Plasticity of functional epithelial polarity

The fundamental characteristics that allow vectorial transport across an epithelial cell are the differential sorting and insertion of transport proteins either in the apical or the basolateral plasma membrane, and the preferential association of endocytosis and exocytosis with one or the other pole of the cell. Asymmetrical cellular structure and function, being manifestations of terminal differentiation, might be expected to be predetermined and invariant. Here we show that the polarity of transepithelial H+ transport, endocytosis and exocytosis in kidney can be reversed by environmental stimuli. The HCO3- secreting cell in the cortical collecting tubule is found to be an intercalated cell possessing a Cl-/HCO3- exchanger in the apical membrane and proton pumps in endocytic vesicles that fuse with the basolateral membrane; the H+-secreting cell in the medullary collecting tubule has these transport functions on the opposite membranes. Further, the HCO3- -secreting cell can be induced to change its functional polarity to that of the H+-secreting cell by acid-loading the animal.     

Irreversible swelling of secretory granules during exocytosis caused by calcium

The fusion of the limiting membrane of a secretory granule with the plasmalemma during exocytosis is equivalent to the fusion and release of contents that occurs when phospholipid vesicles fuse with planar bilayers. Experiments with bilayers demonstrate that phospholipid vesicles must swell if they are to fuse. Also, inhibition of exocytosis in solutions of high osmolarity occurs in several types of secretory cell. We report here experiments on the cortical granule exocytosis of sea-urchin eggs. Exocytosis is prevented when the osmolality of the medium surrounding the eggs is raised from 1 to 2 osmol kg-1. High osmolality also prevents calcium-dependent exocytosis in vitro. Prior treatment with calcium at high osmolality triggers fusion when normal osmolality is restored, even if calcium is removed before dilution. Addition of calcium causes the cortical granules to swell. The large increase in membrane capacitance which normally accompanies fusion is absent in eggs activated in solutions of high osmolarity. Our data are consistent with the idea that a secretory granule must swell to fuse with the plasma membrane and support the hypothesis of an osmotically driven fusion step during exocytosis.     

An H+-ATPase in opposite plasma membrane domains in kidney epithelial cell subpopulations

Vectorial solute transport by epithelia requires the polarized insertion of transport proteins into apical or basolateral plasmalemmal domains. In the specialized intercalated cells of the kidney collecting duct, the selective placement of an apical plasma membrane proton-pumping ATPase (H+-ATPase) and of a basolateral membrane anion-exchange protein results in transepithelial proton secretion. It is currently believed that amino-acid sequences of membrane proteins contain critical signalling regions involved in sorting these proteins to specific membrane domains. Recently, it was proposed that intercalated cells can reverse their direction of proton secretion under different acid-base conditions by redirecting proton pumps from apical to basolateral membranes, and anion exchangers from basolateral to apical membranes. But others have found that antibodies raised against the red cell anion-exchange protein (Band 3) only labelled intercalated cells at the basolateral plasma membrane, providing evidence against the model of polarity reversal. In this report, we have examined directly the distribution of proton pumps in kidney intercalated cells using specific polyclonal antibodies against subunits of a bovine kidney medullary H+-ATPase. We find that some cortical collecting duct intercalated cells have apical plasma membrane proton pumps, whereas others have basolateral pumps. This is the first direct demonstration of neighbouring epithelial cells maintaining opposite polarities of a transport protein. Thus, either subtle structural differences exist between proton pumps located at opposite poles of the cell, or factors other than protein sequence determine the polarity of H+-ATPase insertion.     

Inhibition of endocytic vesicle fusion in vitro by the cell-cycle control protein kinase cdc2

Membrane transport between the endoplasmic reticulum and the plasma membrane, which involves the budding and fusion of carrier vesicles, is inhibited during mitosis in animal cells. At the same time, the Golgi complex and the nuclear envelope, as well as the endoplasmic reticulum in some cell types, become fragmented. Fragmentation of the Golgi is believed to facilitate its equal partitioning between daughter cells. In fact, it has been postulated that both the inhibition of membrane traffic and Golgi fragmentation during mitosis are due to an inhibition of vesicle fusion, while vesicle budding continues. Although less is known about the endocytic pathway, internalization and receptor recycling are also arrested during mitosis. We have now used a cell-free assay to show that the fusion of endocytic vesicles from baby hamster kidney cells is reduced in Xenopus mitotic cytosol when compared with interphase cytosol. We reconstituted this inhibition in interphase cytosol by adding a preparation enriched in the starfish homologue of the cdc2 protein kinase. Inhibition was greater than or equal to 90% when the added cdc2 activity was in the range estimated for that in mitotic Xenopus eggs, which indicates that during mitosis the cdc2 kinase mediates an inhibition of endocytic vesicle fusion, and possibly other fusion events in membrane traffic.     

Inhibition of exocytosis by intracellularly applied antibodies against a chromaffin granule-binding protein

Exocytotic secretion requires the interaction and fusion of secretory vesicles with the plasma membrane. This process could be mediated by specific recognition molecules acting as intracellular, membrane-bound receptors and ligands. One possible component of such a recognition site on the plasma membrane is a protein of relative molecular mass (Mr) 51,000 (51K) that has been isolated from bovine adrenal chromaffin cells. This protein binds strongly to chromaffin granules, the secretory vesicles of these cells. To determine the function of this membrane-anchored chromaffin granule-binding protein in exocytosis, we tested the effect of intracellularly injected antibodies on secretion. Here we show, by two independent techniques in two different cell types, that antibodies against this protein inhibit exocytosis. In rat pheochromocytoma cell cultures, monospecific antibodies, applied by erythrocyte ghost fusion, impair the release of 3H-noradrenaline. The same antibodies, introduced into individual chromaffin cells through a patch pipette, block exocytosis, as revealed by the measurement of membrane capacitance. These results demonstrate the functional involvement in exocytosis of a plasma membrane protein with high affinity for secretory vesicles.     

Exocytotic fusion is activated by Rab3a peptides.

Studies of intracellular traffic in yeast and mammalian systems have implicated members of the Rab family of small GTP-binding proteins as regulators of membrane fusion. We have used the patch clamp technique to measure exocytotic fusion events directly and investigate the role of GTP-binding proteins in regulating exocytosis in mast cells. Intracellular perfusion of mast cells with GTP-gamma S is sufficient to trigger complete exocytotic degranulation in the absence of other intracellular messengers. Here we show that GTP is a potent inhibitor of GTP-gamma S-induced degranulation, indicating that sustained activation of a GTP-binding protein is sufficient for membrane fusion. We have found that synthetic oligopeptides, corresponding to part of the effector domain of Rab3a, stimulate complete exocytotic degranulation, similar to that induced by GTP-gamma S. The response is selective for Rab3a sequence and is strictly dependent on Mg2+ and ATP. This suggests that sustained activation of a Rab3 protein causes exocytotic fusion. The peptide response can be accelerated by GDP-beta S, suggesting that Rab3a peptides compete with endogenous Rab3 proteins for a binding site on a target effector protein, which causes fusion on activation.     

Direct measurement of exocytosis and calcium currents in single vertebrate nerve terminals

The release of neurohormone is widely thought to be exocytotic, involving Ca2(+)-dependent fusion of secretory vesicles with the plasma membrane. The inaccessibility of most nerve ending has so far hampered direct time-resolved measurements of neuronal exocytosis in response to brief depolarization. By using 'whole-terminal' patch-clamp and circuit-analysis techniques to measure membrane capacitance, we have now monitored changes in the surface membrane area of individual nerve terminals isolated from the mammalian neurohypophysis. A single depolarizing pulse leading to Ca2+ entry through voltage-gated calcium channels, rapidly and reproducibly increases the membrane area by an amount corresponding to the fusion of 1-100 secretory vesicles. The magnitude of the capacitance increase depends not only on Ca2+ entry and buffering, but also on the pattern of stimulation revealing facilitation, fatigue and recovery of the release process.     

Single synaptic vesicles fusing transiently and successively without loss of identity

Vesicle fusion and recycling are particularly critical for ongoing neurotransmitter release in the small nerve terminals of the brain, which typically contain about 30 functional vesicles. However, the modes of exocytosis and endocytosis that operate at synapses of the central nervous system are incompletely understood. Here we show real-time visualization of a single vesicle fusing at a small synapse of the central nervous system, made possible by highly intensified charge-coupled device imaging of hippocampal synaptic terminals, in which a single vesicle was labelled with the fluorescent membrane marker FM1-43 (ref. 6). In a small number of cases, full loss of fluorescent membrane dye was elicited by a single action potential, consistent with classical complete collapse. In most cases, however, action potentials triggered only partial loss of fluorescence, suggesting vesicular retention of membrane marker, consistent with 'kiss-and-run' vesicle cycling. An alternative hypothesis of independent fusion of partially stained vesicles arising from endosomal splitting could be excluded by observations on the size and timing of successive fusion events. Thus, our experimental evidence supports a predominance of kiss-and-run fusion events and rapid vesicular re-use.     

The Cl-/H+ antiporter ClC-7 is the primary chloride permeation pathway in lysosomes

Lysosomes are the stomachs of the cell-terminal organelles on the endocytic pathway where internalized macromolecules are degraded. Containing a wide range of hydrolytic enzymes, lysosomes depend on maintaining acidic luminal pH values for efficient function. Although acidification is mediated by a V-type proton ATPase, a parallel anion pathway is essential to allow bulk proton transport. The molecular identity of this anion transporter remains unknown. Recent results of knockout experiments raise the possibility that ClC-7, a member of the CLC family of anion channels and transporters, is a contributor to this pathway in an osteoclast lysosome-like compartment, with loss of ClC-7 function causing osteopetrosis. Several mammalian members of the CLC family have been characterized in detail; some (including ClC-0, ClC-1 and ClC-2) function as Cl--conducting ion channels, whereas others act as Cl-/H+antiporters (ClC-4 and ClC-5). However, previous attempts at heterologous expression of ClC-7 have failed to yield evidence of functional protein, so it is unclear whether ClC-7 has an important function in lysosomal biology, and also whether this protein functions as a Cl- channel, a Cl-/H+ antiporter, or as something else entirely. Here we directly demonstrate an anion transport pathway in lysosomes that has the defining characteristics of a CLC Cl-/H+ antiporter and show that this transporter is the predominant route for Cl- through the lysosomal membrane. Furthermore, knockdown of ClC-7 expression by short interfering RNA can essentially ablate this lysosomal Cl-/H+ antiport activity and can strongly diminish the ability of lysosomes to acidify in vivo, demonstrating that ClC-7 is a Cl-/H+ antiporter, that it constitutes the major Cl- permeability of lysosomes, and that it is important in lysosomal acidification.     

Vasopressin stimulates formation of coated pits in rat kidney collecting ducts

The water permeability of collecting ducts is greatly increased by the antidiuretic hormone, vasopressin. This permeability increase is associated with the appearance of intramembrane particle (IMP) clusters on the luminal plasma membranes of principal cells of the collecting duct epithelium. IMP aggregates have also been related to an increase in water permeability of two other vasopressin-sensitive epithelia, the amphibian urinary bladder and the amphibian epidermis, and it has been proposed that these specialized membrane domains might represent specific water-permeable membrane patches, induced by the hormone in their respective epithelia. Using a cytochemical probe for membrane cholesterol, filipin, we show here that the membrane patches in rat kidney are selectively devoid of filipin-sterol complexes and that when identified in thin sections, they have a cytoplasmic bristle coat: both of these features are characteristics of coated pits which, in other systems, are involved in receptor-mediated endocytosis. We also show that vasopressin induces the appearance of coated pits on collecting duct luminal membranes in Brattleboro homozygous rats, which have hereditary diabetes insipidus.     

The exocyst complex is required for targeting of Glut4 to the plasma membrane by insulin

Insulin stimulates glucose transport by promoting exocytosis of the glucose transporter Glut4 (refs 1, 2). The dynamic processes involved in the trafficking of Glut4-containing vesicles, and in their targeting, docking and fusion at the plasma membrane, as well as the signalling processes that govern these events, are not well understood. We recently described tyrosine-phosphorylation events restricted to subdomains of the plasma membrane that result in activation of the G protein TC10 (refs 3, 4). Here we show that TC10 interacts with one of the components of the exocyst complex, Exo70. Exo70 translocates to the plasma membrane in response to insulin through the activation of TC10, where it assembles a multiprotein complex that includes Sec6 and Sec8. Overexpression of an Exo70 mutant blocked insulin-stimulated glucose uptake, but not the trafficking of Glut4 to the plasma membrane. However, this mutant did block the extracellular exposure of the Glut4 protein. So, the exocyst might have a crucial role in the targeting of the Glut4 vesicle to the plasma membrane, perhaps directing the vesicle to the precise site of fusion.     

Capacitance steps and fusion pores of small and large-dense-core vesicles in nerve terminals

The vesicles that package neurotransmitters fall into two distinct classes, large dense-core vesicles (LDCVs) and small synaptic vesicles, the coexistence of which is widespread in nerve terminals. High resolution capacitance recording reveals unitary steps proportional to vesicle size. Measurements of capacitance steps during LDCV and secretory granule fusion in endocrine and immune cells have provided important insights into exocytosis; however, extending these measurements to small synaptic vesicles has proven difficult. Here we report single vesicle capacitance steps in posterior pituitary nerve terminals. These nerve terminals contain neuropeptide-laden LDCVs, as well as microvesicles. Microvesicles are similar to synaptic vesicles in size, morphology and molecular composition, but their contents are unknown. Capacitance steps of two characteristic sizes, corresponding with microvesicles and LDCVs, were detected in patches of nerve terminal membrane. Both types of vesicles fuse in response to depolarization-induced Ca(2+) entry. Both undergo a reversible fusion process commonly referred to as 'kiss-and-run', but only rarely. Fusion pores seen during microvesicle kiss-and-run have a conductance of 19 pS, 11 times smaller than LDCV fusion pores. Thus, LDCVs and microvesicles use structurally different intermediates during exocytosis.     

Transport, capture and exocytosis of single synaptic vesicles at active zones

To sustain high rates of transmitter release, synaptic terminals must rapidly re-supply vesicles to release sites and prime them for exocytosis. Here we describe imaging of single synaptic vesicles near the plasma membrane of live ribbon synaptic terminals. Vesicles were captured at small, discrete active zones near the terminal surface. An electric stimulus caused them to undergo rapid exocytosis, seen as the release of a fluorescent lipid from the vesicles into the plasma membrane. Next, vesicles held in reserve about 20 nm from the plasma membrane advanced to exocytic sites, and became release-ready 250 ms later. Apparently a specific structure holds vesicles at an active zone to bring v-SNAREs and t-SNAREs, the proteins that mediate vesicle fusion, within striking distance of each other, and then allows the triggered movement of such vesicles to the plasma membrane.     

α-LTX and α-LTXN4C induce [Ca2+]i elevation through different mechanisms in pancreatic β cells

α-latrotoxin (α-LTX) is the only neurotoxin from black-widow spider which has secretagogue effects in the vertebrates. It causes massive neurotransmitter and hormone release via two instinct mechanisms after binding with its high-affinity membrane recep…     

Ca2+-induced fusion of phospholipid vesicles monitored by mixing of aqueous contents

Ca2+ has a central role in various cellular phenomena involving membrane fusion. However, little is known about the mechanisms involved. Model membrane systems such as phospholipid vesicles have been used extensively to study the mechanism of membrane fusion at the molecular level. For example, phosphatidylserine (PS) vesicles have been shown to undergo massive aggregation and structural rearrangements on additon of Ca2+, with eventual formation of large cochleate structures. Although these structures do not retain appreciable internal volume, their formation has been proposed to result from fusion of the initial vesicles. The significance of the PS--Ca2+ system as a model for biological membrane fusion has been questioned recently by Ginsberg. Based on the observation that divalent cations induce the release of contents from PS vesicles but fail to bring about the uptake of a marker from the medium, he proposes that the vesicles are ruptured completely during interaction with divalent cations and reassemble subsequently to form large non-vesicular structures. The present study demonstrates that the question raised by Ginsberg is not particularly relevant to the phenomenon concerned, and that his experimental observations do not allow the exclusive conclusion that Ca2+ induces lysis of PS vesicles rather than fusion.     

Single and multiple vesicle fusion induce different rates of endocytosis at a central synapse

During synaptic transmission, neurotransmitter-laden vesicles fuse with the presynaptic membrane and discharge their contents into the synaptic cleft. After fusion, the vesicular membrane is retrieved by endocytosis for reuse. This recycling mechanism ensures a constant supply of releasable vesicles at the nerve terminal. The kinetics of endocytosis have been measured mostly after intense or non-physiological stimulation. Here we use capacitance measurements to resolve the fusion and retrieval of single and multiple vesicles following mild physiological stimulation at a mammalian central synapse. The time constant of endocytosis after single vesicle fusion was 56 ms; after a single action potential or trains at < or = 2 Hz it was about 115 ms, but increased gradually to tens of seconds as the frequency and the number of action potentials increased. These results indicate that an increase in the rate of exocytosis at the active zone induces a decrease in the rate of endocytosis. Existing models, including inhibition of endocytosis by Ca(2+), could not account for these results our results suggest that an accumulation of unretrieved vesicles at the plasma membrane slows endocytosis. These findings may resolve the debate about the dependence of endocytosis kinetics on the stimulation frequency, and suggest a potential role of regulation of endocytosis in short-term synaptic depression.     

Vesicular restriction of synaptobrevin suggests a role for calcium in membrane fusion

Release of neurotransmitter occurs when synaptic vesicles fuse with the plasma membrane. This neuronal exocytosis is triggered by calcium and requires three SNARE (soluble-N-ethylmaleimide-sensitive factor attachment protein receptors) proteins: synaptobrevin (also known as VAMP) on the synaptic vesicle, and syntaxin and SNAP-25 on the plasma membrane. Neuronal SNARE proteins form a parallel four-helix bundle that is thought to drive the fusion of opposing membranes. As formation of this SNARE complex in solution does not require calcium, it is not clear what function calcium has in triggering SNARE-mediated membrane fusion. We now demonstrate that whereas syntaxin and SNAP-25 in target membranes are freely available for SNARE complex formation, availability of synaptobrevin on synaptic vesicles is very limited. Calcium at micromolar concentrations triggers SNARE complex formation and fusion between synaptic vesicles and reconstituted target membranes. Although calcium does promote interaction of SNARE proteins between opposing membranes, it does not act by releasing synaptobrevin from synaptic vesicle restriction. Rather, our data suggest a mechanism in which calcium-triggered membrane apposition enables syntaxin and SNAP-25 to engage synaptobrevin, leading to membrane fusion.     

Control of transmembrane lipid asymmetry in chromaffin granules by an ATP-dependent protein

The Ca2+-dependent binding of annexin proteins to secretory granule membranes seems to be involved in the early stage of exocytosis. Binding studies have shown that these proteins have a specificity for phosphatidylserine (PtdS) interfaces. Furthermore, aminolipids are necessary for contact and fusion between lipid vesicles or between liposomes and chromaffin granules. Thus, PtdS must be present on the granule outer (cytoplasmic) monolayer. We report here that chromaffin granules possess a mechanism to maintain PtdS orientation, comparable to the ATP-dependent aminophospholipid translocase from human erythrocytes. The translocase, in granules, selectively transports PtdS from the luminal to the cytoplasmic monolayer, provided the incubation medium contains ATP. As this protein shares several properties with the granule vanadate-sensitive ATPase II, we infer that this ATPase, of relative molecular mass 115,000, is the protein responsible for aminophospholipid translocation. This is the first evidence for an ATP-dependent specific phospholipid 'flippase' in intracellular organelles.     

A small GTP-binding protein dissociates from synaptic vesicles during exocytosis.

Low-molecular-weight GTP-binding proteins are strong candidates for regulators of membrane traffic. In yeast, mutations in the sec4 or ypt1 genes encoding small GTP-binding proteins inhibit constitutive membrane flow at the plasma membrane or Golgi complex, respectively. It has been suggested that membrane fusion-fission events are regulated by cycling of small GTP-binding proteins between a membrane-bound and free state, but although most of these small proteins are found in both soluble and tightly membrane-bound forms, there is no direct evidence to support such cycling. In rat brain a small GTP-binding protein, rab3A, is exclusively associated with synaptic vesicles, the secretory organelles of nerve terminals. Here we use isolated nerve terminals to study the fate of rab3A during synaptic vesicle exocytosis. We find that rab3A dissociates quantitatively from the vesicle membrane after Ca2(+)-dependent exocytosis and that this dissociation is partially reversible during recovery after stimulation. These results are direct evidence for an association-dissociation cycle of a small GTP-binding protein during traffic of its host membrane.     

ClC-5 Cl- -channel disruption impairs endocytosis in a mouse model for Dent's disease

Dent's disease is an X-linked disorder associated with the urinary loss of low-molecular-weight proteins, phosphate and calcium, which often leads to kidney stones. It is caused by mutations in ClC-5, a renal chloride channel that is expressed in endosomes of the proximal tubule. Here we show that disruption of the mouse clcn5 gene causes proteinuria by strongly reducing apical proximal tubular endocytosis. Both receptor-mediated and fluid-phase endocytosis are affected, and the internalization of the apical transporters NaPi-2 and NHE3 is slowed. At steady state, however, both proteins are redistributed from the plasma membrane to intracellular vesicles. This may be caused by an increased stimulation of luminal parathyroid hormone (PTH) receptors owing to the observed decreased tubular endocytosis of PTH. The rise in luminal PTH concentration should also stimulate the hydroxylation of 25(OH) vitamin D3 to the active hormone. However, this is counteracted by a urinary loss of the precursor 25(OH) vitamin D3. The balance between these opposing effects, both of which are secondary to the defect in proximal tubular endocytosis, probably determines whether there will be hypercalciuria and kidney stones.