Sorting of cyst wall proteins to a regulated secretory pathway during differentiation of the primitive eukaryote, Giardia lamblia

Giardia lamblia, which belongs to the earliest identified lineage to diverge from the eukaryotic line of descent, is one of many protists reported to lack a Golgi apparatus. Our recent finding of a developmentally regulated secretory pathway in G. lamblia makes it an ideal organism with which to test the hypothesis that the Golgi may be more readily demonstrated in actively secreting cells. These ultrastructural studies now show that a regulated pathway of transport and secretion of cyst wall antigens via a novel class of large, osmiophilic secretory vesicles, the encystation-specific vesicles (ESV), is assembled during encystation of G. lamblia. Early in encystation, cyst antigens are localized in simple Golgi membrane stacks and concentrated within enlarged Golgi cisternae which appear to be precursors of ESV. This would represent an unusual mechanism of secretory vesicle biogenesis. Later in differentiation, cyst antigens are localized within ESV, which transport them to the plasma membrane and release them by exocytosis to the nascent cell wall. ESV are not observed after completion of the cyst wall. In contrast to the regulated transport of cyst wall proteins, we demonstrate a distinct constitutive lysosomal pathway. During encystation, acid phosphatase activity is localized in endoplasmic reticulum, Golgi, and small constitutive peripheral vacuoles which function as lysosomes. However, acid phosphatase activity is not detectable in ESV. These studies show that G. lamblia, an early eukaryote, is capable of carrying out Golgi-mediated sorting of proteins to distinct regulated secretory and constitutive lysosomal pathways.     

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.     

Active and passive mechanisms drive secretory granule biogenesis during differentiation of the intestinal parasite Giardia lamblia

The parasitic protozoan Giardia lamblia undergoes important changes to survive outside the intestine of its host by differentiating into infective cysts. During encystation, three cyst wall proteins (CWPs) are specifically expressed and concentrated within encystation-specific secretory vesicles (ESVs). ESVs are electron-dense secretory granules that transport CWPs before exocytosis and extracellular polymerization into a rigid cyst wall. Because secretory granules form at the trans-Golgi in higher eukaryotes and because Giardia lacks an identifiable Golgi apparatus, the aim of this work was to investigate the molecular basis of secretory granule formation in Giardia by examining the role of CWPs in this process. Although CWP1, CWP2, and CWP3 are structurally similar in their 26-kDa leucine-rich overlapping region, CWP2 is distinguished by the presence of a 13-kDa C-terminal basic extension. In non-encysting trophozoites, expression of different CWP chimeras showed that the CWP2 basic extension is necessary for biogenesis of ESVs, which occurs in a compartment derived from the endoplasmic reticulum. Nevertheless, the CWP2 basic extension per se is insufficient to trigger ESV formation, indicating that other domains in CWPs are also required. We found that CWP2 is a key regulator of ESV formation by acting as an aggregation factor for CWP1 and CWP3 through interactions mediated by its conserved region. CWP2 also acts as a ligand for sorting via its C-terminal basic extension. These findings show that granule biogenesis requires complex interactions among granule components and membrane receptors.     

Simultaneous evanescent wave imaging of insulin vesicle membrane and cargo during a single exocytotic event

The classical model of secretory vesicle recycling after exocytosis involves the retrieval of membrane (the omega figure) at a different site. An alternative model involves secretory vesicles transiently fusing with the plasma membrane (the 'kiss and run' mechanism) [1,2]. No continuous observation of the fate of a single secretory vesicle after exocytosis has been made to date. To study the dynamics of fusion immediately following exocytosis of insulin-containing vesicles, enhanced green fluorescent protein (EGFP) fused to the vesicle membrane protein phogrin [3] was delivered to the secretory vesicle membrane of INS-1 beta-cells using an adenoviral vector. The behaviour of the vesicle membrane during single exocytotic events was then examined using evanescent wave microscopy [4-6]. In unstimulated cells, secretory vesicles showed only slow Brownian movement. After a depolarizing pulse, most vesicles showed a small decrease in phogrin-EGFP fluorescence, and some moved laterally over the plasma membrane for approximately 1 microm. In contrast, secretory vesicles loaded with acridine orange all showed a transient (33-100 ms) increase in fluorescence intensity followed by rapid disappearance. Simultaneous observations of phogrin-EGFP and acridine orange indicated that the decrease in EGFP fluorescence occurred at the time of the acridine orange release, and that the lateral movement of EGFP-expressing vesicles occurred after this. Post-exocytotic retrieval of the vesicle membrane in INS-1 cells is thus slow, and can involve the movement of empty vesicles under the plasma membrane ('kiss and glide').     

AQPs and Control of Vesicle Volume in Secretory Cells

Aquaporins (AQPs) are a family of small, hydrophobic, integral membrane proteins. In mammals, they are expressed in many epithelia and endothelia and function as channels that permit water or small solutes to pass. Although the AQPs reside constitutively at the plasma membrane in most cell types, the presence of AQPs in intracellular organelles such as secretory granules and vesicles has currently been demonstrated. The secretory granules and vesicles contain secretory proteins, migrate to particular locations within the cell close to the plasma membrane and release their contents to the outside. During the process, including exocytosis, regulation of secretory granule or vesicle volume is important. This paper reviews the possible role of AQPs in secretory granules and vesicles.     

Aspects of signal transduction in stimulus exocytosis-coupling in Paramecium

This paper deals with the detailed mechanisms of signal transduction that lead to exocytosis during regulative secretion induced by specific secretagogues in a eukaryotic cell, Paramecium tetraurelia. There are at least three cellular compartments involved in the process: I) the plasma membrane, which contains secretagogue receptors and other transmembrane proteins, II) the cytoplasms, particularly in the region between the cell and secretory vesicle membranes, where molecules may influence interactions of the membranes, and III) the secretory vesicle itself. The ciliated protozoan Paramecium tetraurelia is very well suited for the study of signal transduction events associated with exocytosis because this eukaryotic cell contains thousands of docked secretory vesicles (trichocysts) below the cell membrane which can be induced to release synchronously when triggered with secretagogue. This ensures a high signal-to-noise ratio for events associated with this process. Upon release the trichocyst membrane fuses with the cell membrane and the trichocyst content undergoes a Ca2+-dependent irreversible expansion. Secretory mutants are available which are blocked at different points in the signal transduction pathway. Aspects of the three components mentioned above that will be discussed here include a) the properties of the vesicle content, its pH, and its membrane; b) the role of phosphorylation/dephosphorylation of a cytosolic 63-kilodalton (kDa)Mr protein in membrane fusion; and c) how influx of extracellular Ca2+ required for exocytosis may take place via exocytic Ca2+ channels which may be associated with specific membrane microdomains (fusion rosettes).     

Release of small transmitters through kiss-and-run fusion pores in rat pancreatic beta cells

Exocytosis of secretory vesicles begins with a fusion pore connecting the vesicle lumen to the extracellular space. This pore may then expand or it may close to recapture the vesicle intact. The contribution of the latter, termed kiss-and-run, to exocytosis of pancreatic beta cell large dense-core vesicles (LDCVs) is controversial. Examination of single vesicle fusion pores demonstrated that rat beta cell LDCVs can undergo exocytosis by rapid pore expansion, by the formation of stable pores, or via small transient kiss-and-run fusion pores. Elevation of cAMP shifted LDCV fusion pore openings to the transient mode. Under this condition, the small fusion pores were sufficient for release of ATP, stored within LDCVs together with insulin. Individual ATP release events occurred coincident with amperometric "stand alone feet" representing kiss-and-run. Therefore, the LDCV kiss-and-run fusion pores allow small transmitter release but likely retain the larger insulin peptide. This may represent a mechanism for selective intraislet signaling.     

Two Distinct Modes of Exocytotic Fusion Pore Expansion in Large Astrocytic Vesicles

Formation of the fusion pore is a central question for regulated exocytosis by which secretory cells release neurotransmitters or hormones. Here, by dynamically monitoring exocytosis of large vesicles (2–7 μm) in astrocytes with two-photon microscopy imaging, we found that the exocytotic fusion pore was generated from the SNARE-dependent fusion at a ring shape of the docked plasma-vesicular membrane and the movement of a fusion-produced membrane fragment. We observed two modes of fragment movements, 1) a shift fragment that shifted to expand the fusion pore and 2) a fall-in fragment that fell into the collapsed vesicle to expand the fusion pore. Shift and fall-in modes are associated with full and partial collapses of large vesicles, respectively. The astrocytic marker, sulforhodamine 101, stained the fusion-produced membrane fragment more brightly than FM 1-43. Sulforhodamine 101 imaging showed that double fusion pores could simultaneously occur in a single vesicle (16% of large vesicles) to accelerate discharge of vesicular contents. Electron microscopy of large astrocytic vesicles showed shift and fall-in membrane fragments. Two modes of fusion pore formation demonstrate a novel mechanism underlying fusion pore expansion and provide a new explanation for full and partial collapses of large secretory vesicles.     

Kiss-and-coat and compartment mixing: coupling exocytosis to signal generation and local actin assembly

Regulated exocytosis is thought to occur either by "full fusion," where the secretory vesicle fuses with the plasma membrane (PM) via a fusion pore that then dilates until the secretory vesicle collapses into the PM; or by "kiss-and-run," where the fusion pore does not dilate and instead rapidly reseals such that the secretory vesicle is retrieved almost fully intact. Here, we describe growing evidence for a third form of exocytosis, dubbed "kiss-and-coat," which is characteristic of a broad variety of cell types that undergo regulated exocytosis. Kiss-and-coat exocytosis entails prolonged maintenance of a dilated fusion pore and assembly of actin filament (F-actin) coats around the exocytosing secretory vesicles followed by direct retrieval of some fraction of the emptied vesicle membrane. We propose that assembly of the actin coats results from the union of the secretory vesicle membrane and PM and that this compartment mixing represents a general mechanism for generating local signals via directed membrane fusion.     

Rab27b regulates exocytosis of secretory vesicles in acinar epithelial cells from the lacrimal gland

Tear proteins are supplied by the regulated fusion of secretory vesicles at the apical surface of lacrimal gland acinar cells, utilizing trafficking mechanisms largely yet uncharacterized. We investigated the role of Rab27b in the terminal release of these secretory vesicles. Confocal fluorescence microscopy analysis of primary cultured rabbit lacrimal gland acinar cells revealed that Rab27b was enriched on the membrane of large subapical vesicles that were significantly colocalized with Rab3D and Myosin 5C. Stimulation of cultured acinar cells with the secretagogue carbachol resulted in apical fusion of these secretory vesicles with the plasma membrane. Evaluation of morphological changes by transmission electron microscopy of lacrimal glands from Rab27b(-/-) and Rab27(ash/ash)/Rab27b(-/-) mice, but not ashen mice deficient in Rab27a, showed changes in abundance and organization of secretory vesicles, further confirming a role for this protein in secretory vesicle exocytosis. Glands lacking Rab27b also showed increased lysosomes, damaged mitochondria, and autophagosome-like organelles. In vitro, expression of constitutively active Rab27b increased the average size but retained the subapical distribution of Rab27b-enriched secretory vesicles, whereas dominant-negative Rab27b redistributed this protein from membrane to the cytoplasm. Functional studies measuring release of a cotransduced secretory protein, syncollin-GFP, showed that constitutively active Rab27b enhanced, whereas dominant-negative Rab27b suppressed, stimulated release. Disruption of actin filaments inhibited vesicle fusion to the apical membrane but did not disrupt homotypic fusion. These data show that Rab27b participates in aspects of lacrimal gland acinar cell secretory vesicle formation and release.     

Fine structure of the biogenesis of Giardia lamblia encystation secretory vesicles

Synthesis, transport, and assembly of the extracellular cyst wall is the hallmark of Giardia lamblia encystation. Much is known of the biochemical pathways and their regulation. However, from a cell biology point of view, the biogenesis of the encystation specific vesicles (ESVs) that transport cyst wall proteins to the periphery of the cell is poorly understood. Therefore, we exploited a number of complementary ultrastructural approaches to test the hypothesis that the formation of ESVs utilizes a novel regulated secretory pathway. We analyzed parasites at different stages of encystation in vitro by electron microscopy of thin sections, freeze fracture replicas, and three-dimensional reconstruction from serial sections of cells fixed for cytochemical localization of the endoplasmic reticulum (ER) marker, glucose 6-phosphatase. We also used a stereological approach to determine the area occupied by the ER, clefts, ESVs, and cyst wall. Taken together, our kinetic data suggest that some ER cisternae first dilate to form clefts, which enlarge into the ESVs. Living non-encysting and early-encysting trophozoites were labeled around the periphery of both nuclei with C(6)-NBD-ceramide. At 18-21 h, outward migration of some ESVs frequently caused protrusions at the periphery of encysting trophozoites. The presence of lysosome-like peripheral vesicles between the ESV and plasma membrane of the cell was confirmed using acridine orange, an acidic compartment marker. Our data suggest that G. lamblia has a novel secretory pathway in which certain functions of the ER and Golgi co-localize spatially and temporally. These studies will increase understanding of the evolutionary appearance of regulated secretory pathways for assembly of a primitive extracellular matrix in an early diverging eukaryote.     

Latent acetylcholinesterase in secretory vesicles isolated from adrenal medulla

A new procedure is described for the preparation of highly purified and stable secretory vesicles from adrenal medulla. Two forms of acetylcholinesterase, a membrane bound form as well as a soluble form, were found within these vesicles. The secretory vesicles, isolated by differential centrifugation, were further purified on a continuous isotonic Percoll? gradient. In this way, secretory vesicles were separated from mitochondrial, microsomal and cell membrane contamination. The secretory vesicles recovered from the gradient contained an average of 2.26 μmol adrenalin/mg protein. On incubation for 30 min at 37°C in media differing in ionic strength, pH, Mg²⁺ and Ca²⁺ concentration, the vesicles released less than 20% of total adrenalin. Acetylcholinesterase could hardly be detected in the secretory vesicle fraction when assayed in isotonic media. However, in hypotonic media (<400 mosmol/kg) or in Triton X-100 (0.2% final concentration) acetylcholinesterase activity was markedly higher. During hypotonic treatment or when secretory vesicles were specifically lyzed with 2 mM Mg²⁺ and 2 mM ATP, adrenalin as well as part of acetylcholinesterase was released from the vesicular content. On polyacrylamide gel electrophoresis this soluble enzyme exhibited the same electrophoretic mobility as the enzyme released into the perfusate from adrenal glands upon stimulation. In addition to the soluble enzyme a membrane bound form of acetylcholinesterase exists within secretory vesicles, which sediments with the secretory vesicle membranes and exhibits a different electrophoretic mobility compared to the soluble enzyme. It is concluded, that the soluble enzyme found within isolated secretory vesicles is secreted via exocytosis, whilst the membrane-bound form is transported to the cell membrane during this process, contributing to the biogenesis of the cell membrane.     

The measurement of exocytosis in plant cells

Exocytosis is of vital importance to the growth and developmentof plant cells. It is a dynamic process in which vesicles bearingpolysaccharide precursors and proteins fuse with the plasmamembrane and release their contents. Equally important, newplasma membrane is delivered by exocytosis as secretory vesiclemembrane becomes incorporated. The requirements for polysaccharides,proteins and plasma membrane are very different in differentcell types, so there must be sophisticated mechanisms for ensuringdelivery of these materials to the correct cellular locationsat the appropriate time and, particularly in the case of membrane,their recovery and recycling. Currently, little is known ofthese mechanisms in plants, but new methods for measuring exocytosisare under development, and existing techniques have alreadycontributed data of considerable relevance. Here the methodsfor measuring exocytosis are described and evaluated, with emphasison the electrophysiological measurement of capacitance as arelatively non-invasive method, and on cell-free assays becauseof their potential importance in the identification of proteinsand other factors that control exocytosis in plant cells. Key words: Exocytosis, vesicle traffic, vesicle fusion, polysaccharides, cell wall, cell plate, root cap, secretion, patch-clamping     

A model for exocytosis based on the opening of calcium-activated potassium channels in vesicles

It is proposed that the role of calcium in calcium-induced exocytosis is to open Ca-activated K channels present in vesicle membranes. The opening of these channels coupled with anion transport across the vesicle membranes would result in an influx of K and anions, increasing the osmotic pressure of the vesicles. For those vesicles situated very close to the cell plasma membrane, this would lead to fusion with the membrane and exocytosis of the vesicle contents. This model can account for facilitation and other key properties of transmitter release. In addition, the model predicts that vesicles with a higher transmitter content, and hence higher initial osmotic pressure, would be preferentially discharged. The model also predicts that a faster response can be obtained for small vesicles than for large vesicles, providing a rationale as to why neurotransmitters, which must be released quickly, are packaged in small vesicles.     

Characterization of Yeast Extracellular Vesicles: Evidence for the Participation of Different Pathways of Cellular Traffic in Vesicle Biogenesis

Background

Extracellular vesicles in yeast cells are involved in the molecular traffic across the cell wall. In yeast pathogens, these vesicles have been implicated in the transport of proteins, lipids, polysaccharide and pigments to the extracellular space. Cellular pathways required for the biogenesis of yeast extracellular vesicles are largely unknown.

Methodology/Principal Findings

We characterized extracellular vesicle production in wild type (WT) and mutant strains of the model yeast Saccharomyces cerevisiae using transmission electron microscopy in combination with light scattering analysis, lipid extraction and proteomics. WT cells and mutants with defective expression of Sec4p, a secretory vesicle-associated Rab GTPase essential for Golgi-derived exocytosis, or Snf7p, which is involved in multivesicular body (MVB) formation, were analyzed in parallel. Bilayered vesicles with diameters at the 100–300 nm range were found in extracellular fractions from yeast cultures. Proteomic analysis of vesicular fractions from the cells aforementioned and additional mutants with defects in conventional secretion pathways (sec1-1, fusion of Golgi-derived exocytic vesicles with the plasma membrane; bos1-1, vesicle targeting to the Golgi complex) or MVB functionality (vps23, late endosomal trafficking) revealed a complex and interrelated protein collection. Semi-quantitative analysis of protein abundance revealed that mutations in both MVB- and Golgi-derived pathways affected the composition of yeast extracellular vesicles, but none abrogated vesicle production. Lipid analysis revealed that mutants with defects in Golgi-related components of the secretory pathway had slower vesicle release kinetics, as inferred from intracellular accumulation of sterols and reduced detection of these lipids in vesicle fractions in comparison with WT cells.

Conclusions/Significance

Our results suggest that both conventional and unconventional pathways of secretion are required for biogenesis of extracellular vesicles, which demonstrate the complexity of this process in the biology of yeast cells.     

A Highlights from MBoC Selection: Age-dependent Preferential Dense-Core Vesicle Exocytosis in Neuroendocrine Cells Revealed by Newly Developed Monomeric Fluorescent Timer Protein

Although it is evident that only a few secretory vesicles accumulating in neuroendocrine cells are qualified to fuse with the plasma membrane and release their contents to the extracellular space, the molecular mechanisms that regulate their exocytosis are poorly understood. For example, it has been controversial whether secretory vesicles are exocytosed randomly or preferentially according to their age. Using a newly developed protein-based fluorescent timer, monomeric Kusabira Green Orange (mK-GO), which changes color with a predictable time course, here we show that small GTPase Rab27A effectors regulate age-dependent exocytosis of secretory vesicles in PC12 cells. When the vesicles were labeled with mK-GO–tagged neuropeptide Y or tissue-type plasminogen activator, punctate structures with green or red fluorescence were observed. Application of high [K⁺] stimulation induced exocytosis of new (green) fluorescent secretory vesicles but not of old (red) vesicles. Overexpression or depletion of rabphilin and synaptotagmin-like protein4-a (Slp4-a), which regulate exocytosis positively and negatively, respectively, disturbed the age-dependent exocytosis of the secretory vesicles in different manners. Our results suggest that coordinate functions of the two effectors of Rab27A, rabphilin and Slp4-a, are required for regulated secretory pathway.     

Comparison of proteins expressed on secretory vesicle membranes and plasma membranes of human neutrophils

Secretory vesicles are neutrophil intracellular storage granules formed by endocytosis. Understanding the functional consequences of secretory vesicle exocytosis requires knowledge of their membrane proteins. The current study was designed to use proteomic technologies to develop a more complete catalog of secretory vesicle membrane proteins and to compare the proteomes of secretory vesicle and plasma membranes. A total of 1118 proteins were identified, 573 (51%) were present only in plasma membrane-enriched fractions, 418 (37%) only in secretory vesicle-enriched membrane fractions, and 127 (11%) in both fractions. Gene Ontology categorized 373 of these proteins as integral membrane proteins. Proteins typically associated with other intracellular organelles, including nuclei, mitochondria, and ribosomes, were identified in both membrane fractions. Ingenuity Pathway Knowledge Base analysis determined that the majority of canonical and functional pathways were significantly associated with proteins from both plasma membrane-enriched and secretory vesicle-enriched fractions. There were, however, some canonical signaling pathways that involved proteins only from plasma membranes or secretory vesicles. In conclusion, a number of proteins were identified that may elucidate mechanisms and functional consequences of secretory vesicle exocytosis. The small number of common proteins suggests that the hypothesis that secretory vesicles are formed from plasma membranes by endocytosis requires more critical evaluation.     

Exocytosis in plants

Exocytosis is the final event in the secretory pathway and requires the fusion of the secretory vesicle membrane with the plasma membrane. It results in the release to the outside of vesicle cargo from the cell interior and also the delivery of vesicle membrane and proteins to the plasma membrane. An electrophysiological assay that measures changes in membrane capacitance has recently been used to monitor exocytosis in plants. This complements information derived from earlier light and electron microscope studies, and allows both transient and irreversible fusion of single exocytotic vesicles to be followed with high resolution in protoplasts. It also provides a tool to investigate bulk exocytotic activity in single protoplasts under the influence of cytoplasmic modulators. This research highlights the role of intracellular Ca²⁺, GTP and pressure in the control of exocytosis in plants.In parallel to these functional studies, plant proteins with the potential to regulate exocytosis are being identified by molecular analysis. In this review we describe these electrophysiological and molecular advances, and emphasise the need for parallel biochemical work to provide a complete picture of the mechanisms controlling vesicle fusion at the plasma membrane of plant cells.     

The number of secretory vesicles remains unchanged following exocytosis.

Earlier studies using electron microscopy demonstrate that there is no loss of secretory vesicles following exocytosis. Depletion however, of vesicular contents resulting in the formation of empty or partially empty vesicles is seen in electron micrographs, post exocytosis, in a variety of cells. Our studies using atomic force microscopy (AFM) reveal that following stimulation of secretion, live pancreatic acinar cells having 100-180 nm in diameter fusion pores located at the apical plasma membrane, dilate only 25-35% during exocytosis. Since secretory vesicles in pancreatic acinar cells range in size from 200 nm to 1200 nm in diameter, their total incorporation at the fusion pore, would distend the structure much more then what is observed. These earlier results prompted the current study to determine secretory vesicle dynamics in live pancreatic acinar cells following exocytosis. AFM studies on live acinar cells reveal no loss of secretory vesicle number following exocytosis. Parallel studies using electron microscopy, further confirmed our AFM results. These studies demonstrate that following stimulation of secretion, membrane-bound secretory vesicles transiently dock and fuse to release vesicular contents.     

Compound exocytosis of casein micelles in mammary epithelial cells

Ultrastructure of lactating bovine and rat mammary epithelial cells was studied with emphasis on secretory vesicle interactions. In the apical zone of the cell, adjacent secretory vesicles formed ball and socket configurations at their points of apposition. Similar configurations were formed between plasma membrane and secretory vesicle membrane. These structures may be formed by the diffusion of water between vesicles with different osmotic potentials. Frequently, vesicular chains consisting of 10 or more linked secretory vesicles were observed. Prior to the exocytotic release of casein micelles, adjacent vesicles fused through fragmentation of the ball and socket membrane. These membrane fragments and the casein micelles appeared to be secreted into the alveolar lumen after passing from one vesicle into another and finally through a pore in the apical plasma membrane. Emptied vesicular chains appeared to collapse and fragmentation of their membrane was observed. Based on these observations, we suggest that most vesicular membrane does not directly contact or become incorporated into the plasma membrane during secretion of the nonfat phase of milk.