Apical epidermal growth factor receptor signaling: regulation of stretch-dependent exocytosis in bladder umbrella cells

The apical surface of polarized epithelial cells receives input from mediators, growth factors, and mechanical stimuli. How these stimuli are coordinated to regulate complex cellular functions such as polarized membrane traffic is not understood. We analyzed the requirement for growth factor signaling and mechanical stimuli in umbrella cells, which line the mucosal surface of the bladder and dynamically insert and remove apical membrane in response to stretch. We observed that stretch-stimulated exocytosis required apical epidermal growth factor (EGF) receptor activation and that activation occurred in an autocrine manner downstream of heparin-binding EGF-like growth factor precursor cleavage. Long-term changes in apical exocytosis depended on protein synthesis, which occurred upon EGF receptor-dependent activation of mitogen-activated protein kinase signaling. Our results indicate a novel physiological role for the EGF receptor that couples upstream mechanical stimuli to downstream apical EGF receptor activation that may regulate apical surface area changes during bladder filling.     

Visualization of the exocytosis/endocytosis secretory cycle in cultured adrenal chromaffin cells

Cultured bovine adrenal medullary chromaffin cells were stimulated to secrete catecholamines by addition of veratridine or nicotine. The formation of an exocytotic pit exposes a major secretory granule membrane antigen, the enzyme dopamine beta-hydroxylase, to the external medium. By including antiserum to this enzyme in the medium, we were able to visualize sites of exocytosis by decoration of bound antibody using a fluorescent second antibody. Internalization of this antibody- antigen complex was then followed in chase experiments: approximately half the surface complex was internalized in 15-30 min. In other experiments, secretion was triggered in the absence of antiserum, and surface enzyme was revealed by binding antibodies at various times after secretion had been halted by an antagonist. Surface patches of antigen remained discrete from the bulk of the plasma membrane for at least 30 min, although a substantial proportion of the antigen was internalized within this time. Cell surface concanavalin A receptors were internalized at a roughly similar rate, suggesting that mechanisms may be similar. After internalization, chromaffin granule membranes fused to larger structures, possibly lysosomes, and were transported over a few hours to the perinuclear region of the cell.     

Quantitative analysis of exocytosis and endocytosis in the hydroosmotic response of toad bladder

Summary This study concerns the timing and magnitude of exocytosis and endocytosis in the granular cells of toad bladder during the hydroosmotic response to antidiuretic hormone. Granule exocytosis at the luminal cell surface is extensive within 5 min of the administration of a physiological dose of hormone. Hydroosmosis becomes detectable during this time period. The amount of membrane added to the luminal surface by exocytosis during 60 min of exposure to hormone can be of the same order of magnitude as the extent of the luminal plasma membrane. Endocytosis, demonstrated by peroxidase uptake from the luminal surface, becomes extensive during the period 15–45 min after hormone administration. Thus, maximal endocytic activity occurs later than the period of most extensive exocytosis and seems to correlate with the onset of the decline in water movement. The amount of membrane retrieved from the luminal surface by endocytosis during 60 min of stimulation is at least three quarters of that added by exocytosis. The bulk membrane movement in ADH stimulated preparations does not require the presence of an osmotic gradient. Colchicine inhibits the hydroosmotic response, the exocytosis of granules, and endocytosis at the luminal surface. These results strengthen our view that the bulk circulation of membrane at the cell surface, via exocytosis and endocytosis, is closely related to the permeability changes occuring at the surface.     

The role of calcium in exocytosis and endocytosis in plant cells

The role of calcium in the individual cellular events leading to exocytosis is considered. Both vesicle movement processes and vesicle fusion at the cell surface require calcium for completion of specific events in this pathway. Our knowledge of these events is incomplete. In particular the movement of secretory vesicles by the cytoskeleton in response to added calcium is a key event that is beyond our comprehension at present. At the whole cell level, it is shown that external calcium, at the appropriate concentration, is required to elicit secretion at optimal rates. In both plant and animal cells secretion appears to be dependent on, or is triggered by, a rise in the level of internal free calcium ions from about 10^-7 to 10^-6M or even higher. In these eukaryotes internal organelles take up calcium and maintain a low level of calcium in the cell, offsetting the inflow of calcium from the plasma membrane. In some systems the inflow is restricted to a certain part of the plasma membrane, which then acts as a focus for exocytosis and, thereby, establishes a cellular polarity. In plant tissues there appears to be a requirement for some circulation of calcium within the apoplast, to sustain secretion. Recent papers on endocytosis have confirmed its occurrence in plant cells and made significant advances in isolating and characterising the clathrin coats of the coated vesicles involved in the uptake. There is no evidence, at present, for a direct role for calcium in these events. Indirectly, calcium stimulates exocytosis, and hence the delivery of excess membrane to the cell surface, which may be retrieved by an increase in the rate of endocytosis. Quantitative comparisons of the membrane flow occurring in these pathways are not available. Several plant cellular systems have been employed to study secretion and some of these may prove to be superior model systems for the investigation of certain aspects of the control of exocytosis and endocytosis by calcium ions.     

Calcium control of exocytosis and endocytosis in bovine adrenal medullary cells

Experimental analysis of the mechanisms of exocytosis and endocytosis has hitherto been hampered by the inaccessibility of the intracellular sites at which they are controlled. We have recently developed a technique that overcomes this problem. Cells are subjected to intense electric fields of brief duration; this renders the plasma membrane permeable without impairing its ability to participate in exocytosis and endocytosis. Working with 'leaky' bovine adrenal medullary cells, catecholamine release has a rather specific requirement for Mg-ATP, is activated by micromolar concentrations of ionized Ca and can be inhibited by Mg, detergents, trifluoperazine, high osmotic pressure and various anions. The mechanism of activation by Ca is discussed in some detail.     

A Highlights from MBoC Selection: A Rab11a-Rab8a-Myo5B network promotes stretch-regulated exocytosis in bladder umbrella cells

Multiple Rabs are associated with secretory granules/vesicles, but how these GTPases are coordinated to promote regulated exocytosis is not well understood. In bladder umbrella cells a subapical pool of discoidal/fusiform-shaped vesicles (DFVs) undergoes Rab11a-dependent regulated exocytosis in response to bladder filling. We show that Rab11a-associated vesicles are enmeshed in an apical cytokeratin meshwork and that Rab11a likely acts upstream of Rab8a to promote exocytosis. Surprisingly, expression of Rabin8, a previously described Rab11a effector and guanine nucleotide exchange factor for Rab8, stimulates stretch-induced exocytosis in a manner that is independent of its catalytic activity. Additional studies demonstrate that the unconventional motor protein myosin5B motor (Myo5B) works in association with the Rab8a–Rab11a module to promote exocytosis, possibly by ensuring transit of DFVs through a subapical, cortical actin cytoskeleton before fusion. Our results indicate that Rab11a, Rab8a, and Myo5B function as part of a network to promote stretch-induced exocytosis, and we predict that similarly organized Rab networks will be common to other regulated secretory pathways.     

Kinetic analysis of the triggered exocytosis/endocytosis secretory cycle in cultured bovine adrenal medullary cells

Cultured bovine adrenal medullary cells are an excellent preparation for quantitative analysis of the secretory exocytosis/endocytosis cycle. In this paper we examine the kinetics of endocytosis after stimulation of secretion. Membrane retrieval was monitored by uptake of the fluid phase marker horseradish peroxidase. Horseradish peroxidase was found to be suitable because it can be washed off completely, assayed quantitatively, and its uptake increases linearly with concentration. If this marker is present during stimulation, the rate of uptake is initially slower than catecholamine secretion but faster at a later time, suggesting that the formation of endocytotic vesicles follows exocytosis. To monitor the time-dependent concentration of secretory vesicle-plasma membrane fusion product (omega-profiles), secretion was halted at various time intervals after stimulation and the excess membrane allowed to transform into endocytotic vesicles in the presence of horseradish peroxidase. By adding horseradish peroxidase at various times after inhibition of secretion, the time course of membrane retrieval could be measured directly. All our results are consistent with a two-step kinetic model in which exocytosis and membrane retrieval are consecutive events. The estimated volumes of the compartments involved are roughly equal. The rate of endocytosis is strongly temperature-dependent but unaffected by extracellular calcium in the range of 10(-8)-2.5 X 10(-3) M, suggesting that calcium is not required at the site of endocytotic membrane fusion. Membrane retrieval is also unaffected by Lanthanum (1 mM) but is slowed by hypertonic media.     

Phorbol myristate acetate induces endocytosis as well as exocytosis and hydroosmosis in toad urinary bladder

The induction of the hydroosmotic response in the toad urinary bladder is considered to be associated with membrane addition mediated by exocytosis at the affected luminal membrane and reversed by endocytic retrieval at that surface. The permeability, exocytosis and endocytosis are initiated by antidiuretic hormone (ADH) receptor interaction on the basolateral membrane. In other hormone responsive systems, phorbol ester (phorbol myristate acetate, PMA), a tumor promoter, has been implicated in the regulation of various transport processes through the activation of protein kinase C and cytoskeletal protein phosphorylation. We found that addition of 10(-6) M PMA to the mucosa induces an hydroosmotic response which is gradual and which reaches a maximum within 60 min, equal to about 1/3 the maximal ADH response. Morphologically, PMA causes rapid exocytosis of the granules, endocytosis of horseradish peroxidase from the mucosal medium into tubules and multivesicular bodies and elongation of apical microvilli. Controls treated with mucosal 0.1% dimethylsulfoxide (DMSO) or an inactive PMA isomer on the mucosal surface, or PMA on the serosal surface lack the hydroosmotic, exocytic, endocytic and cytoskeletal changes. Addition of serosal ADH to PMA-treated bladders results in a precocious hydroosmotic and exocytic ADH response, but a lowering of the maximal response. Also pretreatment of bladders with PMA prevented the ADH-induced increase in transepithelial potential difference. Thus, apical events mediating the PMA hydroosmotic response are correlated with exo- and endocytosis and elongation of apical microvilli.     

Temperature dependence of prolactin endocytosis and casein exocytosis in epithelial mammary cells

As previously reported in epithelial mammary cells of lactating rabbit, prolactin exerts a stimulatory effect on casein secretion. After binding to a membrane receptor, the complex hormone-receptor is internalized in mammary cells. Peptide hormone action involves the generation of second messengers. These second messengers can be emitted as soon as hormone is linked to the membrane receptor. However, it is not excluded that endocytosis and transfer of prolactin inside the cell take part in the emission of second messenger and related secretory response. In order to precise intracellular transport pathways in the lactating mammary cell, we have examined the effects of reduced temperature on the one hand on prolactin endocytosis, on the other hand on casein secretion and on the stimulating effect of prolactin on casein secretion. Endocytosed prolactin was cytochemically localized mainly on the plasma membrane at 4 degrees C. At 25 degrees C, the hormone accumulated, during 60 min, in endosomes and multivesicular bodies. At 37 degrees C, prolactin was detectable after 15 and 30 min inside the cells and disappeared after 60 min. Transport and exocytosis of secretory proteins were only partly inhibited at 25 degrees C as attested by autoradiography localization and biochemical assays of newly synthesized caseins. However, at 25 degrees C, prolactin was no more able to stimulate casein exocytosis. These results show that intracellular transport of prolactin and secretagogue effect of the hormone does not proceed at 25 degrees C. However, secretory mechanisms of the cell are always able to be stimulated by exogenous arachidonic acid at this temperature. Low temperature appears as a good means to study intracellular transport in the mammary cell.     

Function of intersectin in endocytosis and exocytosis

Intersectin is an evolutionarily conserved multifunctional adaptor protein with multifunctional domains. These domains interact with components of the endocytic and exocytic pathways, such as the clathrin mediating synaptic vesicle recycling, the protein related to endocytosis via caveolae, the with-no-lysine kinases related to the regulation of renal outer medullar potassium, and the Cdc42 mediating exocytic pathway. Recently, the understanding of intersectin function in the pathogenesis of endocrine tumor and many neurodegenerative diseases such as Down syndrome, Alzheimer disease has been deepened. This article reviewed the structure and roles in endocytosis/exocytosis and diseases of intersectin.     

Compensatory endocytosis in bladder umbrella cells occurs through an integrin‐regulated and RhoA‐ and dynamin‐dependent pathway

Compensatory endocytosis (CE) ensures recycling of membrane components and maintenance of plasma membrane size; however, the mechanisms, regulation, and physiological functions of clathrin‐independent modes of CE are poorly understood. CE was studied in umbrella cells, which undergo regulated exocytosis of subapical discoidal/fusiform vesicles (DFV) during bladder filling, and may then replenish the pool of DFV by internalizing apical membrane during voiding. We found that voiding‐stimulated CE, which depended on β₁ integrin‐associated signalling pathways, occurred by a dynamin‐, actin‐, and RhoA‐regulated mechanism and was independent of caveolins, clathrin, and flotillin. Internalized apical membrane and fluid were initially found in ZO‐1‐positive vesicles, which were distinct from DFV, classical early endosomes, or the Golgi, and subsequently in lysosomes. We conclude that clathrin‐independent CE in umbrella cells functions to recover membrane during voiding, is integrin regulated, occurs by a RhoA‐ and dynamin‐dependent pathway, and terminates in degradation and not recapture of membrane in DFV.     

Involvement of dynamin-2 in formation of discoid vesicles in urinary bladder umbrella cells

Umbrella cells (UCs) of the epithelium of the urinary bladder have the capacity to control bladder volume by regulating exocytosis/endocytosis of their intracellular discoid vesicles (DVs). Dynamin (Dyn) is a GTPase that promotes endocytic processes through scission of cell membranes. We have examined whether Dyn2, the most abundant Dyn form, is expressed in UCs and contributes to their endocytic actions. A specific antibody against Dyn2 was used to localize Dyn2 in human and rodent UCs by immunohistochemistry. To clarify the functional roles of Dyn2, mouse bladders were treated with a Dyn-GTPase inhibitor, dynasore, and its effects on their UC structure were assessed. Since uropathogenic Escherichia coli can be encased into UCs during infection, we used immunohistochemistry to determine whether bacteria-encasing compartments in the infected UCs were also enriched with Dyn2. Light microscopy showed that Dyn2 was abundantly expressed in UCs, especially near the apical cytoplasmic regions. By immunoelectron microscopy, Dyn2 was found on and around DV membranes in UCs. Ultrastructural analysis with a quick-freezing and deep-etching method confirmed these findings and revealed the existence of distinct Dyn2-bound microfilaments in close association with DV membranes. Dynasore treatment of bladders markedly reduced the number of DVs in UCs. In infected UCs, E. coli was encased in compartments enriched in Dyn2. Therefore, Dyn2 is highly enriched in UCs and mostly associated with membranes of DVs and microfilaments in the UCs. Pretreatment of bladders with dynasore inhibits E. coli invasion of UCs. Dyn2 thus contributes to the structural integrity of DVs and to the endocytic activity of UCs.     

Freeze-fracture images of exocytosis and endocytosis in anterior pituitary cells of rabbits and mice

Summary Freeze-fracture images of exocytosis and endocytosis were studied in various kinds of secretory cells of the anterior pituitary of mice and rabbits. Exocytotic figures are frequently observed in thin section of the anterior pituitary cells. In freeze-fracture images, small elevated membrane areas without membrane particles are often seen on the PF of the plasma membrane of the secretory cells. There is a secretory granule in the cytoplasm just beneath the particle-free membrane area, and limiting membrane of the granule is also devoid of the membrane particles at the part facing the plasma membrane. The fusion of membranes for exocytosis may occur at this particle-free area.The limiting membrane of the granule which is continuous with the plasma membrane is almost always coated after release of the granule core. This invagination of coated membrane may be an initiation site for the membrane retrieval after exocytosis. In freeze-fracture images, this depressed region with an accumulation of the membrane particles is observed on the PF of the plasma membrane. This particle-rich depressed region is thought to correspond to the coated area of the plasma membrane observed in thin section. It is thought that the membrane retrieval by pinocytosis initiates at the particle-rich depressed region of the plasma membrane.This study was supported by a grant from the Japan Ministry of Education     

Temporal and spatial coordination of exocytosis and endocytosis

In secretory cells, exocytosis and compensatory endocytosis are tightly coupled membrane trafficking processes that control the surface area and composition of the plasma membrane. While exocytic and endocytic processes have been studied independently in great detail, at present there is much interest in understanding the mode of their coupling. This review discusses emerging insights into the coupling of these processes, both in the chemical synapses of neurons and in non-neuronal cells.     

Cyclic AMP-regulated exocytosis of Escherichia coli from infected bladder epithelial cells

The superficial bladder epithelium is a powerful barrier to urine and also serves as a regulator of bladder volume, which is achieved by apical exocytosis of specialized fusiform vesicles during distension of the bladder. We report that type 1 fimbriated uropathogenic Escherichia coli (UPEC) circumvents the bladder barrier by harboring in these Rab27b/CD63-positive and cAMP-regulatable fusiform vesicles within bladder epithelial cells (BECs). Incorporation of UPEC into BEC fusiform compartments enabled bacteria to escape elimination during voiding and to re-emerge in the urine as the bladder distended. Notably, treatment of UPEC-infected mice with a drug that increases intracellular cAMP and induces exocytosis of fusiform vesicles reduced the number of intracellular E. coli.     

Protein scaffolds in the coupling of synaptic exocytosis and endocytosis

Mechanisms that ensure robust long-term performance of synaptic transmission over a wide range of activity are crucial for the integrity of neuronal networks, for processing sensory information and for the ability to learn and store memories. Recent experiments have revealed that such robust performance requires a tight coupling between exocytic vesicle fusion at defined release sites and endocytic retrieval of synaptic vesicle membranes. Distinct presynaptic scaffolding proteins are essential for fulfilling this requirement, providing either ultrastructural coordination or acting as signalling hubs.     

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.     

The synaptic vesicle: cycle of exocytosis and endocytosis

Synaptic vesicles are clustered at the presynaptic terminal where they fuse and recycle in response to stimulation. Vesicles appear to be sorted into pools, but we do not yet understand how physiologically defined pools relate to morphological pools. The advent of dynamic imaging approaches has led to an appreciation of the regulation of vesicle mobility. Newly endocytosed vesicles are highly mobile but appear to become transiently trapped as they re-enter the recycling pool. Recent experiments indicate that endocytosis might have a constant rate, but limited capacity. How endocytosis is linked to exocytosis remains unclear, although calcium emerges as an important player.     

受体介导内吞引起的巨噬细胞胞质酸化和胞吐作用

The effects of Con A, WGA, Zymosan A on macrophage cytosolic pH and outflow of lysosomal content through exocytosis were studied with SNAFL-calcein and FITC-Dextran on ACAS570. The results showed all three ligands could induce macrophage cytosolic acidification in about 10 min and kept at the same level hereafter; outflow of lysosomal fluorescent probe through exocytosis appeared in 15-20 min. In resting conditions, macrophage lysosomes mainly distributed in cell center; after stimulated for 15 min by three ligands, the number of lysosomes increased in membrane periphery, in 25-30 min lysosomes moved back toward cell center. We proposed that ligands induced lysosomal pH rises was a basic factor for outflow of lysosomal content through exocytosis, cytosolic acidification inhibited receptor-mediated endocytosis. Cytosolic acidification and outflow of lysosomal content through exocytosis were the results of cellular self-regulation and self-protection during receptor-mediated endocytosis.