Giardia intestinalis is a parasitic protozoan that inhabits its vertebrate hosts' upper small intestine and is the most common cause of waterborne diarrhoea worldwide. Giardia trophozoites present few organelles, and among them, they possess peripheral vesicles (PVs), which are considered an endosomal–lysosomal system. All experimental procedures carried out until now indicate that Giardia ingests macromolecules by fluid-phase and receptor-mediated endocytic pathways. Still, there is no description concerning the interaction and ingestion of large materials. Here, we tested Giardia's capacity to interact with large particles; once, in vivo, it inhabits an environment with a microbiota. We tested protozoan interaction with yeasts, bacteria, latex beads, ferritin and albumin, in different times of interaction and used several microscopy techniques (light microscopy, scanning electron microscopy and transmission electron microscopy) to follow their fate. Giardia interacted with all of the materials we tested. Projections of the plasma membrane similar to pseudopods were seen. As albumin, small markers were found in the PVs while the larger materials were not seen there. Large vacuoles containing large latex beads were detected intracellularly. Thus, we observed that: (1) Giardia interacts with large materials; (2) Giardia can display an amoeboid shape and exhibit membrane projections when in contact with microorganisms and large inorganic materials; (3) the region of the exit of the ventral flagella is very active when in contact with large materials, although all cell surface also present activity in the interactions; (4) intracellular vacuoles, which are not the PVs, present ingested large beads.

Skin schistosomula can be prepared by collecting them after isolated mouse skin have been penetrated by cercariae in vitro. The schistosomula can also migrate out of isolated mouse skin penetrated by cercariae in vitro and from mouse skin penetrated by cercariae in vivo. Schistosomula can also be produced from cercariae applied through a syringe or in a vortex. When certain surface properties of the different forms of schistosomula were compared, those migrating from mouse skin penetrated by cercariae in vivo or in vitro had greatly increased permeability to membrane impermeant molecules such as Lucifer yellow and high molecular weight dextrans. These migrating forms also possessed surfaces which showed greatly enhanced uptake into internal membrane vesicles of the dye FM 143, a marker for endocytosis. This greatly enhanced activity and permeability of the surfaces of tissue migrating schistosomula is likely to be of great importance in the adaptation to the new host.

Release from rods is triggered by the opening of L-type Ca2+ channels that lie beneath synaptic ribbons. After exocytosis, vesicles are retrieved by compensatory endocytosis. Previous work showed that endocytosis is dynamin-dependent in rods but dynamin-independent in cones. We hypothesized that fast endocytosis in rods may also differ from cones in its dependence upon the amount of Ca2+ influx and/or endocytic load. We measured exocytosis and endocytosis from membrane capacitance (Cm) changes evoked by depolarizing steps in voltage clamped rods from tiger salamander retinal slices. Similar to cones, the time constant for endocytosis in rods was quite fast, averaging <200 ms. We manipulated Ca2+ influx and the amount of vesicle release by altering the duration and voltage of depolarizing steps. Unlike cones, endocytosis kinetics in rods slowed after increasing Ca2+ channel activation with longer step durations or more strongly depolarized voltage steps. Endocytosis kinetics also slowed as Ca2+ buffering was decreased by replacing BAPTA (10 or 1 mM) with the slower Ca2+ buffer EGTA (5 or 0.5 mM) in the pipette solution. These data provide further evidence that endocytosis mechanisms differ in rods and cones and suggest that endocytosis in rods is regulated by both endocytic load and local Ca2+ levels.

Defining the ultrastructure of endocytic sites and localization of endocytic proteins in Saccharomyces cerevisiae by immunoelectron microscopy is central in understanding the mechanisms of membrane deformation and scission during endocytosis. We show that an improved sample preparation protocol based on high-pressure freezing, freeze substitution, and low-temperature embedding allows us to maintain the cellular fine structure and to immunolabel green fluorescent protein–tagged endocytic proteins or actin in the same sections. Using this technique we analyzed the stepwise deformation of endocytic membranes and immunolocalized the endocytic proteins Abp1p, Sla1p, Rvs167p, and actin, and were able to draw a clear ultrastructural distinction between endocytic sites and eisosomes by immunolocalizing Pil1p. In addition to defining the geometry and the fine structure of budding yeast endocytic sites, we observed associated actin filaments forming a cage-like meshwork around the endocytic membrane.

Soya is considered to be one of the eight most significant food allergens. Among the allergenic soya proteins determined to date, P34 has been identified as one of the immunodominant soya antigens. Sensitisation to a specific food antigen like P34 generally follows the transit of intact antigens across the intestinal barrier and usually occurs in infants, who are most susceptible to food allergies. In the present study, we used the intestinal epithelial cell line IPEC-J2, which was originally derived from the jejunum of a neonatal piglet, to recapitulate the infant intestinal epithelium and study the binding and uptake of P34 protein. P34 was partially resistant to degradation in an in vitro proteolysis assay. IPEC-J2 cells were able to endocytose intact P34, as shown by immunofluorescence and immunoelectronmicroscopy methods. P34 associated with lipid raft microdomains of IPEC-J2 cells, and disruption of caveolae/lipid raft microdomains using methyl-β-cyclodextrin abolished P34 endocytosis, indicating that the observed endocytosis was mediated by caveolae. Using IPEC-J2 cells grown on Transwell filters, we further demonstrated that P34 is transported through the epithelial monolayer by transcytosis. Piglets frequently show hypersensitivity to soya antigens, and in this study, we show that healthy adult pigs with dietary exposure to soya protein mount an antibody response to soyabean protein P34, suggesting that this protein has entered the body, probably through gastrointestinal uptake. In summary, our data suggest that soya P34 resists proteolysis in the gastrointestinal tract and is transported through the intestinal epithelial barrier, thereby allowing sensitisation of immune cells in the sub-epithelial compartment.

Bipolar cells play a vital role in the transfer of visual information across the vertebrate retina. The synaptic output of these neurons is regulated by factors that are extrinsic and intrinsic. Relatively little is known about the intrinsic factors that regulate neurotransmitter exocytosis. Much of what we know about intrinsic presynaptic mechanisms that regulate glutamate release has come from the study of the unusually large and accessible synaptic terminal of the goldfish rod-dominant bipolar cell, the Mb1 bipolar cell. However, over the past several years, examination of presynaptic mechanisms governing neurotransmitter release has been extended to the mammalian rod bipolar cell. In this review, we discuss the recent advances in our understanding of synaptic vesicle dynamics and neurotransmitter release in rodent rod bipolar cells and consider how these properties help to shape the synaptic output of the mammalian retina.

Gap junction (GJ) endocytosis appears to be part of a cycle of GJ renewal in horizontal cells of the teleost fish retina. At least three stages of GJ endocytosis in these neurons have been identified using conventional electron microscopy (EM): invagination of GJ membranes (GJ blebs); free GJ vesicles; and GJ vesicle fusion with mature lysosomes (Vaughan & Lasater, 1990a). In the present study, EM-level acid phosphatase (AP) histochemistry of white bass retina was used to determine at what stage enzymatic degradation of endocytosed GJs begins. Electron-dense AP reaction product was observed within the trans face of the Golgi apparatus, mature lysosomes, and occasional, internal GJ vesicles. In contrast, GJ blebs, peripheral GJ vesicles, and most internal GJ vesicles lacked AP reaction product. These results support the idea that at least some of the GJ vesicles observed within these retinal neurons arise from endocytosis, are on a degradative pathway, and can be termed GJ “endosomes.” Furthermore, GJ vesicles appear to be initially free of AP, but some later acquire it (presumably from transport vesicles bearing degradative enzymes). It is still unclear whether our previous report of GJ vesicle fusion with mature lysosomes is a subsequent step in GJ degradation or part of a different degradative pathway altogether.

To better understand synaptic signaling at the mammalian rod bipolar cell terminal and pave the way for applying genetic approaches to the study of visual information processing in the mammalian retina, synaptic vesicle dynamics and intraterminal calcium were monitored in terminals of acutely isolated mouse rod bipolar cells and the number of ribbon-style active zones quantified. We identified a releasable pool, corresponding to a maximum of ≈35 vesicles/ribbon-style active zone. Following depletion, this pool was refilled with a time constant of ≈7 s. The presence of a smaller, rapidly releasing pool and a small, fast component of refilling was also suggested. Following calcium channel closure, membrane surface area was restored to baseline with a time constant that ranged from 2 to 21 s depending on the magnitude of the preceding Ca2+ transient. In addition, a brief, calcium-dependent delay often preceded the start of onset of membrane recovery. Thus, several aspects of synaptic vesicle dynamics appear to be conserved between rod-dominant bipolar cells of fish and mammalian rod bipolar cells. A major difference is that the number of vesicles available for release is significantly smaller in the mouse rod bipolar cell, both as a function of the total number per neuron and on a per active zone basis.

Enteral exposure of suckling rats to phytohaemagglutinin (PHA) has been shown to induce growth and precocious functional maturation of the gastrointestinal tract. The aim of the present study was to explore the mechanism of this action. Suckling rats, 14d old, were fed a single dose of PHA (0·05mg/g body weight) or saline. The binding of PHA to the gut epithelium and its effect on the morphology and functional properties of the gut and pancreas were studied up to 3d after treatment. Initially, at 1–24h, the PHA bound along the gut mucosal lining, resulting in disturbed gut morphology with villi shortening and rapid decreases in disaccharidase activities and macromolecular absorption capacity. During a later phase, between 1 and 3d, the PHA binding had declined, and an uptake by enterocytes was observed. An increase in crypt cell proliferation and gut growth became evident during this period, together with a functional maturation, as indicated by increases in disaccharidase (maltase and sucrase) activities and the low macromolecular absorption capacity. Pancreas growth also increased, as did its content of digestive enzymes. We conclude that enteral exposure to PHA in suckling rats temporarily causes mucosal disarrangement and functional impediment of the gut, which may be explained by binding to and disruption of the gut mucosa and a two-fold increase in the plasma corticosterone concentration. These findings may lead to a better understanding of the role of diet in gastrointestinal maturation and may constitute a basis for the treatment of mammals having an immature gut.

Reservosomes are endocytic compartments found in the posterior region of epimastigotes of Trypanosoma cruzi. In the differentiation from trypomastigotes to epimastigotes (reverse metacyclogenesis in vitro), one has the rare opportunity of following the biogenesis of an endocytic compartment. Metacyclic trypomastigotes incubated in LIT medium highly enriched with fetal calf serum differentiated directly to epimastigotes. In recently differentiated epimastigotes, acidic organelles were found in round compartments spread along the cell body, whereas in control epimastigotes they were found in reservosomes located in the posterior region. Ultrastructural analysis of intermediate forms showed that the cytostome and reservosomes appeared before differentiation to epimastigotes was completed. Many polymorphic reservosomes, with or without lipid inclusions, were observed from the anterior portion of the cell body, in close relationship with the Golgi complex, to the posterior region. Endocytic tracers were observed in the cytostome, flagellar pocket, vesicles, and newly formed reservosomes. Cruzipain, the main protease of T. cruzi, was localized in newly formed reservosomes and in vesicles budding from the trans-Golgi network that seem to fuse with reservosomes. Ingested gold-labeled albumin and cruzipain colocalized in recently formed reservosomes. Endocytosis and immunocytochemical analysis suggested that the endocytic and the secretory pathways may contribute to reservosome formation.

Protozoan parasites are fearsome pathogens responsible for a substantial proportion of human mortality, morbidity, and economic hardship. The principal disease agents are members of the orders Apicomplexa (Plasmodium, Toxoplasma, Eimeria) and Kinetoplastida (Trypanosomes, Leishmania). The majority of humans are at risk from infection from one or more of these organisms, with profound effects on the economy, social structure and quality of life in endemic areas; Plasmodium itself accounts for over one million deaths per annum, and an estimated 4 × 107 disability-adjusted life years (DALYs), whereas the Kinetoplastida are responsible for over 100,000 deaths per annum and 4 × 106 DALYs. Current control strategies are failing due to drug resistance and inadequate implementation of existing public health strategies. Trypanosoma brucei, the African Trypanosome, has emerged as a favored model system for the study of basic cell biology in Kinetoplastida, because of several recent technical advances (transfection, inducible expression systems, and RNA interference), and these advantages, together with genome sequencing efforts are widely anticipated to provide new strategies of therapeutic intervention. Here we describe a suite of methods that have been developed for the microscopic analysis of T. brucei at the light and ultrastructural levels, an essential component of analysis of gene function and hence identification of therapeutic targets.

Reservosomes are large membrane-bound organelles found at the posterior end of epimastigote forms of Trypanosoma cruzi, but absent in amastigotes and trypomastigotes. We have transferred bloodstream trypomastigotes to LIT medium supplemented with gold-labelled transferrin in order to analyse, at the ultrastructural level, the occurrence of reservosomes and endocytosis during the trypomastigote to epimastigote differentiation. After 24 h, the trypomastigotes differentiated into amastigotes, which adhered to each other forming large clusters. Electron-dense vesicles were detected close to the Golgi complex in cells with intermediary characteristics between amastigotes and epimastigotes, but typical reservosomes at the posterior cell tip were still absent. Transferrin–gold complexes were observed only bound to the surface of clustered cells. After 72 h, epimastigotes were observed being released from the clusters and free-swimming epimastigotes appeared, containing electron-dense vesicles at their posterior region. Typical reservosomes, labelled with transferrin–gold, were observed only in free-swimming epimastigotes. When fully differentiated epimastigotes were incubated with transferrin–gold complexes and then processed for the immunocytochemical detection of cysteine proteinase, all reservosomes were positive for the enzyme, but co-localization of both markers did not occur in all organelles. Our data demonstrate that in T. cruzi epimastigotes endocytosis is strongly related to reservosome biogenesis during the trypomastigote to epimastigote differentiation process.

The primary function of cobalamin (Cbl; vitamin B12) is the formation of red blood cells and the maintenance of a healthy nervous system. Before cells can utilise dietary Cbl, the vitamin must undergo cellular transport using two distinct receptor-mediated events. First, dietary Cbl bound to gastric intrinsic factor (IF) is taken up from the apical pole of ileal epithelial cells via a 460 kDa receptor, cubilin, and is transported across the cell bound to another Cbl-binding protein, transcobalamin II (TC II). Second, plasma TC II–Cbl is taken up by cells that need Cbl via the TC II receptor (TC II-R), a 62 kDa protein that is expressed as a functional dimer in cellular plasma membranes. Human Cbl deficiency can develop as a result of acquired or inherited dysfunction in either of these two transmembrane transport events. This review focuses on the biochemical, cellular and molecular aspects of IF and TC II and their cell-surface receptors.

Limulus lateral eyes shed and renew a portion of their photosensitive membrane (rhabdom) daily. Shedding, in many species including Limulus, is regulated by complex interactions between circadian rhythms and light. Little is known about how circadian clocks and photoreceptors communicate to regulate shedding. Limulus photoreceptors do not contain an endogenous circadian oscillator, but rely upon efferent outflow from a central clock for circadian timing. To investigate whether the putative efferent neurotransmitter octopamine (OA) communicates circadian rhythms that prime the lateral eye for transient rhabdom shedding, we decoupled photoreceptors from the clock by transecting the lateral optic nerve (contains the retinal efferent fibers). Overnight (6 h) intraretinal injections of 40 μM OA restored transient shedding to lateral eyes with transected nerves to levels comparable to those of intact internal control eyes. To determine whether OA acts alone in communicating circadian rhythms that prime the lateral eye for transient shedding, we “primed” eyes with intact nerves for transient shedding with exogenous OA during subjective day. In nature, lateral eyes shed their rhabdoms only once a day at dawn following overnight efferent priming. Eyes in animals placed in darkness during subjective day, when the retinal efferents are quiescent, and injected for 6 h with 40 μM OA shed their rhabdoms in response to a second introduction to light. Untreated control eyes of the same animals did not. The same results were observed in vitro in lateral eyes treated similarly. Octopamine is the only efferent neurotransmitter/messenger required to make lateral eyes competent for transient shedding. Phentolamine, an OA receptor antagonist, reduced the number of photoreceptors primed for transient shedding and the amount of rhabdom shed in those photoreceptors suggesting that OA acts via a specific OA receptor.

Foam cells in the atherosclerotic lesion have substantial cholesterol stores within large, swollen lysosomes. This feature is mimicked by incubating THP-1 macrophages with mildly oxidized low density lipoprotein (LDL). Incubation of THP-1 cells with acetylated LDL produces cytoplasmic cholesteryl ester accumulation rather than lysosomal storage. The differences could be due to differences in uptake and delivery of lipoprotein to lysosomes or to lysosomal and post-lysosomal processing events. We compared uptake and lysosomal trafficking of acetylated and oxidized LDL using colloidal gold-labeled lipoproteins. Labeling did not alter cellular cholesterol accumulation. We found that uptake and delivery to lysosomes are not different for acetylated and oxidized LDL. In fact, both oxidized and acetylated LDL can be delivered to the same lysosomes. Sequential incubation with oxidized LDL followed by acetylated LDL showed that the lipid-engorged lysosomes are long-lived structures, continuously accepting newly ingested lipoprotein. Comparison of acetylated and oxidized LDL in mouse peritoneal macrophages, a cell which does not accumulate substantial lysosomal lipid, also revealed no differences in uptake. This indicates that in THP-1 cells, the differences in metabolism of oxidized and acetylated LDL are due to cell-specific lysosomal or post-lysosomal events not present in B6C3F1 mouse macrophages.

The long tubular branching hypha is the most important fungal growth form, although some fungi grow as discrete spherical or ellipsoidal yeast cells. Vacuoles are ubiquitous in yeast cells as well as in hyphae of filamentous fungi and can be readily stained by simple methods of dye uptake. They appear as spherical or tubular organelles of extremely variable size. Vacuoles are known or suspected to play several crucial roles in the physiology of vegetative growth. Only some of these roles are related to the activity of lytic intravacuolar enzymes. Thus, vacuoles can degrade redundant organelles, presumably recycling macromolecules, especially under conditions of nutrient starvation. They are also the destination of endocytotic vesicles originating by inward budding of the plasma membrane, thereby providing a mechanism of ‘face-lifting’ of the plasma membrane and a means of communication with the extracellular environment. Further, vacuoles store organic and inorganic nutrients, and they may detoxify the cytoplasm by sequestration of toxic substances.

The pulmonary intravascular macrophages (PIMs) of domestic ungulates are recognised by their specific surface coat, consisting of linearly arranged globules along the external leaf of the plasma membrane. The coat is sensitive to in vitro digestion with lipolytic lipase (LPL), intravenous heparin and clinical exposure to halothane anaesthesia. The sensitivity to these experimental manipulations suggests that the globules of the coat are predominantly composed of lipoproteins (LDL). The present administration of oestradiol proprionate in castrated male calves potentiated the translocation of the surface coat into the endocytotic pathway of the PIMs. Concurrently with mobilisation of the coat, the plasma membrane was thrown into prominent arrays of lamellipodial extensions. The sprawling macrophages made extensive adhesive contacts with the lining endothelium of the capillaries. Consequently, the endothelial cells were highly attenuated and precariously maintained the integrity of the vascular wall. At some focal points, the vascular wall was penetrated by the filopodial processes of PIMs, which protruded into the perivascular space. Furthermore, there were signs of neovascularisation in the form of overt mitotic changes, sprouting and precursor capillary formation. It is conceivable that the evolving profile of angiogenesis is due to the vascular endothelial growth factor (VEGF) paracrine function of PIMs. Endothelial cell specificity has been considered an important advantage of VEGF for neovascularisation. It allows pleotrophic response of endothelial cells to proliferate and to assemble into endothelial tubes.

CONTENTS

Summary 389

I. INTRODUCTION 389

II. 1. The SNARE hypothesis 393

2. SNAREs and associates 395

(a) NSF 395

(b) SNAP 395

(c) SNARE 395

(d) Rab and Rab-associated proteins 397

(e) Other interacting proteins 398

(f) Clostridial neurotoxins 399

3. New perspectives on SNARE function 400

(a) Revisiting the SNARE hypothesis 400

(b) Roles for SNARE proteins 401

III. VESICLE TRAFFICKING IN PLANTS 402

1. Identifying secretory proteins in plants 402

2. Placing syntaxins in order 403

3. Functional analysis of plant SNAREs in vesicle trafficking 404

4. Small GTPases 405

5. Searching for a vesicle–fusion complex 405

IV. REGULATION OF VESICLE TRAFFICKING IN PLANT CELLS 406

1. Hormonal control of secretion 406

(a) Auxin-evoked secretion and cell growth 406

(b) Gibberellic acid and secretion by barley aleurone 407

(c) Abscisic acid control of stomatal aperture 407

2. Coupling secretion to cytosolic-free Ca+ concentration 408

3. Stress-evoked secretion 409

(a) Mechanical stress 409

(b) Wounding and pathogen attack 409

V. CONCLUSION 410

Acknowledgements 411

References 411

Eukaryotic cells share a set of secretory pathways for the flux of membrane and protein material. In 1993, ideas about the functioning of three major proteins of the neurosecretory complex were consolidated in the SNARE hypothesis, which proposed that the interaction of these proteins provides both the specificity for vesicle targeting and the molecular machinery for fusion between vesicle and target membranes. Subsequetly, the organization, molecular mechanics and control of vesicle trafficking have become topics of intense research, and the hypothesis has evolved to accommodate new discoveries from the analysis of secretion in yeast and mammals. It is likely to be challenged again as more information comes to light about secretory processes in plants. New tools for measuring and manipulating vesicle traffic and secretion are now being used, drawing on in vivo fluorescence and capacitance recording as well as genetic engineering. These new technologies have already begun to yield details wholly unexpected from past studies. Here we focus on recent findings relating to the mechanisms of vesicle trafficking and the background to these developments, highlighting both current understanding of the molecular events of secretion and the gaps therein, as well as discussing emerging themes from work with plants.

We have identified a ∼140 amino acid domain that is shared by a variety of proteins in budding and fission yeast, nematode, rat, mouse, frog, oat, and man. Typically, this domain is located within 20 residues of the N-terminus of the various proteins. The percent identity among the domains in the 12 proteins ranges from 42 to 93%, with 16 absolutely conserved residues: N-x11–13-V-x2-A-T-x34–36-R-x7–8-W-R-x3-K-x12-G-x-E-x15-L-x11–12-D-x-G-R-x11-D-x7-R. Even though these proteins share little beyond their segment of homology, data are emerging that several of the proteins are involved in endocytosis and or regulation of cytoskeletal organization. We have named this protein segment the ENTH domain, for Epsin N-terminal Homology domain, and hypothesize that it is a candidate for binding specific ligands and/or enzymatic activity in the cell.

Concern about the cholesterol content of the human diet has led to many attempts to reduce the cholesterol content of eggs. Although this has involved a number of different approaches, including genetic selection and nutritional or pharmacological manipulation, the overwhelming evidence is that egg yolk cholesterol level is very resistant to change. The present review argues that this is because of the particular mechanisms involved in yolk formation. Yolk precursors are synthesized in the liver of the laying hen and transported in the plasma to the ovary where they are taken up into the developing follicles by receptor-mediated endocytosis. As a consequence, the cholesterol content of the yolk is primarily dependent on the cholesterol content of triglyceride-rich lipoproteins. Studies in mammals have shown that inhibition of cholesterol synthesis can reduce the rate of synthesis and secretion of lipoproteins by the liver, but has little effect on the composition of the lipoproteins that are secreted. This response, and the key role of cholesterol in the synthesis of steroid hormones, would seem to preclude the possibility of any substantial reductions in egg yolk cholesterol levels through manipulation of cholesterol metabolism. Possible alternative approaches are briefly discussed.