analyzed by morphometry. After stimulation exocytosis membranes newly inserted into the plasma membrane labeled with gold particles for glycoprotein III were found to be endocytosed via coated vesicles and finally found in organelles devoid of chromogranin A, the major secretory granule protein. At intervals between 30 min and 24 h after cell stimulation and immunolabeling, most labeled structures were identified by two different morphological approaches as prelysosomes and lysosomes. In contrast with results obtained on freshly isolated chromaffin cells, it is thus concluded that in cultured cells granule membrane recycling into new granules does not occur. It is suggested that the fate of granule membrane endocytosed after cell stimulation may be influenced by the external conditions to which cells are previously exposed.     

Bovine chromaffin granule membranes undergo Ca(2+)-regulated exocytosis in frog oocytes

We have devised a new method that permits the investigation of exogenous secretory vesicle function using frog oocytes and bovine chromaffin granules, the secretory vesicles from adrenal chromaffin cells. Highly purified chromaffin granule membranes were injected into Xenopus laevis oocytes. Exocytosis was detected by the appearance of dopamine-beta-hydroxylase of the chromaffin granule membrane in the oocyte plasma membrane. The appearance of dopamine-beta-hydroxylase on the oocyte surface was strongly Ca(2+)-dependent and was stimulated by coinjection of the chromaffin granule membranes with InsP3 or Ca2+/EGTA buffer (18 microM free Ca2+) or by incubation of the injected oocytes in medium containing the Ca2+ ionophore ionomycin. Similar experiments were performed with a subcellular fraction from cultured chromaffin cells enriched with [3H]norepinephrine-containing chromaffin granules. Because the release of [3H]norepinephrine was strongly correlated with the appearance of dopamine-beta-hydroxylase on the oocyte surface, it is likely that intact chromaffin granules and chromaffin granule membranes undergo exocytosis in the oocyte. Thus, the secretory vesicle membrane without normal vesicle contents is competent to undergo the sequence of events leading to exocytosis. Furthermore, the interchangeability of mammalian and amphibian components suggests substantial biochemical conservation of the regulated exocytotic pathway during the evolutionary progression from amphibians to mammals.     

A cytochemical study of the calcium-activated adenosinetriphosphatase in hamster adrenal medulla: its occurrence in the golgi region of chromaffin cells

Summary Several different fixation procedures and incubation media were used in order to demonstrate the ultrastructural localisation of Ca²⁺-activated adenosinetriphosphatase (ATPase) in the hamster adrenal medulla. Fixation by perfusion with 2.5% glutaraldehyde gave the best preservation of fine structure without markedly inhibiting the enzymic activity. The localisation of Ca²⁺-activated ATPase was different from that of Mg²⁺-activated ATPase: the Mg²⁺-dependent enzyme was confined to plasma membranes. Ca²⁺-dependent ATPase also occurred on the plasma membranes of neurons and of some chromaffin cells, but the most prominent site of this enzyme was in the Golgi apparatus of chromaffin cells. Most of the reaction product was localised between Golgi lamellae, but some was found in Golgi vesicles and in prosecretory granules. The nucleus, mature chromaffin granules, roughsurfaced endoplasmic reticulum and mitochondria were usually free of reaction product. Rarely, some precipitate was found in the matrix of mitochondria and in lysosomes.Wellcome Research Fellow.J. H. Burn Research Scholar.This work was supported by a grant from the Medical Research Council.     

High-resolution three-dimensional views of membrane-associated clathrin and cytoskeleton in critical-point-dried macrophages

We obtained high-resolution topographical information about the distribution of clathrin and cytoskeletal filaments on cytoplasmic membrane surfaces of macrophages spreading onto glass coverslips by both critical-point drying of broken-open cells and preparation of rotary platinum replicas. Irregular patches of the adherent ventral surface of the plasma membrane were exposed in these cells, and large areas of these exposed membranes were covered with clathrin-coated patches, pits, and vesicles. Various amounts of cytoskeleton were attached to the plasma membranes of these spreading cells, either as distinct starlike foci, or as individual filaments and bundles radiating out from the cytoskeletal meshwork. In newly adherent cells a well developed Golgi-GERL complex, characterized by smooth, dish-like cisternae associated with rough endoplasmic reticulum, was observed. There were many coated vesicles budding off from the Golgi cisternae, and these were predominantly of the large type (150 nm) usually associated with the plasma membrane. In critical-point-dried samples, both cytoskeleton and membranes were preserved in detail comparable to that of quick-frozen samples, after appropriate fixation. Rotary replication of critical-point-dried cells provides a rapid, easily controlled, and generally easy to perform method for obtaining samples of exposed membrane large enough to permit quantification of membrane- associated clathrin and cytoskeleton under various experimental conditions.     

Electron microscopic studies of the endocytotic process of cationized ferritin in cultured human retinal pigment epithelial cells

Summary The endocytotic process in cultured human RPE cells was observed after 1 min, 20 min, and 2 h incubation with cationized ferritin. Within 1 min the ferritin particles were seen to attach to the cell membrane, especially between microvilli. Uncoated and coated pits could be recognized on the cell membranes, and uncoated and coated endocytotic vesicles were found in the cytoplasm after 20 min of incubation. These vesicles were surrounded by abundant microfilaments and had no visible membranes. Loss of membrane may be an initial step in the process of developing into the irregular clumps of ferritin particles found inside the plasma membrane. With time, more irregular clumps of ferritin, smaller than the particles introduced during incubation, appeared just beneath the cell membrane. Lysosomes were adjacent to the clumps of ferritin particles associated with microtobules and finally degraded these particles. The phagolysosomes containing many particles were surrounded by many microtubules. Small ferritin particles surrounded but had not entered the rough endoplasmic reticulums, and no particles were seen either around or in the Golgi apparatus. Presented at the 7th International Congress of Eye Research, Nagoya, Japan, 27 September 1986.     

ASSEMBLY OF LIPIDS INTO MEMBRANES IN ACANTHAMOEBA PALESTINENSIS : II. The Origin and Fate of Glycerol3H-Labeled Phospholipids of Cellular Membranes

The membranes of Acanthamoeba palestinensis were studied by examination in fixed cells, and then by following the movements of glycerol-³H-labeled phospholipids by cell fractionation. Two previously undescribed structures were observed: collapsed cytoplasmic vesicles of cup shape, and plaques in food vacuole and plasma membrane similar in size to the collapsed vesicles. It appeared that the plaques formed by insertion of collapsed vesicles into membranes and/or that collapsed vesicles formed by pinching off of plaques. Fractions were isolated, enriched with nuclei, rough endoplasmic reticulum (RER), plasma membrane, Golgi-like membranes, and collapsed vesicles. The changes in specific activity of glycerol-³H-labeled phospholipids in these membranes during incorporation, turnover, and after pulse-labeling indicated an ordered sequence of appearances of newly synthesized phospholipids, first in nuclei and RER, then successively in Golgi membranes, collapsed vesicles, and finally, plasma membrane. In previous work we had found no large nonmembranous phospholipid pool in A. palestinensis. These observations are consistent with the hypothesis that membrane phospholipids are synthesized, perhaps as integral parts of membranes, in RER and nuclei. Subsequently, some of the newly synthesized phospholipids are transported to the Golgi complex to become integrated into the membranes of collapsed vesicles, which are precursors of the plasma membrane. Collapsed vesicles from the plasma membrane by inserting into it as plaques. When portions of the plasmalemma from food vacuoles, collapsed vesicles pinch off from their membranes and are recycled back to the cell surface.     

Asymmetrical distribution of clathrin in the Golgi apparatus of polypeptide-secreting cells

Using an antibody revealed by the protein A-gold technique, we have studied the distribution of clathrin antigenic sites in the Golgi area of pancreatic B-cells. Golgi compartments showing an immunolabelling comprised extensive segments of cisternae, typical coated vesicles, dilated extremities of cisternae with condensing secretory material, and newly formed secretory granules. Most of the labelled membranes were observed at the trans Golgi pole while little immunoreactivity was found on the cis pole.     

Rapid redistribution of clathrin onto macrophage plasma membranes in response to Fc receptor-ligand interaction during frustrated phagocytosis

We have observed increases in assembled clathrin on the plasma membrane during "frustrated phagocytosis," the spreading of macrophages on immobilized immune complexes. Resident macrophages freshly harvested from the peritoneal cavity of mice and attached to bovine serum albumin (BSA)-anti-BSA-coated surfaces at 4 degrees C had almost no clathrin basketworks on their adherent plasma membrane (less than 0.01 coated patch/micron 2), as observed by immunofluorescence, immunoperoxidase, and platinum-carbon replica techniques, although abundant assembled clathrin was observed in the perinuclear Golgi region. When the cells were warmed to 37 degrees C they started to spread by 4 min and reached their maximum extent by 20 min. Spreading preceded clathrin assembly at the plasma membrane. Clathrin-coated patches were first observed on the adherent plasma membrane at 6 min. Between 12 and 20 min assembled clathrin coats appeared on both adherent and nonadherent plasma membranes with a concomitant decrease in identifiable clathrin in the perinuclear region. A new steady state emerged by 2 h, as perinuclear clathrin began to reappear. At 20 min at 37 degrees C the adherent plasma membranes of macrophages spreading on BSA alone had 0.9 coated patch/micron 2, whereas in cells spread on immune complex-coated surfaces, the clathrin patches increased, dependent on ligand concentration, to a maximum of 2.1 coated patches/micron 2. Because frustrated phagocytosis of immune complex-coated surfaces at 37 degrees C increased the area of adherent plasma membrane, the total area coated by clathrin basket-works increased 5-fold (28 micron 2/cell) as compared with cells plated on BSA alone (5.6 micron 2/cell) and 200-fold as compared with cells adhering to immune complexes at 4 degrees C. We then determined that macrophages cultured on BSA-coated coverslips for 24 h already have abundant surface clathrin. When immune complexes were formed by the addition of anti-BSA IgG to already spread macrophages cultured on BSA-coated coverslips for 24 h, clathrin assembled at the sites of ligand-receptor interaction even at 4 degrees C, before spreading, and a 2.6-fold increase in assembled clathrin was observed on the adherent plasma membrane of cells on immune complexes as compared with cells on BSA alone. Clathrin was reversibly redistributed to the Golgi region, returning to the steady state by 2 h.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cholinergic stimulation of chromaffin cells induces rapid coating of the plasma membrane

Stimulation of primary bovine adrenal chromaffin cells by carbachol produced a 6-fold increase in cell surface coated pits within 30 s. This coat appeared not to be recruited from a preformed pool at the plasma membrane, but from some pool transparent to electron microscopy. The number of coated pits appeared to decrease rapidly after 1 to 2 min stimulation, but processing for electron microscopy using tannic acid to enhance contrast indicated that both coated pits and closed coated vesicles were increased relative to unstimulated cells for up to 30 min. Analyses of purified adrenal medulla coated vesicles showed a lipid composition close to that expected for cell surface membrane, but there were only trace levels of plasma membrane marker enzymes. Coated vesicles contained significant amounts of both membrane and content proteins characteristic of the chromaffin granule, suggesting that medulla coated vesicles preferentially carry secretion granule proteins. The kinetics of stimulus-dependent formation of coated membrane in the cortical zone of chromaffin cells is closely similar to that observed for secretion granule membrane retrieval.     

Morphological effects of somatostatin on rat somatotrophs previously activated by growth hormone-releasing factor

Summary Correlative morphological and physiological analysis was carried out in order to clarify the role of somatostatin in the inhibition of the secretion of growth hormone (GH) from somatotrophs of the rat anterior pituitary gland in vivo. Transmission electron microscopy combined with immunogold labelling showed an increased number of exocytotic GH-containing secretory granules in somatotrophs fixed between 2 and 10 min after injection of GH-releasing factor (GRF). Injection of GRF also induced the appearance of immunopositive material in cisternae of the Golgi apparatus, many coated vesicles and multivesicular bodies. Microtubules were observed more frequently throughout the cytoplasm, particularly in and near the Golgi region. At 2 and 10 min after injection of somatostatin (SRIF), both the number of exocytotic figures in the somatotrophs previously stimulated by GRF and the amount of radioimmunoassayable GH in the plasma were clearly decreased. Undulation of the plasma membrane (PM) induced by GRF rapidly disappeared, and the number of granules just beneath the plasma membrane was significantly reduced. After injection of SRIF, parallel bundles of microfilaments were often observed in the space between the granules and the plasma membrane. SRIF did not cause a noticeable decrease in the amount of immunopositive material, coated vesicles and multivesicular bodies in the Golgi areas or any significant changes in the distribution of microtubules. SRIF therefore appears to inhibit hormone release mainly at the level of the plasma membrane, probably through changes in the distribution of microfilaments.     

Intracellular vesicles involved in the transport of Semliki Forest virus membrane proteins to the cell surface.

The route of transport of Semliki Forest virus (SFV) membrane glycoproteins to the plasma membrane was studied using immunoperoxidase electron microscopy. SFV glycoproteins were localized in cultured BHK-21 fibroblasts infected with a temperature-sensitive mutant ts-1 of SFV, which shows a temperature-dependent, reversible defect in the transport of membrane glycoproteins to the cell surface. At 39 degrees C (restrictive temperature) the viral proteins were retained in the endoplasmic reticulum and the nuclear membrane. After shift of the infected cultures to 28 degrees C (permissive temperature) the proteins were synchronously transported to the Golgi complex. In the Golgi complex the labeled proteins were first (at 2.5 min) detected in large Golgi-associated vacuoles (GAV). Subsequently, i.e., at 5-30 min, the viral glycoproteins appeared in the cisternal stack: at 5 min the label was found in one or two of the proximal cisternae whereas at 15 or 30 min also the more distal cisternae were partially or uniformly labeled. At all time points examined after the temperature-shift, peroxidase label was found in 50 nm vesicles which were frequently coated. At 30 min, in addition to the 50 nm vesicles, larger 80 nm vesicles, which often had a cytoplasmic coat were labeled in the Golgi region. These results identify two major size classes of both coated and smooth vesicles which appear to function in the transport of the viral membrane proteins from the endoplasmic reticulum via distinct GAV and the stacked Golgi cisternae to the plasma membrane.     

Post Golgi apparatus structures and membrane removal in plants

Summary In nongrowing secretory cells of plants, large quantities of membrane are transferred from the Golgi apparatus to the plasma membrane without a corresponding increase in cell surface area or accumulation of internal membranes. Movement and/or redistribution of membrane occurs also in trans Golgi apparatus cisternae which disappear after being sloughed from the dictyosome, and in secretory vesicles which lose much of their membrane in transit to the cell surface. These processes have been visualized in freeze-substituted corn rootcap cells and a structural basis for membrane loss during trafficking is seen. It involves three forms of coated membranes associated with the trans parts of the Golgi apparatus, with cisternae and secretory vesicles, and with plasma membranes. The coated regions of the plasma membrane were predominantly located at sites of recent fusion of secretory vesicles suggesting a vesicular mechanism of membrane removal. The two other forms of coated vesicles were associated with the trans cisternae, with secretory vesicles, and with a post Golgi apparatus tubular/vesicular network not unlike the TGN of animal cells. However, the trans Golgi network in plants, unlike that in animals, appears to derive directly from the trans cisternae and then vesiculate. The magnitude of the coated membrane-mediated contribution of the endocytic pathway to the formation of the TGN in rootcap cells is unknown. Continued formation of new Golgi apparatus cisternae would be required to maintain the relatively constant form of the Golgi apparatus and TGN, as is observed during periods of active secretion.     

Clathrin-coated vesicular transport of secretory proteins during the formation of ACTH-containing secretory granules in AtT20 cells

We have studied by electron microscopy and immunocytochemistry the formation of secretory granules containing adrenocorticotropic hormone (ACTH) in murine pituitary cells of the AtT20 line. The first compartment in which condensed secretory protein appears is a complex reticular network at the extreme trans side of the Golgi stacks beyond the TPPase-positive cisternae. Condensed secretory protein accumulates in dilated regions of this trans Golgi network. Examination of en face and serial sections revealed that "condensing vacuoles" are in fact dilations of the trans Golgi network and not detached vacuoles. Only after presumptive secretory granules have reached an advanced stage of morphological maturation do they detach from the trans Golgi network. Frequently both the dilations of the trans Golgi network containing condensing secretory protein and the detached immature granules in the peri-Golgi region have surface coats which were identified as clathrin by immunocytochemistry. Moreover both are the site of budding (or fusion) of coated vesicles, some of which contain condensed secretory protein. The mature granules below the plasma membrane do not, however, have surface coats. Immunoperoxidase labeling with an antiserum specific for ACTH and its precursor polypeptide confirmed that many of the coated vesicles associated with the trans Golgi network contain ACTH. The involvement of the trans Golgi network and coated vesicles in the formation of secretory granules is discussed.     

Quantitative aspects of membrane shifts in rat parathyroid cells initiated by decrease in serum calcium

The secretory activity of parathyroid glands in rats was stimulated by decreasing the serum Ca++ concentration through constant intravenous infusion of EGTA. The morphometric analysis of the nuclear and cytoplasmic volume and of the surface area of the rough endoplasmic reticulum, Golgi complex, secretory granules and plasma membrane revealed a membrane shift from secretory granules and Golgi complex to the plasma membrane within 1 hr of calcium depression. Subsequently, between 1 and 3 hr of calcium depression, the membrane shift was from the plasma membrane to the Golgi complex. It is considered likely that these membrane shifts are related to a rise in release of parathyroid hormone by exocytosis and a subsequent increase in retrieval of plasma membrane by endocytosis—probably through the compartment of coated pits and coated and uncoated vesicles.     

Surface binding, uptake and fate of cationic ferritin in a steroid producing ovarian cell

Ovarian granulosa cells (GC) were exposed to cationic ferritin (CF) in an effort to determine the binding, intracellular fate of endocytosed negatively charged plasma membrane. Following labeling at zero degrees or after pre-fixation, CF accumulated in patches over the cell surface. Exposure to methylamine (MA) resulted in an even distribution of CF over the GC surface. Endocytosis occurred in non-clathrin coated regions of the GC surface and CF was subsequently observed in a variety of smooth surfaced vesicles. Following a 60 min exposure to CF many of the CF containing vesicles appeared to fuse with each other forming larger vesicles. Numerous examples of small CF containing vesicles surrounding large CF containing vesicles were observed. Also observed at 60 min were CF containing multivcsicular and vesicular bodies. Tubular evaginations of the large vesicular structures were often observed; some containing CF. Acid phosphatase activity was observed in multivesicular bodies and the large CF filled vesicles. CF-containing vesicles were also observed in the Golgi region, but CF was never observed in the saccules of this organelle. Our study suggests that endocytosed CF does not pass through the Golgi complex. Many of the internalized vesicles become associated with the lysosomal system. Since GC's secrete progesterone in culture, these observations may indicate that membrane recycling in steroid secreting cells differs from protein secreting cells.     

Renewal of goblet cell mucus granules during the cell migration along the crypt-villus axis in rabbit jejunum: an immunolabeling study

A monoclonal antibody against intestinal mucins (5H7) was obtained and used immunolabel thin frozen sections and Epon-embedded sections of rabbit jejunum. It recognized a mucin oligosaccharide, the synthesis of which increased during goblet cell migration along the crypt-villus axis. During the earliest steps in their differentiation, the goblet cells located at the bottom of crypts synthesized mucins devoid of the 5H7 epitope, thus generating unlabeled granules. These unlabeled granules were gradually replaced by more and more labeled granules during the cell maturation. During goblet cell migration along the middle half of the villi, the mucus granules were found to be totally renewed twice. However, some newly formed labeled granules were observed to reach the apical pole of the cells before older unlabeled ones and might have had a faster turnover. At least one glycoconjugate of the goblet cell microvillar membrane also bore the 5H7 epitope. It was rapidly carried from the Golgi apparatus to the apical plasma membrane domain by a transport process that was independent of baseline mucin secretion.     

Incorporation of galactose from UDP-galactose into microsomal and golgi membranes of rat liver

Rough and smooth microsomes and Golgi membranes were incubated with UDP[¹⁴C]galactose and the incorporation of radioactivity into the lipid extract and into endogenous protein acceptors were measured. Antagonistic pyrophosphatases were inhibited with ATP and interference from β-galactosidase activity was greatly decreased by carrying out the incubation at pH 7.8. After incubation the particles were centrifuged to remove free oligosaccharide residues. Radioactivity was found in the lipid extract from Golgi membranes but not from rough and smooth microsomes. This radioactivity, however, was not associated with dolichol or retinyl phosphates. The incorporation of radioactivity into proteins of the Golgi fraction was more than double than that of the microsomal fractions. In addition, the transferases in these two types of particles exhibited different properties. Trypsin treatment of intact rough microsomal vesicles, smooth vesicles and Golgi membranes removed about 5, 15 and 50%, respectively, of newly incorporated protein-bound galactose, indicating that the proportion of the newly galactosylated proteins, which are localized at the cytoplasmic surface of the membrane, is lowest in rough microsomes, intermediate in smooth, and highest in Golgi membranes.     

Ultrastructural and cytochemical characterization of adrenal medullary plasma membrane vesicles and their interaction with chromaffin granules

Plasma membrane vesicles obtained by density gradient centrifugation of bovine adrenal medullary homogenates were analyzed by electron microscopic methods, including negative staining, ultrathin sections and freeze-fracture replicas. Rapid freezing showed the intramembrane structure of plasma membrane vesicles to be distinct from that of other organelle membranes, such as chromaffin granules. Cytochemical demonstration of acetylcholinesterase (EC 3.1.1.7) activity on most membrane profiles confirmed that plasma membrane vesicles are derived predominantly from plasma membranes. About half of the plasma membrane vesicles were smaller than 0.15 micron and almost none larger than 0.55 micron. Practically all were composed of single shells. Most vesicles were impermeable to cytochemical markers of the size of Ruthenium red (Mr 800) and none were permeable to markers larger than 40 kDa. Surface charge probes, concanavalin A binding and endogenous actin decoration with heavy meromyosin indicated that the major fraction of plasma membrane vesicles is oriented right-side-out. A minor population with opposite orientation could also be detected. Isotonic ionic media caused vesicle aggregation in suspensions of plasma membrane vesicles and chromaffin granules. Freeze-fracturing always revealed clusters of membrane-intercalated particles at the sites of contact between aggregated membranes.     

Dictyosome polarity and membrane differentiation in outer cap cells of the maize root tip

Outer rootcap cells of maize produce large numbers of secretory vesicles that ultimately fuse with the plasma membrane to discharge their product from the cell. As a result of the fusion, these vesicles contribute large quantities of membrane to the cell surface. In the present study, this phenomenon has been investigated using sections stained with phosphotungstic acid at low pH (PACP), a procedure in plant cells that specifically stains the plasma membrane. In the maize root tip, the PACP also stains the membranes of the secretory vesicles derived from Golgi apparatus to about the same density that it stains the plasma membrane. Additionally, the membranes of the secretory vesicles acquire the staining characteristic while still attached to the Golgi apparatus. The staining progresses across the dictyosome from the forming to the maturing pole, thus confirming the marked polarity of these dictyosomes. Interestingly, the PACP staining of Golgi apparatus is confined to the membranes of the secretory vesicles. It is largely absent from the central plates or peripheral tubules and provides an unambiguous example of lateral differentiation of membranes orthogonal to the major polarity axis. In the cytoplasm we could find no vesicles other than secretory vesicles bearing polysaccharide that were PACP positive. Even the occasional coated vesicle seen in the vicinity of the Golgi apparatus did not stain. Thus, if exocytotic vesicles are present in the maize root cap cell, they are formed in a manner where the PACP-staining constituent is not retained by the internalized membrane. The findings confirm dictyosome polarity in the maize root cap, provide evidence for membrane differentiation both across and at right angles to the major polarity axis, and suggest that endocytotic vesicles, if present, exclude the PACP-staining component.     

Conformational change and localization of calpactin I complex involved in exocytosis as revealed by quick-freeze, deep-etch electron microscopy and immunocytochemistry

Calpactin I complex, a calcium-dependent phospholipid-binding protein, promotes aggregation of chromaffin vesicles at physiological micromolar calcium ion levels. Calpactin I complex was found to be a globular molecule with a diameter of 10.7 +/- 1.7 (SD) nm on mica. When liposomes were aggregated by calpactin, quick-freeze, deep-etching revealed fine thin strands (6.5 +/- 1.9 [SD] nm long) cross-linking opposing membranes in addition to the globules on the surface of liposomes. Similar fine strands were also observed between aggregated chromaffin vesicles when they were mixed with calpactin in the presence of Ca2+ ion. In cultured chromaffin cells, similar cross-linking short strands (6-10 nm) were found between chromaffin vesicles and the plasma membrane after stimulation with acetylcholine. Plasma membranes also revealed numerous globular structures approximately 10 nm in diameter on their cytoplasmic surface. Immunoelectron microscopy on frozen ultrathin sections showed that calpactin I was closely associated with the inner face of the plasma membranes and was especially conspicuous between plasma membranes and adjacent vesicles in chromaffin cells. These in vivo and in vitro data strongly suggest that calpactin I complex changes its conformation to cross-link vesicles and the plasma membrane after stimulation of cultured chromaffin cells.