The nature of cnidarian desmocytes

The electron microscope reveals that the cnidarian desmocyte is an ectodermal cell which forms acidophil protein tonofibrillae intracellularly. One end of the cell is bound to mesogleal fibrils; the other becomes embedded in the thickening cuticle. The bundle of tonofibrillae later becomes rivetshaped and the cell dies, but still the mesoglea remains bound to the cuticle by means of the rivet. The histochemistry and formation of the rivet as well as the comparative cytology of cnidarian desmocytes are discussed.     

Determination of four sequential stages during microautophagy in vitro

Microautophagy is the transfer of cytosolic components into the lysosome by direct invagination of the lysosomal membrane and subsequent budding of vesicles into the lysosomal lumen. This process is topologically equivalent to membrane invagination during multivesicular body formation and to the budding of enveloped viruses. Vacuoles are lysosomal compartments of yeasts. Vacuolar membrane invagination can be reconstituted in vitro with purified yeast vacuoles, serving as a model system for budding of vesicles into the lumen of an organelle. Using this in vitro system, we defined different reaction states. We identified inhibitors of microautophagy in vitro and used them as tools for kinetic analysis. This allowed us to characterize four biochemically distinguishable steps of the reaction. We propose that these correspond to sequential stages of vacuole invagination and vesicle scission. Formation of vacuolar invaginations was slow and temperature-dependent, whereas the final scission of the vesicle from a preformed invagination was fast and proceeded even on ice. Our observations suggest that the formation of invaginations rather than the scission of vesicles is the rate-limiting step of the overall reaction.     

The role of dynamin and its binding partners in coated pit invagination and scission

Plasma membrane clathrin-coated vesicles form after the directed assembly of clathrin and the adaptor complex, AP2, from the cytosol onto the membrane. In addition to these structural components, several other proteins have been implicated in clathrin-coated vesicle formation. These include the large molecular weight GTPase, dynamin, and several Src homology 3 (SH3) domain-containing proteins which bind to dynamin via interactions with its COOH-terminal proline/arginine-rich domain (PRD). To understand the mechanism of coated vesicle formation, it is essential to determine the hierarchy by which individual components are targeted to and act in coated pit assembly, invagination, and scission.To address the role of dynamin and its binding partners in the early stages of endocytosis, we have used well-established in vitro assays for the late stages of coated pit invagination and coated vesicle scission. Dynamin has previously been shown to have a role in scission of coated vesicles. We show that dynamin is also required for the late stages of invagination of clathrin-coated pits. Furthermore, dynamin must bind and hydrolyze GTP for its role in sequestering ligand into deeply invaginated coated pits.We also demonstrate that the SH3 domain of endophilin, which binds both synaptojanin and dynamin, inhibits both late stages of invagination and also scission in vitro. This inhibition results from a reduction in phosphoinositide 4,5-bisphosphate levels which causes dissociation of AP2, clathrin, and dynamin from the plasma membrane. The dramatic effects of the SH3 domain of endophilin led us to propose a model for the temporal order of addition of endophilin and its binding partner synaptojanin in the coated vesicle cycle.     

Morphology of coral desmocytes, cells that anchor the calicoblastic epithelium to the skeleton

 We used light, scanning, and electron microscopy to investigate the ultrastructure of desmocytes in the scleractinian Stylophora pistillata from the Red Sea. Desmocytes are abundant on the calicoblastic epithelium, numbering up to 150 per mm² in the coenosarc. The surface of the skeleton bears shallow pits which may represent desmocyte attachment scars. Previously described as cell remnants or extracellular products, coral desmocytes appear to be bona fide cells as they manifest plasma membranes, organelles, and nuclei. Desmocytes attach to the mesoglea in mortise and tenon fashion. A field of 40 or more tenons protrude fingerlike from the proximal surface of the desmocyte and interdigitate with the mesoglea. Each tenon is coated extracellularly with short fibers which are joined to fibers of the mesoglea. The arrangement resembles previously described “fascial” hemidesmosomes. The short fibers pass through the plasma membrane and connect with relatively long intracellular fibers which occupy the center of each tenon. The long fibers extend distally and attach to structures resembling vertebrate hemidesmosomes. These, in turn, attach to the skeleton. The fiber arrangement and orientation seems designed to resist tensile forces. The dynamic adhesion potentially provided by the distal hemidesmosomes may enable desmocytes to detach and reattach to the skeleton during episodes of mineral accretion. Accepted: 15 April 1997     

Attachment to the substrate by soft coral fragments: desmocyte development, structure, and function

Abstract. Pieces cut from colonies of the soft coral Dendronephthya hemprichi exhibited rapid and effective attachment to hard surfaces. Attachment involved development of root-like processes (RLPs), which appeared at the basal part of the fragment 4 days after its removal from the colony. The fine structural changes and cascade of cellular events occurring in the RLP before and after attachment were studied using SEM, TEM, and LM. The epidermis of the RLPs is actively involved in the attachment process and several distinct phases are documented: appearance of numerous oval vesicles, extrusion of these vesicles resulting in the formation of an outer layer composed of extracellular organic matrix and organellar debris, which functions as an adhesive device leading to initial attachment. The latter phase was followed by the formation of desmocytes, which develop in the RLP epidermis and function as anchoring devices, mediating the firm attachment of the fragment to the substrate. This is the first evidence among anthozoans that desmocytes play an active role in anchoring tissue to substrate and thus extends the range of functions exhibited by desmocytes among anthozoans.     

Induction of mutant dynamin specifically blocks endocytic coated vesicle formation

Dynamin is the mammalian homologue to the Drosophila shibire gene product. Mutations in this 100-kD GTPase cause a pleiotropic defect in endocytosis. To further investigate its role, we generated stable HeLa cell lines expressing either wild-type dynamin or a mutant defective in GTP binding and hydrolysis driven by a tightly controlled, tetracycline- inducible promoter. Overexpression of wild-type dynamin had no effect. In contrast, coated pits failed to become constricted and coated vesicles failed to bud in cells overexpressing mutant dynamin so that endocytosis via both transferrin (Tfn) and EGF receptors was potently inhibited. Coated pit assembly, invagination, and the recruitment of receptors into coated pits were unaffected. Other vesicular transport pathways, including Tfn receptor recycling, Tfn receptor biosynthesis, and cathepsin D transport to lysosomes via Golgi-derived coated vesicles, were unaffected. Bulk fluid-phase uptake also continued at the same initial rates as wild type. EM immunolocalization showed that membrane-bound dynamin was specifically associated with clathrin-coated pits on the plasma membrane. Dynamin was also associated with isolated coated vesicles, suggesting that it plays a role in vesicle budding. Like the Drosophila shibire mutant, HeLa cells overexpressing mutant dynamin accumulated long tubules, many of which remained connected to the plasma membrane. We conclude that dynamin is specifically required for endocytic coated vesicle formation, and that its GTP binding and hydrolysis activities are required to form constricted coated pits and, subsequently, for coated vesicle budding.     

Anchoring cells (Desmocytes) in the hydrozoan polyp Cordylophora.

Desmocytes or anchoring cells are present on the upright stolons of the athecate hydroid Cordylophora caspia and function to support the soft coenosarc within the rigid tube of perisarc by linking the perisarc with the mesoglea. These cells are characterized by accumulations of 70 A filaments which aggregate into dense rods at the apical end and contact the perisarc. At the base of the desmocytes the filaments are distributed within large cytoplasmic processes which interdigitate with an extension of the mesoglea. Desmocytes in Cordylophora are temporally and spatially formed in sequence as the upright elongates. Depending on their location and structure they can be categorized as forming, functional, or remnant desmocytes. The youngest, forming desmocytes are found in the distal end of the stolon 0.5-1.0 mm from the base of the hydranth. In this region coenosarc is just beginning to separate from the perisarc. Functional desmocytes are scattered 1-3 mm from the base of the hydranth and are associated with perpendicular extensions of the mesoglea. Remnants have lost their mesogleal connection and are located in more proximal, older regions of upright stolon. Support provided by the desmocytes to the upright stolon is limited by three factors that characterize the athecate hydroid: distribution of perisarc, pattern of growth, and extent of movement. The distal location of forming desmocytes is coincident with the hardening of new perisarc. The temporary nature of attachment sites is directly related to upright elongation. It is probable that the orientation of filaments within the cell and the mesogleal extension provide an addition feature of flexibility necessary to permit feeding, growth, and rhythmic pulsation movements characteristic of these hydroids.     

Multivesicular body morphogenesis requires phosphatidyl-inositol 3-kinase activity

Multivesicular bodies are endocytic compartments containing multiple small vesicles that originate from the invagination and ‘pinching off’ of the limiting membrane into the luminal space [1], [2], [3]. The molecular mechanisms responsible for the formation of these compartments are unknown. In the human melanoma cell line Mel JuSo, newly synthesised major histocompatibility complex (MHC) class II molecules accumulate in multivesicular early lysosomes [4]. The phosphatidylinositol (PI) 3-kinase inhibitor wortmannin induced the transient vacuolation of early MHC class II compartments, but also of early and late endosomes. We demonstrate that endocytic membrane influx is required for the wortmannin-induced swelling of vesicles. The wortmannin-induced vacuoles contained a reduced number of intraluminal vesicles that were linked to the limiting membrane by membraneous connections. These data suggest that wortmannin inhibits the invagination and/or pinching off of intraluminal vesicles and provide evidence of a role for PI 3-kinase in multivesicular body morphogenesis. We propose that the wortmannin-induced vacuolation occurs as a result of the inability of multivesicular bodies to store endocytosed membranes as intraluminal vesicles thereby causing the formation of large ‘empty’ vacuoles.     

Membrane vesicles of A431 cells contain one class of epidermal growth factor binding sites

Epidermoid carcinoma A431 cells exhibit two classes of epidermal growth factor (EGF) receptors as deduced from Scatchard analysis. Steady-state binding of EGF to isolated A431 membranes indicated, however, the presence of only one class of EGF binding sites. The apparent dissociation constant (Kd) of these sites was approx. 0.45 nM which is similar to that of the high-affinity receptor of intact A431 cells. These results suggest that the vesicle receptor population consists only of high-affinity receptors. However, further studies indicated that the binding sites were similar to the low-affinity class, since binding of EGF could be blocked entirely by 2E9, a monoclonal anti-EGF receptor antibody which is able to inhibit specifically EGF binding to low-affinity receptors in A431 cells. The difference in affinity of the receptors in membrane vesicles as compared to intact cells may be explained by differences in biophysical parameters such as diffusion-limited EGF binding and receptor distribution. Based upon these considerations, it is concluded that membrane vesicles of A431 cells contain one class of EGF receptors which are apparently identical to the low-affinity receptors of intact cells.     

Distribution of [3H]Dihydrotetrabenazine Binding in Bovine Striatal Subsynaptic Fractions: Enrichment of Higher Affinity Binding in a Synaptic Vesicle Fraction

[3H]Dihydrotetrabenazine bound to a single class of binding sites in bovine striatal synaptic vesicles with an apparent dissociation constant of 3-9 nM. This is comparable to the inhibitory potency of dihydrotetrabenazine in catecholamine transport assays. In contrast to these results, [3H]dihydrotetrabenazine bound to at least two classes of sites in all other subsynaptic fractions investigated. The higher affinity class of sites was comparable in affinity to that of synaptic vesicles, whereas the lower affinity sites exhibited an apparent dissociation constant of 95-400 nM. Higher affinity sites were most abundant in the synaptic vesicle fraction, and little higher affinity binding was observed in mitochondrial and myelin fractions, or in highly purified synaptic plasma membranes. Lower affinity binding was not enriched in any subsynaptic fraction and was the only class of binding sites detected in homogenates of liver and diaphragm. The distribution of the presynaptic vesicle marker synaptophysin corresponded with that of higher affinity but not lower affinity binding. These results are consistent with the expectation that the higher affinity sites are associated primarily with synaptic vesicles and other neuronal entities that are in communication with these organelles.     

Molecular assemblies and membrane domains in multivesicular endosome dynamics

Along the degradation pathway, endosomes exhibit a characteristic multivesicular organization, resulting from the budding of vesicles into the endosomal lumen. After endocytosis and transport to early endosomes, activated signaling receptors are incorporated into these intralumenal vesicles through the action of the ESCRT machinery, a process that contributes to terminate signaling. Then, the vesicles and their protein cargo are further transported towards lysosomes for degradation. Evidence also shows that intralumenal vesicles can undergo “back-fusion” with the late endosome limiting membrane, a route exploited by some pathogens and presumably followed by proteins and lipids that need to be recycled from within the endosomal lumen. This process depends on the late endosomal lipid lysobisphosphatidic acid and its putative effector Alix/AIP1, and is presumably coupled to the invagination of the endosomal limiting membrane at the molecular level via ESCRT proteins. In this review, we discuss the intra-endosomal transport routes in mammalian cells, and in particular the different mechanisms involved in membrane invagination, vesicle formation and fusion in a space inaccessible to proteins known to control intracellular membrane traffic.     

Binding of antigen-bearing fluorescent liposomes to the murine myeloma tumor MOPC 315.

Small unilamellar lipid vesicles bearing the DNP-hapten on their surfaces and containing the water-soluble fluorescent dye carboxyfluorescein were formed by sonication. These vesicles were incubated with cells from the murine myeloma tumor MOPC 315, which secrete and also bear on the cell surface an immunoglobulin with affinity for the nitrophenyl hapten. At 0 degrees C the cells bound an average of several thousand vesicles at saturation. This binding was specific for the nitrophenyl hapten on the vesicle since it was abolished by an excess of soluble nitrophenyl derivative, by omission of the hapten from the vesicle, or by substitution for MOPC 315 of a tumor lacking receptors for the nitrophenyl hapten. Specific binding of vesicles was greater when cells were incubated at 37 degrees C. The study suggests that ligand-bearing vesicles can be a useful marker for cell surface immunoglobulin. However, in spite of the ability to "target" vesicles to cell surface determinants, binding did not result in increased delivery of vesicle contents to the cytoplasm.     

CTP:phosphocholine cytidylyltransferase binds anionic phospholipid vesicles in a cross-bridging mode

CTP:phosphocholine cytidylyltransferase (CCT) catalyzes the rate-limiting step in phosphatidylcholine (PC) synthesis, and its activity is regulated by reversible association with membranes, mediated by an amphipathic helical domain M. Here we describe a new feature of the CCTalpha isoform, vesicle tethering. We show, using dynamic light scattering and transmission electron microscopy, that dimers of CCTalpha can cross-bridge separate vesicles to promote vesicle aggregation. The vesicles contained either class I activators (anionic phospholipids) or the less potent class II activators, which favor nonlamellar phase formation. CCT increased the apparent hydrodynamic radius and polydispersity of anionic phospholipid vesicles even at low CCT concentrations corresponding to only one or two dimers per vesicle. Electron micrographs of negatively stained phosphatidylglycerol (PG) vesicles confirmed CCT-mediated vesicle aggregation. CCT conjugated to colloidal gold accumulated on the vesicle surfaces and in areas of vesicle-vesicle contact. PG vesicle aggregation required both the membrane-binding domain and the intact CCT dimer, suggesting binding of CCT to apposed membranes via the two M domains situated on opposite sides of the dimerization domain. In contrast to the effects on anionic phospholipid vesicles, CCT did not induce aggregation of PC vesicles containing the class II lipids, oleic acid, diacylglycerol, or phosphatidylethanolamine. The different behavior of the two lipid classes reflected differences in measured binding affinity, with only strongly binding phospholipid vesicles being susceptible to CCT-induced aggregation. Our findings suggest a new model for CCTalpha domain organization and membrane interaction, and a potential involvement of the enzyme in cellular events that implicate close apposition of membranes.     

OBSERVATIONS ON THE STRUCTURE OF SOME FORMS OF GOMPHONEMA PARVULUM KÜTZ. III. FRUSTULE FORMATION1

Gomphonema parvulum Kütz. was investigated by electron microscopy for details of frustule formation. An expansion of the cell along the pervalvar plane occurs prior to cell division. After nuclear division the organelles are, separated into 2 entities, either by division or by dispersion. The cell divides into 2 halves by the invagination of the plasmalemma which is derived from Golgi vesicular activity. When cytoplasmic cleavage, is complete, the Golgi actively produces electronlucent vesicles which collect and coalesce beneath the. plasmalemma to form the silicalemma around the silicon deposition vesicle. The endoplasmic reticulum is also closely associated with this vesicular activity. The vesicle gradually expands and becomes extremely electron dense as silica is deposited within it—first in the region, followed by the mantle edge. When the valve is mature, Golgi vesicles collect and fuse to form the silicalemma of the first girdle band. The first girdle band becomes aligned against the mantle edge on completion, by the “sloughing off” of the external silicalemma and plasmalemma. The second and third bands are formed, individually in a similar manner. Separation of the 2 daughter cells commences at the apical pole and progresses to the basal pole. The plasmalemma and external silicalemma are “sloughed off” so that the 2 cells can separate. The inner segment of the silicalemma becomes the new plasmalemma of the daughter cell.     

The ultrastructure of the anal vesicle of the parasitoid,Microplitis croceipes (Hymenoptera:Braconidae)

The fine structural characteristics of epithelial cells of the anal vesicle in the hymenopteran parasitoid, Microplitis croceipes (Cresson), are similar to those of transport cells. Apical and basal infoldings, an abundance of mitochondria, ribosomes, rough endoplasmic reticulum, Golgi complexes and pinocytotic vesicles all indicate a transport function for these epithelial cells. The medial portions of both Malpighian tubules located within the anal vesicle also were examined and on the basis of morphology appear to be active. These observations support earlier physiological data which indicate that the anal vesicle functions in absorption of nutrients and excretion.     

Probing intracellular vesicle trafficking and membrane remodelling by cryo-EM

Protein transport between the membranous compartments of the eukaryotic cells is mediated by the constant fission and fusion of the membrane-bounded vesicles from a donor to an acceptor membrane. While there are many membrane remodelling complexes in eukaryotes, COPII, COPI, and clathrin-coated vesicles are the three principal classes of coat protein complexes that participate in vesicle trafficking in the endocytic and secretory pathways. These vesicle-coat proteins perform two key functions: deforming lipid bilayers into vesicles and encasing selective cargoes. The three trafficking complexes share some commonalities in their structural features but differ in their coat structures, mechanisms of cargo sorting, vesicle formation, and scission. While the structures of many of the proteins involved in vesicle formation have been determined in isolation by X-ray crystallography, elucidating the proteins' structures together with the membrane is better suited for cryogenic electron microscopy (cryo-EM). In recent years, advances in cryo-EM have led to solving the structures and mechanisms of several vesicle trafficking complexes and associated proteins.     

Role of coated vesicles, microfilaments, and calmodulin in receptor- mediated endocytosis by cultured B lymphoblastoid cells

Cell surface receptor IgM molecules of cultured human lymlphoblastoid cells (WiL2) patch and redistribute into a cap over the Golgi region of the cell after treatment with multivalent anti-IgM antibodies. During and after the redistribution, ligand-receptor clusters are endocytosed into coated pits and coated vesicles. Morphometric analysis of the distribution of ferritin-labeled ligand at EM resolution reveals the following sequence of events in the endocytosis of cell surface IgM: (a) binding of the multivalent ligand in a diffuse cell surface distribution, (b) clustering of the ligand-receptor complexes, (c) recruitment of clathrin coats to the cytoplasmic surface of the cell membrane opposite ligand-receptor clusters, (d) assembly and (e) internalization of coated vesicles, and (f) delivery of label into a large vesicular compartment, presumably partly lysosomal. Most of the labeled ligand enters this pathway. The recruitment of clathrin coats to the membrane opposite ligand-receptor clusters is sensitive to the calmodulin-directed drug Stelazine (trifluoperazine dihydrochloride). In addition, Stelazine inhibits an alternate pathway of endocytosis that does not involve coated vesicle formation. The actin-directed drug dihydrocytochalasin B has no effect on the recruitment of clathrin to the ligand-receptor clusters and the formation of coated pits and little effect on the alternate pathway, but this drug does interfere with subsequent coated vesicle formation and it inhibits capping. Cortical microfilaments that decorate with heavy meromyosin with constant polarity are observed in association with the coated regions of the plasma membrane and with coated vesicles. SDS-polyacrylamide gel electrophoresis analysis of a coated vesicle preparation isolated from WiL2 cells demonstrates that the major polypeptides in the fraction are a 175-kdalton component that comigrates with calf brain clathrin, a 42- kdalton component that comigrates with rabbit muscle actin and a 18.5- kdalton minor component that comigrates with calmodulin as well as 110- , 70-, 55-, 36-, 30-, and 17-kdalton components. These results clarify the pathways of endocytosis in this cell and suggest functional roles for calmodulin, especially in the formation of clathrin-coated pits, and for actin microfilaments in coated vesicle formation and in capping.     

Non-covalent cross-linking of lipid bilayers by myelin basic protein: a possible role in myelin formation.

Myelin basic protein associates with bilayer vesicles of pure egg phosphatidylcholine, L-alpha-dimyristoyl phosphatidylcholine and DL-alpha-dipalmitoyl phosphatidylcholine. Under optimum conditions the vesicles contain 15-18% of protein by weight. The binding to dipalmitoyl phosphatidylcholine is facilitated above its gel-to-liquid crystalline transition temperature. At low ionic strength the protein provokes a large increase in vesicle size and aggregation of these enlarged vesicles. Above a sodium chloride concentration of 0.07 M vesicle fusion is far less marked but aggregation persists. The pH- and ionic strength-dependence of this aggregation follows that of the protein alone; in both cases it occurs despite appreciable electrostatic repulsion between the associated species. A similar interaction was observed with diacyl phosphatidylserine vesicles. These observations, which contrast with earlier reports in the literature of a lack of binding of basic protein to phosphatidylcholine-containing lipids, demonstrate the ability of this protein to interact non-ionically with lipid bilayers. The strong cross-linking of lipid bilayers suggests a role for basic protein in myelin, raising the possibility that the protein is instrumental in collapsing the oligodendrocyte cell membrane and thus initiating myelin formation.     

Interaction of guanine-nucleotide-binding regulatory proteins with chemotactic peptide receptors in differentiated human leukemic HL-60 cells.

Human leukemic HL-60 cells were differentiated into neutrophil-like cells by treatment with dimethylsulfoxide (Me2SO) or N6,O2'-dibutyryladenosine 3',5'-phosphate (Bt2cAMP), and membrane fractions were prepared from the differentiated cells. Receptors for fMLF (fM,N-formylmethionine) and guanine-nucleotide-binding regulatory proteins (G proteins) serving as the substrate for pertussis toxin (islet-activating protein; IAP) were extracted from cell membranes then reconstituted into phospholipid vesicles. The binding of fMLF to the reconstituted vesicles (or the membranes) was determined with 10 nM [3H] fMLF. In both cases, high-affinity binding to vesicle preparations from the Me2SO- and Bt2cAMP-induced cells was abolished following treatment with IAP, suggesting that fMLF receptors were functionally coupled to IAP-sensitive G proteins in each of the two vesicle types. However, the high-affinity fMLF binding was much higher in vesicle preparations originating from Bt2cAMP-induced cells than in those from Me2SO-induced cells, although the amount of IAP-substrate G protein reconstituted into the each phospholipid vesicles preparation was not significantly different from the other. The G proteins of the two differentiated cells were both identified as inhibitory forms (Gi-2) based on their electrophoretic mobilities and immunoblot analyses. When purified Gi-2 from rat brain was reconstituted into the two IAP-treated vesicles, high-affinity fMLF binding was restored in a similar manner in both. IAP-substrate G proteins partially purified from the two differentiated HL-60 cells were also effective in restoring high-affinity fMLF binding to the IAP-treated vesicles. However, a significant difference was observed that the reconstituted binding was higher with the G-protein-rich fraction from Bt2cAMP-induced cells than with that from Me2SO-induced cells, with each of the two IAP-treated vesicle types. These results suggest that the different high-affinity binding of fMLF observed in the two differentiated HL-60 cells are due to a difference in the property of endogenous G proteins rather than fMLF receptors, though the two G proteins are indistinguishable from each other in terms of the subtype of G protein, Gi-2.     

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.