Triggered exocytosis and endocytosis have different requirements for calcium and nucleotides in permeabilized bovine chromaffin cells

The intracellular requirements for membrane recapture in permeabilized chromaffin cells were compared to the requirements for exocytosis from the same cells.In permeabilized bovine chromaffin cells, calcium-driven exocytosis also triggers, with a short delay, uptake of extracellular horseradish peroxidase (HRP). This internalized HRP remains compartmentalized within the cell and migrates to a low density band on a Percoll gradient which is distinct from the heavier chromaffin granules.The amount of horseradish peroxidase internalized is similar in intact and leaky cells and is approximately equivalent to the volumes secreted. Endocytosis in both preparations is blocked by botulinum toxin, operates in a collapsed membrane potential, and is inhibited by low temperature. In permeabilized cells, exocytosis and coupled endocytosis are activated by the same concentrations of Ca²⁺ and MgATP. Although secretion requires Ca²⁺ and MgATP, once exocytosis has occurred the subsequent endocytosis can proceed in the virtual absence of Ca²⁺ or MgATP, and is largely unaffected by a variety of nucleotide triphosphates (including nonhydrolyzable analogues), and cyclic nucleotides.These data suggest that endocytosis can proceed, once exocytosis has been triggered, under conditions that are quite different from those necessary to support exocytosis, and that the specific requirements for Ca²⁺ and MgATP in secretion are for the exocytotic limb of the secretory cycle rather than for the associated endocytotic pathway.We are grateful to Mr. John Gibbs for excellent technical assistance, and to the Medical Research Council (UK) for financial support.     

Freeze-fracture study of the rat parathyroid gland under hypo- and hypercalcemic conditions,with special reference to secretory granules

Summary Freeze-fracture images of the parenchymal cells in the parathyroid gland of rats were observed after vitamin D₂ plus calcium chloride-suppression and EGTA-activation of secretion. In cells of the suppressed glands, large bulges protruded from the Golgi cisternae, and large granules with a stalk, which are identified as storage granules, suggest that, during maturation, some storage granules may be connected by long tubules with the Golgi cisternae and supplied with secretory products from the Golgi cisternae via these tubules.In the activated glands, presumptive exocytotic and endocytotic specializations of intramembranous particles of the parenchymal cell plasma membrane were frequently observed. In addition, elevations and complementary shallow depressions of various shape and extent were occasionally encountered in the intercellular space. From their morphological characteristics it was concluded that these originated from secretory granule cores, which are discharged from the parenchymal cells into the intercellular space by exocytosis, and it was suggested that discharged granule cores may retain their spherical shape until they fuse to form a flat conglomerate.     

Freeze-fracture study of the chromaffin cell during exocytosis: Evidence for connections between the plasma membrane and secretory granules and for movements of plasma membrane-associated particles

Summary Exocytosis was studied in acetylcholine-stimulated bovine adrenal medulla. During a pre-exocytotic stage, chromaffin granules are found in juxtaposition to the plasma membrane and separated from it by an electron dense space 25–27 Å in width. Freeze-fracture studies show this stage to be characterized by connections between the granules and the plasma membrane. These connections are apparently cytoplasmic but bridge both membranes; they are presumably proteinaceous, but their exact nature remains to be elucidated. Later stages of exocytosis were also studied by the freeze-fracture technique; a typical feature is the lack of intramembrane particles around the fusion site. Both connections and membrane particle movement are discussed in terms of recent biochemical findings.     

Quantitative analysis of the distribution of organelles in tobacco pollen tubes: implications for exocytosis and endocytosis

Summary The present study provides the first quantitative analysis on the distribution of organelles in pollen tubes ofNicotiana tabacum L. Organelles were studied on living pollen tubes by means of fluorescence confocal laser scanning microscopy and on cryo-fixed, freeze-substituted and serially sectioned material by electron microscopy. In the tip a 300 nm to 400 nm thick wall was secreted that proximately gradually separated into a wall with an opaque inner side and a more translucent, layered outer side. Tubular endoplasmic reticulum was particularly abundant in the tip of the tube, surrounding the region where secretory vesicles (SV) accumulated. Mitochondria were randomly distributed throughout the cytoplasm, no accumulations were present. Dictyosomes, however, showed an increased abundance at 25–30 m behind the tip. The accumulation of coated pits (CP) in a zone 6–15 m behind the tip identifies this zone as the major site of endocytosis: 50% of all CP occur in this zone. Quantification of exo- and endocytosis showed that only part of the membrane material of the SV can be retrieved after exocytosis. The typical zonation in endocytotic activity may serve to maintain a difference in membrane protein composition between the tip and the tube.     

L-type calcium channels are preferentially coupled to endocytosis in bovine chromaffin cells

Exocytosis and endocytosis are Ca(2+)-dependent processes. The contribution of high-voltage activated Ca(2+) channels subtypes to exocytosis has been thoroughly studied in chromaffin cells. However, similar reports concerning endocytosis are unavailable. Thus, we studied here the effects of blockers of L (nifedipine), N (omega-conotoxin GVIA) and P/Q (omega-agatoxin IVA) Ca(2+) channel on Ca(2+) currents (I(Ca)), Ca(2+) entry (Q(Ca)), as well as on the changes in membrane capacitance (C(m)) in perforated-patch voltage-clamped bovine adrenal chromaffin cells. Using 500-ms pulses to 0 or +10 mV, given from a holding potential of -80 mV and 2 mM Ca(2+) we found that omega-conotoxin GVIA affected little the exo-endocytotic responses while omega-agatoxin IVA markedly blocked those responses. However, nifedipine blocked little exocytosis but almost completely inhibited endocytosis. We conclude that L-type Ca(2+) channels seem to be selectively coupled to endocytosis.     

Freeze-fracture of sperm of Plumbago zeylanica L. in pollen and in vitro

 Sperm of Plumbago zeylanica are dimorphic with regard to numbers of mitochondria and plastids. In most cases examined, the plastid-rich sperm fused with the egg while the sperm with fewer plastids fused with the central cell. However, plastids cannot be directly responsible for fusion because fusion occurs between the plasma membranes of egg and sperm. The question is whether sperm cell membranes are distinctive and possibly dimorphic. Sperm in whole pollen grains and isolated sperm were freeze-fractured. In pollen, freeze-fractured sperm appeared only in cross fractures. No extended membrane fracture faces of sperm were found. Among isolated sperm, two sizes of sperm with different organelles were observed. Isolated sperm were assigned to two categories based on cell diameter and on size and density of organelles. Membrane particles on most sperm were arranged without distinctive pattern. Some hexagonal arrays were observed. In sperm that had been maintained at 4°C, particle-free areas, a probable consequence of lipid phase separations, appeared on plasma membrane fracture faces. No unique fracture patterns and no patterns of dimorphism were detected on freeze-fractured plasma membranes of Plumbago sperm. Received: 14 January 1997 / Revision accepted: 6 June 1997     

Paradoxical facilitation of exocytosis by inhibition of L-type calcium channels of bovine chromaffin cells

Ca²⁺ entry through the L-subtype (α_1D, Ca_v1,3) of voltage-dependent calcium channels (VDCCs) seems to selectively regulate the endocytotic response after the application of a single depolarizing pulse to voltage-clamped bovine chromaffin cells. Here we have found that L channel blockade with nifedipine transformed the exocytotic responses elicited by a double-pulse protocol, from depression to facilitation. This apparent paradoxical effect was mimicked by pharmacological interventions that directly block endocytosis namely, dynasore, calmidazolium, GTP-γS and GDP-βS. This reinforces our view that Ca²⁺ entry through PQ channels (α_1A; Ca_v2.1) regulates fast exocytosis while Ca²⁺ entry through L channels preferentially controls rapid endocytosis.     

Dysferlin is essential for endocytosis in the sea star oocyte

Dysferlin is a calcium-binding transmembrane protein involved in membrane fusion and membrane repair. In humans, mutations in the dysferlin gene are associated with muscular dystrophy. In this study, we isolated plasma membrane-enriched fractions from full-grown immature oocytes of the sea star, and identified dysferlin by mass spectrometry analysis. The full-length dysferlin sequence is highly conserved between human and the sea star. We learned that in the sea star Patiria miniata, dysferlin RNA and protein are expressed from oogenesis to gastrulation. Interestingly, the protein is highly enriched in the plasma membrane of oocytes. Injection of a morpholino against dysferlin leads to a decrease of endocytosis in oocytes, and to a developmental arrest during gastrulation. These results suggest that dysferlin is critical for normal endocytosis during oogenesis and for embryogenesis in the sea star and that this animal may be a useful model for studying the relationship of dysferlin structure as it relates to its function.     

Freeze-etch study of the teleostean pituitary

Summary The structure of membranes in the pituitary of several teleostean species was studied with the freeze-etching technique. Nuclear pore density is higher in hormone-secreting than in stellate cells, suggesting a higher rate of nucleo-cytoplasmic interactions in the secretory cells. The perinuclear endoplasmic reticulum (ER) in the MSH-secreting cells has a large number of fenestrae, whereas in the ER whorls of the prolactin-secreting cells the fenestration occurs only rarely. Three different types of membrane specializations are described, presumably related to exo-endocytotic events at the level of the plasmalemma.The Following Abbreviations are used in the Present Study AH adenohypophysis - ER endoplasmic reticulum - ES extravascular spaces - FE freeze-etching - HRP horseradish peroxidase - NH neurohypophysis - NS neurosecretory - SG secretory granule This work was supported by grants from the Swiss National Science Foundation Nos. 3.636.75, 3.611.75 and the Dr. Eric Slack-Gyr Stiftung in Zürich     

A simulation study on the Ca2+-independent but voltage-dependent exocytosis and endocytosis in dorsal root ganglion neurons

In patch-clamped somata of dorsal root ganglion (DRG) neurons, two types of secretion have been proposed: Ca²⁺-dependent secretion and Ca²⁺-independent but voltage-dependent secretion (CIVDS). The Ca²⁺-induced and the depolarization-induced membrane capacitance (C_m) increases contribute 80 and 20% to the total C_m increase, respectively (Zhang and Zhou in Nat Neurosci 5:425, 2002). In order to explore the mechanism of the voltage-dependent C_m change (C_m), we constructed a model with sequential states. The simulation with this model closely approximates all the experimental data. The model predicts that the majority of fusion events (approximately 80%) are so-called kiss-and-run events, which account for the fast recovery or the rapid retrieval feature of the signals. The remaining 20% are attributed to full fusion events, which account for a slow retrieval feature. On the basis of the model, one mechanism of the activity-dependent endocytosis has revealed a differential distribution of vesicles between the kiss-and-run and full fusion states at different stimulation frequencies. The quantitative model presented in this study may help us to understand the mechanism of the CIVDS and the tightly coupled endocytosis found in mammalian DRG neurons.     

Profilin II regulates the exocytosis of kainate glutamate receptors

The trafficking of ionotropic glutamate receptors to and from synaptic sites is regulated by proteins that interact with their cytoplasmic C-terminal domain. Profilin IIa (PfnIIa), an actin-binding protein expressed in the brain and recruited to synapses in an activity-dependent manner, was shown previously to interact with the C-terminal domain of the GluK2b subunit splice variant of kainate receptors (KARs). Here, we characterize this interaction and examine the role of PfnIIa in the regulation of KAR trafficking. PfnIIa directly and specifically binds to the C-terminal domain of GluK2b through a diproline motif. Expression of PfnIIa in transfected COS-7 cells and in cultured hippocampal neurons from PfnII-deficient mice decreases the level of extracellular of homomeric GluK2b as well as heteromeric GluK2a/GluK2b KARs. Our data suggest a novel mechanism by which PfnIIa exerts a dual role on the trafficking of KARs, by a generic inhibition of clathrin-mediated endocytosis through its interaction with dynamin-1, and by controlling KARs exocytosis through a direct and specific interaction with GluK2b.     

Quick-freeze,deep-etch visualization of exocytosis in anterior pituitary secretory cells: localization and possible roles of actin and annexin II

The exocytotic process in the anterior pituitary secretory cells was studied using quick-freeze deep-etch electron microscopy, fluorescein-isothiocyanate-phalloidin staining, heavy meromyosin decoration, and immuno-electron microscopy. The subcortical actin filaments are distributed unevenly in the peripheral cytoplasm. Few secretory granules are seen beneath the plasma membrane in the region where the peripheral cytoplasm is occupied by numerous subcortical actin filaments. On the contrary, in the region free of the subcortical actin filaments, many secretory granules lie in contact with the plasma membrane. Thus, the subcortical actin filaments may control the approach of the secretory granules to the plasma membrane in these cells. The granule and plasma membranes that lie in close proximity are linked by intervening strands. Unfused portions of both membranes remain linked by these strands during membrane fusion and opening. These strands may be involved in membrane contact, fusion and opening during exocytosis. Annexin II (calpactin I) has been demonstrated immunocytochemically to be localized at the contact sites between the granule and plasma membranes, and is therefore a possible component of the intervening strands. Membrane fusion starts within focal regions of both membranes less than 50 nm in diameter. The plasma membrane shows inward depressions toward the underlying granules immediately before fusion. The disappearance of intramembranous particles from the exocytotic site of the membrane has not been observed.     

Freeze-fracture study of heart mitochondria in the condensed or orthodox state

Rat heart mitochondria were isolated and forced in a well-defined metabolic state. After freeze-fracturing, the intramembrane particle dimension and density on both fracture faces of the inner mitochondrial membrane were measured. No significant differences could be calculated between the diameter of the membrane particles in the five different states. However, the particle density on the fracture faces of the inner mitochondrial membrane in the condensed configuration is significantly smaller than in the orthodox configuration on the 99.5% level of confidence. These results are compared with the literature, where conflicting data have been published about these particle densities.     

Ouabain evokes exocytosis dependent on ryanodine and mitochondrial calcium stores that is not followed by compensatory endocytosis at the neuromuscular junction

Ouabain is a cardiotonic glycoside that inhibits the sodium potassium ATPase pump leading to sodium accumulation in nerve terminals. At the frog neuromuscular junction, ouabain induces acetylcholine release and a rapid depletion of synaptic vesicles. In the present work, we used FM1–43 vital labeling to dissect the effect of ouabain on synaptic vesicles recycling. We first examined images of nerve-muscle preparations that were stained with FM1–43 by electrical stimulation of the nerve and destained with ouabain. We observed that ouabain induced exocytosis of synaptic vesicles independently of extracellular calcium, implying a mechanism of exocytosis that can bypass the requirement for extracellular calcium. We therefore tested the hypothesis that ouabain induces exocytosis by mobilizing intracellular calcium and we report that calcium release from endoplasmic reticulum through ryanodine receptors is necessary for ouabain-evoked exocytosis. In addition, the ouabain-evoked exocytosis was dependent on calcium released from mitochondria. We also investigated if exocytosis evoked by ouabain is followed by compensatory endocytosis. We observed that muscles incubated with FM1–43 in the presence of ouabain did not present significant staining. In conclusion, our data demonstrate that exocytosis evoked by ouabain is independent on extracellular calcium but dependent on calcium release from endoplasmic reticulum and mitochondrial stores. In addition, we suggest that ouabain can be used as a pharmacological tool to uncouple synaptic vesicles exocytosis from endocytosis at the neuromuscular junction.     

Freeze-fracture studies on isolated sperm cells ofSpinacia oleracea L.

Summary With freeze-fracturing sperm cells appear to be fractured preferentially through the plasma membranes. Only few fracture planes through the cytoplasm are found. Both the PF as well as the EF side of the sperm cell plasma membranes show a slightly undulating surface and contain intramembrane particles. The particle distribution is irregular and does not show any clustering. The EF side of the plasmamembrane contains approximately 3 times more particles per m² than the PF side.Abbreviations EF extraplasmatic fracture face - IMP intramembrane particles - FDA fluorescein diacetate - PF plasmatic fracture face     

Freeze-fracture study of plasma membranes in wild type and daunorubicin-resistant Ehrlich ascites tumor and P388 leukemia cells

The plasma membrane is considered to play a major role in the development and maintenance of the multidrug resistance (MDR) phenotype, a role which may in part be mediated by an inducible 170 kD transmembrane protein (P-170). The present freeze-fracture study of plasma membranes of daunorubicin-resistant Ehrlich ascites and P388 leukemia cells demonstrated a significant increase in the density of intramembrane particles (IMP) in the P-face, but not the E-face, of resistant sublines compared with wild type cells. Furthermore, a three-dimensional histogram plot of the diameters of P-face IMPs in Ehrlich ascites tumor cells showed the emergence of a subpopulation of 9 × 11 nm IMPs not found in wild type cells. The size of these IMPs would be consistent with a MW of approximately 340 kD, thus indicating that P-170, shown to be present in both resistant cell lines by Western blot analysis and immunohistochemical staining, exists as a dimer in the plasma membrane. Incubation with the calcium channel blocker verapamil, in concentrations known to inhibit daunorubicin efflux in resistant cells, showed evidence of membrane disturbance in the form of IMP clustering in both wild type and resistant Ehrlich ascites tumor cells. However, incubation with daunorubicin itself did not alter the freeze-fracture morphology of the plasma membranes.     

Linking exocytosis and endocytosis during phagocytosis

Phagocytosis is used by macrophages, dendritic cells and neutrophils to capture and destroy pathogens and particulate antigens. Although localized assembly of actin filaments is the driving force for particle internalization, exocytosis of intracellular compartments, and in particular endocytic compartments, has been shown recently to be required for the early steps of phagosome formation. Here we report on the different compartments undergoing exocytosis during phagocytosis, with a special focus on late endosomes. We then compare this process with secretion from lysosomes or lysosome-related organelles in specialized cells. Finally, we discuss how some of the molecular mechanisms responsible for lysosome-related organelle secretion could also be implicated in phagosome formation.     

Mobilizing store Ca(2+) in the presence of La(3+) evokes exocytosis in bovine chromaffin cells

The effect on exocytosis of La(3+), a known inhibitor of plasma membrane Ca(2+)-ATPases and Na(+)/Ca(2+) exchangers, was studied using cultured bovine adrenal chromaffin cells. At high concentrations (0.3-3 mM), La(3+) substantially increased histamine-induced catecholamine secretion. This action was mimicked by other lanthanide ions (Nd(3+), Eu(3+), Gd(3+), and Tb(3+)), but not several divalent cations. In the presence of La(3+), the secretory response to histamine became independent of extracellular Ca(2+). La(3+) enhanced secretion evoked by other agents that mobilize intracellular Ca(2+) stores (angiotensin II, bradykinin, caffeine, and thapsigargin), but not that due to passive depolarization with 20 mM K(+). La(3+) still enhanced histamine-induced secretion in the presence of the nonselective inhibitors of Ca(2+)-permeant channels SKF96365 and Cd(2+), but the enhancement was abolished by prior depletion of intracellular Ca(2+) stores with thapsigargin. La(3+) inhibited (45)Ca(2+) efflux from preloaded chromaffin cells in the presence or absence of Na(+). It also enhanced and prolonged the rise in cytosolic [Ca(2+)] measured with fura-2 during mobilization of intracellular Ca(2+) stores with histamine in Ca(2+)-free buffer. The results suggest that the efficacy of intracellular Ca(2+) stores in evoking exocytosis is enhanced dramatically by inhibiting Ca(2+) efflux from the cell.     

Clathrin-independent endocytosis: A cargo-centric view

Clathrin-independent endocytosis occurs in all cells and interest in this mode of cellular entry has grown. Although this form of endocytosis was first described for entry of bacterial toxins, here we focus our attention on the endogenous cell surface “cargo” proteins that enter cells by this mechanism. The cargo proteins entering by this mechanism are varied and include nutrient transporters, ion channels, cell adhesion molecules and proteins associated with the immune system. Despite the apparent lack of selection at the cell surface, we provide some examples of specific sorting of these cargo proteins after entry, leading to distinct itineraries and cellular fates.