Ca2+ triggers a novel clathrin-independent but actin-dependent fast endocytosis in pancreatic beta cells

The existence of clathrin-independent recycling of secretory vesicles has been controversial. By combining patch-clamp capacitance recording, optical methods and specific molecular interventions, we dissect two types of mechanistically different endocytosis in pancreatic β cells, both of which require GTP and dynamin. The fast one is a novel clathrin-independent but actin-dependent endocytosis that is triggered by high cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). Large fluorescent dextran (10 nm in diameter) was able to be internalized by this pathway, indicating that it was not likely to be 'kiss and run'. The slow endocytosis is a clathrin-dependent process in which actin plays a complementary role. For the first time, we show that the rate constants for both types of endocytosis exhibit supralinear dependence on increase in [Ca²⁺]_i. Compared with the slow endocytosis, higher [Ca²⁺]_i level was required to fully accelerate the fast one, indicative of distinct Ca²⁺ sensors for different endocytosis. In the end, we show that physiologically relevant stimulation induces clathrin-independent endocytosis in intact β cells, implying that it may contribute to the normal recycling of secretory vesicles in vivo .     

Regulation of the Ca2+ Sensitivity of Exocytosis by Rab3a

Abstract: Ca²⁺ ions trigger the release of hormones and neurotransmitters and contribute to making the secretory vesicles competent for fusion. Here, we present evidence for the involvement of the GTP-binding protein Rab3a in the sensitivity of the exocytotic process to internal [Ca²⁺]. The secretory activity of bovine adrenal chromaffin cells was elicited by Ca²⁺ dialysis through a patch-clamp pipette and assayed by monitoring changes in cell membrane capacitance. Microinjection of antisense oligonucleotides directed to rab3a mRNA increased the secretory activity observed at low (0.2–4 µ M ) [Ca²⁺], but did not change the maximal activity observed at 10 µ M free [Ca²⁺]. Moreover, after a train of depolarizing stimuli, the secretory activity of antisense-injected cells dialyzed with 10 µ M [Ca²⁺] was increased significantly compared with that of control cells. This result suggests that the activity of either Rab3a or its partners might change upon stimulation. We conclude that Rab3a, together with its partners, participates in the Ca²⁺ dependence of exocytosis and that its activity is modulated further in a stimulus-dependent manner. These findings should provide some clues to elucidate the role of Rab3a in synaptic plasticity.     

Control of Granule Mobility and Exocytosis by Ca2+-Dependent Formation of F-Actin in Pancreatic Duct Epithelial Cells

Elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) triggers exocytosis of secretory granules in pancreatic duct epithelia. In this study, we find that the signal also controls granule movement. Motions of fluorescently labeled granules stopped abruptly after a [Ca²⁺]_i increase, kinetically coincident with formation of filamentous actin (F-actin) in the whole cytoplasm. At high resolution, the new F-actin meshwork was so dense that cellular structures of granule size appeared physically trapped in it. Depolymerization of F-actin with latrunculin B blocked both the F-actin formation and the arrest of granules. Interestingly, when monitored with total internal reflection fluorescence microscopy, the immobilized granules still moved slowly and concertedly toward the plasma membrane. This group translocation was abolished by blockers of myosin. Exocytosis measured by microamperometry suggested that formation of a dense F-actin meshwork inhibited exocytosis at small Ca²⁺ rises <1 μ m . Larger [Ca²⁺]_i rises increased exocytosis because of the co-ordinate translocation of granules and fusion to the membrane. We propose that the Ca²⁺-dependent freezing of granules filters out weak inputs but allows exocytosis under stronger inputs by controlling granule movements.     

Sidedness of (Calcium, Magnesium) Adenosine Triphosphatase of Purified Torpedo Synaptic Vesicles

Abstract: Purified Torpedo synaptic vesicles contain ouabain-insensitive Mg²⁺_τ and Ca²⁺-stimulated ATPase activity. The sidedness of the ATPase on the vesicular membranes was investigated. Addition of ATP and Mg²⁺ or Ca²⁺ to intact vesicles results in activation of the ATPase. Exposure of the vesicles to low concentrations of Lubrol-PX and Triton X-100, which do not solubilize the activity, results in the concurrent release of the vesicular contents and in an increase of the Mg²⁺-ATPase activity, whereas the Ca²⁺-dependent activity is drastically decreased. p -Chloromercuribenzene sulphonate (PCMBS) almost completely inhibits the activity of detergent-treated vesicles whereas that of the native material is only slightly affected. Tryptic digestion of intact vesicles and of vesicular ghosts results in partial reduction of the ATPase activity. These results suggest that the vesicles contain an outward oriented Ca²⁺/Mg²⁺ ATPase activity which can be modulated by detergents.     

Role of Calcineurin in Ca2+-Induced Release of Catecholamines and Neuropeptides

Abstract: Neurotransmission requires rapid docking, fusion, and recycling of neurotransmitter vesicles. Several of the proteins involved in this complex Ca²⁺-regulated mechanism have been identified as substrates for protein kinases and phosphatases, e.g., the synapsins, synaptotagmin, rabphilin3A, synaptobrevin, munc18, MARCKS, dynamin I, and B-50/GAP-43. So far most attention has focused on the role of kinases in the release processes, but recent evidence indicates that phosphatases may be as important. Therefore, we investigated the role of the Ca²⁺/calmodulin-dependent protein phosphatase calcineurin in exocytosis and subsequent vesicle recycling. Calcineurin-neutralizing antibodies, which blocked dynamin I dephosphorylation by endogenous synaptosomal calcineurin activity, but had no effect on the activity of protein phosphatases 1 or 2A, were introduced into rat permeabilized nerve terminals and inhibited Ca²⁺-induced release of [³H]noradrenaline and neuropeptide cholecystokinin-8 in a specific and concentration-dependent manner. Our data show that the Ca²⁺/calmodulin-dependent phosphatase calcineurin plays an essential role in exocytosis and/or vesicle recycling of noradrenaline and cholecystokinin-8, transmitters stored in large dense-cored vesicles.     

Cellular Differentiation in Submerged Monolayers of Dictyostelium discoideum: Possible Functions of Cytoplasmic Ca2+and DIF

Cytoplasmic calcium ion (Ca²⁺) has generally been proposed to be a key factor of numerous cellular processes. Among several agents which might be expected to alter cytoplasmic Ca²⁺-concentration ([Ca²⁺]_i), unexpectedly Ca²⁺-antagonist TMB-8 was found to raise considerably [Ca²⁺]_i, and inhibited not only the formation of prespore cells, but also their maintenance in the monolayer cultures of Dictyostelium discoideum . This seems to indicate that higher [Ca²⁺]_i is unfavorable to the prespore differentiation. In this study, we adopted the monolayer culture technique to monitor cell differentiation. However, in high density monolayers there arised a number of unique cells which was highly vacuolated and morphologically intermediate between the stalk and spore cells. These vacuolated cells having both cellulosic wall and spore coat were also induced by differentiation inducing factor (DIF). Thus the monolayer culture system used might be not necessarily qualified to monitor the terminal differentiation of Dictyostelium cells. Nevertheless, the data presented here have strongly suggested that DIF have two physiologically valued roles: 1) Induction of the membrane fusion of vesicles and/or vacuoles (vacuolization), and 2) Induction of the fusion between the cell membrane and vacuole (or vesicle) membrane (exocytosis).     

Effects of Somatostatin on Intracellular Calcium Concentration in PC12 Cells

Abstract: Somatostatin (SS) is a neuropeptide that is distributed in various regions of the CNS, where it may act as a neurotransmitter or neuromodulator. SS produces multiple effects in the CNS through interactions with membrane receptors. In particular, SS inhibits various secretory responses in different cell types. In the present study, we have investigated the effects of exogenous application of SS on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in PC12 cells, a rat pheochromocytoma cell line. SS did reduce the magnitude of the secondary, maintained Ca²⁺ influx brought about by K⁺ depolarization. Similar effects were obtained with the application of SS analogues, such as d -Trp⁸-SS, d -Trp⁸- d -Cys¹⁴-SS, CGP-23996, and SMS-201995. In addition, treatment with cyclo-SS, a SS antagonist, did not alter [Ca²⁺]_i. Experiments with selective blockers of different voltage-dependent Ca²⁺ channels, such as methoxyverapamil (D600) and Ω-conotoxin GVIA, demonstrated that the effects of SS on [Ca²⁺]_i were mediated by voltage-dependent Ca²⁺ channels of the L type. Control experiments with a membrane potential indicator, i.e., the fluorescent dye bisoxonol, excluded that SS influenced the level of the membrane potential. SS effects on PC12 cells suggest the possibility that this neuropeptide plays a role in the modulation of cell functional activity by altering Ca²⁺ influx.     

Function of caveolae in Ca2+ entry and Ca2+-dependent signal transduction

The correct spatial and temporal control of Ca²⁺ signaling is essential for such cellular activities as fertilization, secretion, motility, and cell division. There has been a long-standing interest in the role of caveolae in regulating intracellular Ca²⁺ concentration. In this review we provide an updated view of how caveolae may regulate both Ca²⁺ entry into cells and Ca²⁺-dependent signal transduction     

Tyrosine Kinases Are Required for Catecholamine Secretion and Mitogen-Activated Protein Kinase Activation in Bovine Adrenal Chromaffin Cells

Abstract: Nicotine-induced catecholamine secretion in bovine adrenomedullary chromaffin cells is accompanied by rapid tyrosine phosphorylation of multiple cellular proteins, most notably the mitogen-activated protein kinases (MAPKs). The requirement for activation of tyrosine kinases and MAPKs in chromaffin cell exocytosis was investigated using a panel of tyrosine kinase inhibitors. Genistein and tyrphostin 23, two compounds that inhibit tyrosine kinases by distinct mechanisms, were found to inhibit secretion by >90% in cells stimulated by nicotine, 55 m M KCI, or the Ca²⁺ ionophore A23187. Inhibition of secretion induced by all three secretagogues correlated with a block in both protein tyrosine phosphorylation and activation of the MAPKs and their activators (MEKs) in situ. However, neither genistein nor tyrphostin 23 inhibited the activities of the MAPKs or MEKs in vitro. These results indicate that the target(s) of inhibition lie down-stream of Ca²⁺ influx and upstream of MEK activation. This Ca²⁺-activated tyrosine kinase activity could not be accounted for entirely by c-Src or Fyn (two nonreceptor tyrosine kinases that are expressed abundantly in chromaffin cells), because their in vitro kinase activities were not inhibited by tyrphostin 23 and only partially inhibited by genistein. These results demonstrate that an unidentified Ca²⁺-activated tyrosine kinase(s) is required for MAPK activation and exocytosis in chromaffin cells and suggest that MAPK participates in the regulation of secretion.     

Amphetamine reduces vesicular dopamine content in dexamethasone-differentiated PC12 cells only following l-DOPA exposure

Amphetamine (AMPH) increases brain dopamine (DA) levels via reversal of the membrane DA transporter. Additional mechanisms have been suggested, including inhibition of vesicular monoamine transporters and vesicular leakage of DA and Ca²⁺. According to the widely-accepted weak base theory, AMPH disrupts the proton gradient required for filling vesicles with DA. As a result, DA and Ca²⁺ will leak from vesicles, giving rise to exocytosis of less-filled vesicles. As several contradictions have been described, the aim of the present study was to re-examine this theory using amperometry and Fura-2 imaging to measure AMPH-induced changes in exocytosis and intracellular Ca²⁺ levels, respectively, in PC12 and chromaffin cells. Unexpectedly, 15 min exposure to AMPH (20–200 μM) does not affect the amount of DA released per vesicle, the frequency of exocytosis or intracellular Ca²⁺ levels in PC12 cells or chromaffin cells. Comparable results were found following prolonged exposure to AMPH (45 min) or at 37°C. When cells were pre-treated with the DA precursor l -DOPA, vesicle content increased to ∼150%. When these pre-treated cells are exposed to AMPH, vesicle content is strongly reduced. These results indicate that in dexamethasone-differentiated PC12 cells AMPH-induced vesicle leakage occurs only under specific conditions, therefore arguing for re-evaluation of the theory of AMPH-induced vesicular DA leakage.     

Multisite Interactions Between Pb2+ and Protein Kinase C and Its Role in Norepinephrine Release from Bovine Adrenal Chromaffin Cells

Abstract: We investigated the interaction between Pb²⁺ and protein kinase C (PKC) in the Pb²⁺-induced release of norepinephrine (NE) from permeabilized adrenal chromaffin cells. Our analysis of endogenous PKC activity in permeabilized cells suggests that Pb²⁺ interacts with the adrenal enzyme at multiple sites. Pb²⁺ activates the enzyme through high-affinity ( K _A(Pb) = 2.4 × 10⁻¹² M ) interactions and inhibits the enzyme by competitive and noncompetitive interactions with nanomolar-( K _i = 7.1 × 10⁻⁹ M ) and micromolar- ( K '_i = 2.8 × 10⁻⁷ M ) affinity sites, respectively. Activation of PKC by 12- O -tetradecanoylphorbol 13-acetate (TPA) in Ca²⁺-deficient, Pb²⁺-containing medium, enhances the Pb²⁺-induced NE release from permeabilized chromaffin cells by lowering the concentration of Pb²⁺ required for half-maximal activation of the secretory response from 7.5 × 10⁻¹⁰ to 5.7 × 10⁻¹¹ M . The PKC inhibitors staurosporine and pseudosubstrate PKC (19–36) abolish the effect of TPA without affecting the Pb²⁺-induced secretion in the absence of TPA. These results indicate that (a) Pb²⁺ is a partial agonist of PKC, capable of both activating and inhibiting the enzyme and (b) synergistic activation of PKC by TPA and Pb²⁺ results in increased sensitivity of exocytosis to Pb²⁺ but is not obligatory for Pb²⁺-triggered secretion.     

Regulation of exocytotic protein expression and Ca2+-dependent peptide secretion in astrocytes

Vesicular transmitter release from astrocytes influences neuronal development, function and plasticity. However, secretory pathways and the involved molecular mechanisms in astroglial cells are poorly known. In this study, we show that a variety of SNARE and Munc18 isoforms are expressed by cultured astrocytes, with syntaxin-4, Munc18c, SNAP-23 and VAMP-3 being the most abundant variants. Exocytotic protein expression was differentially regulated by activating and differentiating agents. Specifically, proteins controlling Ca²⁺-dependent secretion in neuroendocrine cells were up-regulated after long-term 8Br-cAMP administration in astrocytes, but not by proinflammatory cytokines. Moreover, 8Br-cAMP treatment greatly increased the cellular content of the peptidic vesicle marker secretogranin-2. Release assays performed on cAMP-treated astrocytes showed that basal and stimulated secretogranin-2 secretion are dependent on [Ca²⁺]_i. As shown release of the chimeric hormone ANP.emd from transfected cells, cAMP-induced differentiation in astrocytes enhances Ca²⁺-regulated peptide secretion. We conclude that astroglial cells display distinctive molecular components for exocytosis. Moreover, the regulation of both exocytotic protein expression and Ca²⁺-dependent peptide secretion in astrocytes by differentiating and activating agents suggest that glial secretory pathways are adjusted in different physiological states.     

Silencing of NtMPK4 impairs CO-induced stomatal closure, activation of anion channels and cytosolic Casignals in Nicotiana tabacum guard cells

Light-induced stomatal opening in C3 and C4 plants is mediated by two signalling pathways. One pathway is specific for blue light and involves phototropins, while the second pathway depends on photosyntheticaly active radiation (PAR). Here, the role of Nt MPK4 in light-induced stomatal opening was studied, as silencing of this MAP kinase stimulates stomatal opening. Stomata of Nt MPK4-silenced plants do not close in elevated atmospheric CO₂, and show a reduced response to PAR. However, stomatal closure can still be induced by abscisic acid. Measurements using multi-barrelled intracellular micro-electrodes showed that CO₂ activates plasma membrane anion channels in wild-type Nicotiana tabacum guard cells, but not in Nt MPK4-silenced cells. Anion channels were also activated in wild-type guard cells after switching off PAR. In approximately half of these cells, activation of anion channels was accompanied by an increase in the cytosolic free Ca²⁺ concentration. The activity of anion channels was higher in cells showing a parallel increase in cytosolic Ca²⁺ than in those with steady Ca²⁺ levels. Both the darkness-induced anion channel activation and Ca²⁺ signals were repressed in Nt MPK4-silenced guard cells. These data show that CO₂ and darkness can activate anion channels in a Ca²⁺-independent manner, but the anion channel activity is enhanced by parallel increases in the cytosolic Ca²⁺ concentration. Nt MPK4 plays an essential role in CO₂- and darkness-induced activation of guard-cell anion channels, through Ca²⁺-independent as well as Ca²⁺-dependent signalling pathways.     

Catecholamine Secretion from Bovine Adrenal Chromaffin Cells: The Role of the Na+/Ca2+ Exchanger and the Intracellular Ca2+ Pool

Abstract: The role of the Na⁺/Ca²⁺ exchanger and intracellular nonmitochondrial Ca²⁺ pool in the regulation of cytosolic free calcium concentration ([Ca²⁺]_i) during catecholamine secretion was investigated. Catecholamine secretion and [Ca²⁺]_i were simultaneously monitored in a single chromaffin cell. After high-K⁺ stimulation, control cells and cells in which the Na⁺/Ca²⁺ exchange activity was inhibited showed similar rates of [Ca²⁺]_i elevation. However, the recovery of [Ca²⁺]_i to resting levels was slower in the inhibited cells. Inhibition of the exchanger increased the total catecholamine secretion by prolonging the secretion. Inhibition of the Ca²⁺ pump of the intracellular Ca²⁺ pool with thapsigargin caused a significant delay in the recovery of [Ca²⁺]_i and greatly enhanced the secretory events. These data suggest that both the Na⁺/Ca²⁺ exchanger and the thapsigargin-sensitive Ca²⁺ pool are important in the regulation of [Ca²⁺]_i and, by modulating the time course of secretion, are important in determining the extent of secretion.     

Two Distinct ATP Receptors Activate Calcium Entry and Internal Calcium Release in Bovine Chromaffin Cells

Abstract: ATP, an established neurotransmitter, causes elevation of cytosolic Ca²⁺ and catecholamine secretion when applied to chromaffin cells in the intact adrenal gland. The ATP-induced rise in Ca²⁺ is due both to release from internal stores and to entry across the plasma membrane. The latter source of Ca²⁺ causes secretion; the primary role of Ca²⁺ released from internal stores remains undetermined. In this article, we have studied the nucleotide specificity for activating the two types of Ca²⁺ increases. The agonist potency order for the increase in fluorescence from fura-2-loaded chromaffin cells due to release of Ca²⁺ from internal stores is ATP = UTP > ADP > 2-methylthio-ATP, α,β-methylene ATP, identifying the receptor as a P_2U purinoceptor. The potency order for secretion is 2-methylthio-ATP > ATP > α,β-methylene ATP, ADP, UTP, placing the receptor in the P_2Y subtype. Thus, two distinct receptors are responsible for Ca²⁺ release and secretion. Agonists were more effective in the absence of extracellular Mg²⁺, suggesting that ATP uncomplexed with divalent cations binds preferentially to both receptors. The low response of both receptors to ADP distinguishes them from the ATP receptor on these cells that inhibits voltage-dependent Ca²⁺ current and secretion.     

Adenylyl Cyclases: mRNA and Characteristics of Enzyme Activity in Three Areas of Brain

Abstract: Arachidonic acid and oleoylacetylglycerol enhance depolarization-evoked glutamate release from hippocampal mossy fiber nerve endings. It was proposed this is a Ca²⁺-dependent effect and that protein kinase C is involved. Here we report that arachidonic acid and oleoylacetylglycerol synergistically potentiate the glutamate release induced by the Ca²⁺ ionophore ionomycin. The Ca²⁺ dependence of this effect was established, as removal of Ca²⁺ eliminated evoked release and the lipid-dependent potentiation. Also, Ca²⁺ channel blockers attenuated ionomycin- and KCI-evoked exocytosis, as well as the facilitating effects of the lipid mediators. Although facilitation required Ca²⁺, it may not involve an enhancement of evoked Ca²⁺ accumulation, because ionomycin-dependent glutamate release was potentiated under conditions that did not increase ionomycin-induced Ca²⁺ accumulation. Also, the facilitation may not depend on inhibition of K⁺ efflux, because enhanced release was observed in the presence of increasing concentrations of 4-aminopyridine and diazoxide did not reduce the lipid-dependent potentiation of exocytosis. In contrast, disruption of cytoskeleton organization with cytochalasin D occluded the lipid-dependent facilitations of both KCI- and ionomycin-evoked glutamate release. In addition, arachidonic acid plus glutamatergic or cholinergic agonists enhanced glutamate release, whereas a role for protein kinase C in the potentiation of exocytosis was substantiated using kinase inhibitors. It appears that the lipid-dependent facilitation of glutamate release from mossy fiber nerve endings requires Ca²⁺ and involves multiple presynaptic effects, some of which depend on protein kinase C.     

Roles for Potassium and Calcium Channels in the Initiation of Sperm Motility in Rainbow Trout.

It is well known that the motility of spermatozoa in rainbow trout is suppressed by K⁺. We showed here that although trout sperm are completely immotile in medium containing 5 mM K⁺, motility was initiated by the subsequent addition of several mM Ca²⁺, suggesting that both K⁺and Ca²⁺are related to the process of the initiation of sperm motility. It was further found that K⁺channel blockers tetraethylammonium, nonyltriethylammonium, Ba²⁺and Cs⁺, as well as the Ca²⁺channel blocker verapamil, inhibited the initiation of sperm motility at doses at which these reagents inhibit chnnel-related functions in other cells. However, Na⁺channel blocker, tetrodotoxin and anion channel blocker 4, 4-diisothiocyatatostilbene-2, 2'-disulfonic acid inhibited the motility only at extremely high doses. These results suggest that transport of K⁺and Ca²⁺through ion channels at the plasma membrane of spermatozoa is the first event that triggers the initiation of sperm motility in rainbow trout.     

Direct Observation of Serotonin 5-HT3 Receptor-Induced Increases in Calcium Levels in Individual Brain Nerve Terminals

Abstract: Confocal microscopy was used to assess internal calcium level changes in response to presynaptic receptor activation in individual, isolated nerve terminals (synaptosomes) from rat corpus striatum, focusing, in particular, on the serotonin 5-HT₃ receptor, a ligand-gated ion channel. The 5-HT₃ receptor agonist-induced calcium level changes in individual synaptosomes were compared with responses evoked by K⁺ depolarization. Using the fluorescent dye fluo-3 to measure relative changes in internal free Ca²⁺ concentration ([Ca²⁺]_i), K⁺-induced depolarization resulted in variable but rapid increases in apparent [Ca²⁺]_i among the individual terminals, with some synaptosomes displaying large transient [Ca²⁺]_i peaks of varying size (two- to 12-fold over basal levels) followed by an apparent plateau phase, whereas others displayed only a rise to a sustained plateau level of [Ca²⁺]_i (two- to 2.5-fold over basal levels). Agonist activation of 5-HT₃ receptors induced slow increases in [Ca²⁺]_i (rise time, 15–20 s) in a subset (∼5%) of corpus striatal synaptosomes, with the increases (averaging 2.2-fold over basal) being dependent on Ca²⁺ entry and inhibited by millimolar external Mg²⁺. We conclude that significant increases in brain nerve terminal Ca²⁺, rivaling that found in response to excitation by depolarization but having distinct kinetic properties, can therefore result from the activation of presynaptic ligand-gated ion channels.     

Calcium and Cyclic AMP Mediate Sperm Activation, but Ca2+Alone Contributes Sperm Chemotaxis in the Ascidian, Ciona savignyi

Sperm-activating and -attracting factor (SAAF) released from the ascidian, Ciona savignyi , was partially purified from egg seawater with ethanol extraction and separation with the two-phase system of chloroform and water. SAAF did not activate sperm motility and cAMP synthesis in calcium-free seawater (CaFSW), but activated the both in the presence of Ca²⁺. Sperm activation by SAAF in Ca²⁺-containing medium was inhibited by flunarizine, a T-type Ca²⁺channel antagonist, but L-type Ca²⁺channel specific antagonists had no effect. Theophylline, a phosphodiesterase inhibitor, induced the increase of cAMP level and sperm activation in CaFSW without SAAF. On the other hand, the theophylline-activated sperm in CaFSW did not exhibit chemotaxis toward the tip of glass capillary containing SAAF, but upon the addition of Ca²⁺they were attracted toward SAAF in the same manner as chemotaxis in normal artificial seawater. These results suggest that sperm activation is induced by the increased cAMP level caused by Ca²⁺influx through T-type Ca²⁺channel, and that Ca²⁺alone mediates the sperm chemotaxis in Ciona .     

Change in Membrane Fluidity of Sand Dollar Egg Cortices Caused by Ca2+-Induced Exocytosis: Microscopic Analysis with Fluorescence Anisotropy

The surface membrane fluidity of sand dollar eggs after exocytosis was investigated by using a specially designed video-microscope system to measure the fluorescence depolarization of isolated cortices stained with hexadecanoylaminofluorescein. When unfertilized eggs were stained before isolation, the plasma membranes became labeled with fluorescent dye, but cortical vesicles did not. The fluorescence anisotropy of the isolated cortices increased from 0.256 to 0.285 during exocytosis induced by Ca²⁺. The increased anisotropy was not changed by lowering the Ca²⁺concentration after exocytosis. Dislodging of cortical vesicles by shearing with a stream of solution had no affect on the anisotropy. These results suggest that the fluidity of the plasma membrane decreases after exocytosis. When cortices were stained after isolation, both plasma membranes and cortical membrane organelles became labeled. These cortices possessed an anisotropy of 0.215. After dislogding the cortical organelles the anisotropy increased to 0.232. These results indicate that the fluidity of the cytoplasmic membrane leaflets of cortical organelles is higher than that of the plasma membrane. Therefore, it was suggested that only the outer leaflet of the plasma membrane becomes less fluid after exocytosis.