Cortical Vesicles Research Articles

Reproductive plasticity of Hypostomus affinis (Siluriformes: Loricariidae) as a mechanism to adapt to a reservoir with poor habitat complexity

In this work we describe the gonad morphology and spawning season of Hypostomus affinis (Steindachner, 1877) in a tropical reservoir based on 55 males and 125 females. Our aim was to assess eventual adaptations in reproductive tactics developed by this riverine species inhabiting an oligotrophic reservoir with low habitat complexity, few rocks and few other preferred consolidated substrata. We described the stages of cells of reproductive lineage, gonadal development and some reproductive traits which were compared with information in the available literature. Cells from the spermatogenic lineage were spermatogonia, spermatocytes, spermatids and spermatozoa, and cells from the oocytarian lineage were primary oocytes, previtelogenic oocytes, cortical vesicle oocytes and yolk globules or vitellogenic. Five stages were described for the males/females according to the distribution of oocytes and spermatogenic lineage cells: resting; initial maturation; advanced maturation; partially spent/spawned; totally spent/spawn. Females outnumbered males and reached larger size. Synchronic ovary development in two groups was found with diameter of mature oocytes ranging from 2 to 3.35 mm. Indication of early maturation, a longer reproductive period and the production of smaller eggs in small clutches seems to be features of the reservoir population not found in riverine systems. Such changes in tactics may indicate a shift to an opportunistic strategy, helping the population to withstand environmental constraints and to succeed in this oligotrophic and poorly structured reservoir.

A new approach to the molecular analysis of docking, priming, and regulated membrane fusion

Studies using isolated sea urchin cortical vesicles have proven invaluable in dissecting mechanisms of Ca(2+)-triggered membrane fusion. However, only acute molecular manipulations are possible in vitro. Here, using selective pharmacological manipulations of sea urchin eggs ex vivo, we test the hypothesis that specific lipidic components of the membrane matrix selectively affect defined late stages of exocytosis, particularly the Ca(2+)-triggered steps of fast membrane fusion. Egg treatments with cholesterol-lowering drugs resulted in the inhibition of vesicle fusion. Exogenous cholesterol recovered fusion extent and efficiency in cholesterol-depleted membranes; α-tocopherol, a structurally dissimilar curvature analogue, selectively restored fusion extent. Inhibition of phospholipase C reduced vesicle phosphatidylethanolamine and suppressed both the extent and kinetics of fusion. Although phosphatidylinositol-3-kinase inhibition altered levels of polyphosphoinositide species and reduced all fusion parameters, sequestering polyphosphoinositides selectively inhibited fusion kinetics. Thus, cholesterol and phosphatidylethanolamine play direct roles in the fusion pathway, contributing negative curvature. Cholesterol also organizes the physiological fusion site, defining fusion efficiency. A selective influence of phosphatidylethanolamine on fusion kinetics sheds light on the local microdomain structure at the site of docking/fusion. Polyphosphoinositides have modulatory upstream roles in priming: alterations in specific polyphosphoinositides likely represent the terminal priming steps defining fully docked, release-ready vesicles. Thus, this pharmacological approach has the potential to be a robust high-throughput platform to identify molecular components of the physiological fusion machine critical to docking, priming, and triggered fusion.

Dissecting the mechanism of Ca2+‐triggered membrane fusion: Probing protein function using thiol reactivity

1. Ca(2+)-triggered membrane fusion involves the coordinated actions of both lipids and proteins, but the specific mechanisms remain poorly understood. The urchin cortical vesicle model is a stage-specific native preparation fully enabling the directly coupled functional-molecular analyses necessary to identify critical components of fast triggered membrane fusion. 2. Recent work on lipidic components has established a direct role for cholesterol in the fusion mechanism via local contribution of negative curvature to readily enable the formation of transient lipidic fusion intermediates. In addition, cholesterol- and sphingomyelin-enriched domains regulate the efficiency of fusion by focally organizing other components to ensure an optimized response to the triggering Ca(2+) transient. 3. There is less known about the identity of proteins involved in the Ca(2+)-triggering steps of membrane fusion. Thiol reagents can be used as unbiased tools to probe protein functions. Comparisons of several thiol-reactive reagents have identified different effects on Ca(2+) sensitivity and the extent of fusion, suggesting that there are at least two distinct thiol sites that participate in the fusion mechanism: one that regulates the efficiency of Ca(2+) sensing/triggering and one that may function during the membrane merger event itself. 4. To identify the proteins that regulate Ca(2+) sensitivity, the fluorescent thiol reagent Lucifer yellow iodoacetamide was used to potentiate fusion and simultaneously tag the proteins involved. Ongoing work involves the isolation of cholesterol-enriched membrane fractions to reduce the complexity of the labelled proteome, narrowing the number of candidate proteins.

346 ULTRASTRUCTURE OF A HUMAN GIANT MII OOCYTE: CASE REPORT

The occurrence of giant oocytes has been reported with considerable frequency during IVF procedures in women of all ages and was associated with an increased response to gonadotrophin therapy. Previous studies suggested that giant oocytes, larger in diameter than normal oocytes and either at the GV, MI, or MII stage, might be a source of human digynic triploidy. However, to our knowledge there are no previous reports concerning the ultrastructure of human giant oocytes, and therefore the objective of this study was to characterize a human giant mature metaphase II oocyte from the ultrastuctural point of view. A giant oocyte with a visible polar body was used under informed consent, after controlled superovulation during a IVF treatment cycle. The oocyte was fixed with Karnovsky’s fixative, post-fixed in 2% OsO4 in 0.15 M cacodylate buffer, pH 7.2, dehydrated and embedded in Epon Semithin and ultrathin sections were cut with a diamond Diatome knife (Diatome, Hatfield, PA, USA) in a LKB ultramicrotome. Semithin sections were stained with aqueous azur II and methylene blue (1:1). Ultrathin sections were collected on 200 mesh copper grids (Taab Laboratories Equipment Ltd, Aldermaston, UK) and stained with 3% aqueous uranyl acetate (20 min) and Reynolds lead citrate (10 min). Contrast ultrathin sections were observed in a JEOL 100XII transmission electron microscope (JEOL Ltd., Tokyo, Japan) operated at 60 Kv. Cytogenetic analysis was not performed in this oocyte. At the time of collection, the giant oocyte was 1.4-fold larger than average size oocytes and contained a fragmented polar body. The zona pellucida had a normal loosely fibrillar structure and the perivitelline space was normal containing remnants of follicular cells. The cytoplasmic organelles were no uniformly dispersed with a large organelle-free zone (lake) observed in the periphery of the oocyte cortex and smaller focal lakes visible in the subcortex. Within the cortex the dense cortical vesicles were reduced in number and did not form one or two continuous rows beneath the oolemma. Smooth endoplasmic reticulum (SER) aggregates of tubules were also scarce but underdeveloped SER aggregates of tubules were present in the cortex. The distribution of the SER large and middle vesicles was abnormal: these organelles were nearly absent in the cortex but present in the inner cytoplasm. The content of some vesicles was denser than normal. We also observed the presence of several large secondary lysosomes filled with multiple small and medium lipid droplets (lipofuscin bodies), corresponding to retractile bodies located in the cortex. Large dense vesicles of different densities and volumes were found in the cortex and subcortex. The polar body showed very few cortical vesicles and did not contain mitochondria and SER large or small vesicles. The condensed chromosomes were aligned on the metaphase II plate, in the cortex of the oocyte. The ultrastructural alterations observed in this oocyte corroborated the conclusions of previous genetic studies and confirmed that giant oocytes are immature cytoplasmatically and would be associated with a higher frequency of abnormal development following fertilization. Thus, giant oocytes should not be used in IVF treatments. ASL was supported by FCT, Portugal.

Sodium-hydrogen exchange isoform expression in blood cells: implications for studies in diabetes mellitus.

There have been many reports of increased Na-H exchange (NHE) activity in the peripheral blood cells (erythrocytes, lymphocytes and platelets) of patients with diabetes mellitus compared to nondiabetic controls. This raised NHE activity has been hypothesized to reflect increased NHE activity in kidney and vascular smooth muscle. Raised NHE activity in these tissues could play a pathophysiological role in mediating hypertension, vascular smooth muscle cell proliferation and progressive renal impairment. It is now known that there are at least five NHE isoforms, but a specific study examining expression of NHE isoforms in peripheral blood cells has not been reported. This study used specific antisera to NHE isoforms 1, 3 and 4 to examine NHE expression by immunoblot analysis. Erythrocyte, lymphocyte and platelet membranes from both rabbit and rat were separated by standard methods. A monoclonal antibody to NHE-1 reacted with a 100-110 kDa band in rabbit and rat platelets and lymphocytes (identical to that observed in basolateral-enriched renal cortical vesicles) and a 100 kDa band in rabbit and rat erythrocytes. In both species, the intensity of the staining was greatest in platelet membranes. A polyclonal antibody to NHE-3, the isoform present on the apical membranes of renal proximal tubule, showed no evidence of staining in any of the peripheral blood cell preparations. Similarly there was no evidence of expression of NHE-4 in the peripheral blood cell preparations. Peripheral blood cells express NHE-1, which likely accounts for amiloride-sensitive Na-H exchange in these cells, playing a role in cell volume and pH regulation. However, there is no evidence that there is expression of NHE-3 or NHE-4 in peripheral blood cells. These data have implications for studies in hypertension and diabetes mellitus which measure peripheral blood cell Na-H exchange and hypothesize regarding a direct pathophysiological role for this increased activity.

Using Thiol-Reactivity to Identify Proteins Involved in the Ca2+-Triggering Steps of Native Membrane Fusion

Ca2+-triggered membrane fusion, the defining step of exocytosis, enables temporal/spatial control over the release of biologically active compounds. The mechanism by which Ca2+ triggers and modulates native membrane fusion is still poorly understood. As an unbiased approach to investigating this process, the effects of several thiol-reactive reagents on the homotypic fusion of isolated cortical vesicles (a stage-specific preparation for analyses of native Ca2+-triggered fusion) have been characterized. Such reagents have been consistently shown to inhibit the Ca2+-sensitivity, rate and extent of triggered fusion. However, we recently showed that iodoacetamide can also potentiate the Ca2+-sensitivity and rate of release [1]. This implicates two distinct thiol sites in the fusion process - one involved in the ability of vesicles to fuse (extent) and one that modulates fusion efficiency (Ca2+-sensitivity and kinetics). Capitalizing on this potentiating effect, we have now identified other fluorescent thiol-reactive reagents with similar effects: treatment with Lucifer yellow iodoacetamide, monobromobimane or dibromobimane resulted in an average leftward shift in EC50 from 17.2±1.6μM to 8.9±1.9μM [Ca2+]free. These fluorescent reagents can be used to enhance fusion and label proteins involved in the Ca2+-sensing mechanism. The lipid matrix at or near the fusion site can also modulate the fusion process, specifically via cholesterol- and sphingomyelin-enrichment that is thought to regulate the Ca2+-sensitivity and rate of fusion through spatial organization of critical lipids and proteins [2,3]. Proteins involved in Ca2+-sensing are thus likely to be situated within such areas of the membrane. Isolation of fluorescently labeled proteins from cholesterol-enriched vesicle membrane fractions by 2-dimesional electrophoresis is now being used to identify proteins potentially involved in the Ca2+-triggering steps of membrane fusion.1) J. Chem. Biol., Epub 3/10/08. 2) J. Cell Sci. 2005, 118:4833. 3) J. Cell Sci. 2006, 119:2688.

Identifying Critical Components of Native Ca2+‐triggered Membrane Fusion

Ca(2+)-triggered membrane fusion is the defining step of exocytosis. Despite realization that the fusion machinery must include lipids and proteins working in concert, only of late has work in the field focused more equally on both these components. Here we use isolated sea urchin egg cortical vesicles (CV), a stage-specific preparation of Ca(2+)-sensitive release-ready vesicles that enables the tight coupling of molecular and functional analyses necessary to dissect molecular mechanisms. The stalk-pore hypothesis proposes that bilayer merger proceeds rapidly via transient, high-negative curvature, intermediate membrane structures. Consistent with this, cholesterol, a major component of the CV membrane, contributes to a critical local negative curvature that supports formation of lipidic fusion intermediates. Following cholesterol depletion, structurally dissimilar lipids having intrinsic negative curvature greater than or equal to cholesterol recover the ability of CV to fuse but do not recover fusion efficiency (Ca(2+) sensitivity and kinetics). Conversely, cholesterol- and sphingomyelin-enriched microdomains regulate the efficiency of the fusion mechanism, presumably by contributing spatial and functional organization of other critical lipids and proteins at the fusion site. Critical proteins are thought to participate in Ca(2+) sensing, initiating membrane deformations, and facilitating fusion pore expansion. Capitalizing on a novel effect of the thiol-reactive reagent iodoacetamide (IA), potentiation of the Ca(2+) sensitivity and kinetics, a fluorescently tagged IA has been used to enhance fusion efficiency and simultaneously label the proteins involved. Isolation of cholesterol-enriched CV membrane fractions, using density gradient centrifugation, is being used to narrow the list of protein candidates potentially critical to the mechanism of fast Ca(2+)-triggered membrane fusion.

Enhancement of the Ca2+-triggering steps of native membrane fusion via thiol-reactivity

Ca(2+)-triggered membrane fusion is the defining step of exocytosis. Isolated urchin cortical vesicles (CV) provide a stage-specific preparation to study the mechanisms by which Ca(2+) triggers the merger of two apposed native membranes. Thiol-reactive reagents that alkylate free sulfhydryl groups on proteins have been consistently shown to inhibit triggered fusion. Here, we characterize a novel effect of the alkylating reagent iodoacetamide (IA). IA was found to enhance the kinetics and Ca(2+) sensitivity of both CV-plasma membrane and CV-CV fusion. If Sr(2+), a weak Ca(2+) mimetic, was used to trigger fusion, the potentiation was even greater than that observed for Ca(2+), suggesting that IA acts at the Ca(2+)-sensing step of triggered fusion. Comparison of IA to other reagents indicates that there are at least two distinct thiol sites involved in the underlying fusion mechanism: one that regulates the efficiency of fusion and one that interferes with fusion competency.

Ischemia enhances activation by Ca2+ and redox modification of ryanodine receptor channels from rat brain cortex.

Cerebral ischemia stimulates Ca2+ influx and thus increases neuronal intracellular free [Ca2+]. Using a rat model of cerebral ischemia without recirculation, we tested whether ischemia enhances the activation by Ca2+ of ryanodine receptor (RyR) channels, a requisite feature of RyR-mediated Ca2+-induced Ca2+ release (CICR). To this aim, we evaluated how single RyR channels from endoplasmic reticulum vesicles, fused into planar lipid bilayers, responded to cytoplasmic [Ca2+] changes. Endoplasmic reticulum vesicles were isolated from the cortex of rat brains incubated without blood flow for 5 min at 37 degrees C (ischemic) or at 4 degrees C (control). Ischemic brains displayed increased oxidative intracellular conditions, as evidenced by a lower ratio (approximately 130:1) of reduced/oxidized glutathione than controls (approximately 200:1). Single RyR channels from ischemic or control brains displayed the same three responses to Ca2+ reported previously, characterized by low, moderate, or high maximal activity. Relative to controls, RyR channels from ischemic brains displayed with increased frequency the high activity response and with lower frequency the low activity response. Both control and ischemic cortical vesicles contained the RyR2 and RyR3 isoforms in a 3:1 proportion, with undetectable amounts of RyR1. Ischemia reduced [3H]ryanodine binding and total RyR protein content by 35%, and increased at least twofold endogenous RyR2 S-nitrosylation and S-glutathionylation without affecting the corresponding RyR3 endogenous levels. In vitro RyR S-glutathionylation but not S-nitrosylation favored the emergence of high activity channels. We propose that ischemia, by enhancing RyR2 S-glutathionylation, allows RyR2 to sustain CICR; the resulting amplification of Ca2+ entry signals may contribute to cortical neuronal death.

Copper (II) sulfate charring for high sensitivity on-plate fluorescent detection of lipids and sterols: quantitative analyses of the composition of functional secretory vesicles

A wide range of methods exist for the on-plate detection of lipids resolved by thin layer chromatography. Fluorescence generally offers improvements in sensitivity over methods that use colorimetric or simple densitometric detection. In this paper, we report that a classic cupric sulfate charring protocol produces a fluorescent signal that sensitively and quantitatively detects a wide range of phospholipids, neutral lipids, and sterols after automated, multi-development high performance thin layer chromatography. The measured lower limits of detection and quantification, respectively, were, on average, 80 and 210 pmol for phospholipids and 43 fmol and 8.7 pmol for sterols. The simple, inexpensive, and highly sensitive approach described here was used to quantitatively analyze the lipid and sterol composition of sea urchin cortical vesicles, a stage-specific model system used to study the mechanism of regulated membrane fusion.

Two independent forms of endocytosis maintain embryonic cell surface homeostasis during early development

Eukaryotic cells have multiple forms of endocytosis which maintain cell surface homeostasis. One explanation for this apparent redundancy is to allow independent retrieval of surface membranes derived from different types of vesicles. Consistent with this hypothesis we find that sea urchin eggs have at least two types of compensatory endocytosis. One is associated with retrieving cortical vesicle membranes, and formed large endosomes by a mechanism that was inhibited by agatoxin, cadmium, staurosporine and FK506. The second type is thought to compensate for constitutive exocytosis, and formed small endosomes using a mechanism that was insensitive to the above mentioned reagents, but was inhibited by phenylarsine oxide (PAO), and by microinjection of mRNA encoding Src kinase. Both mechanisms could act concurrently, and account for all of the endocytosis occurring during early development. Inhibition of either form did not trigger compensation by the other form, and phorbol ester treatment rescued the endocytotic activity blocked by agatoxin, but not the retrieval blocked by PAO.

Fertilization and Nicotinic Acid Adenine Dinucleotide Phosphate Induce pH Changes in Acidic Ca2+ Stores in Sea Urchin Eggs

The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) releases Ca(2+) from the acidic Ca(2+) stores of many organisms, including those of the sea urchin egg. We investigated whether the pH within the lumen of these acidic organelles changes in response to stimuli. Fertilization activates the egg by Ca(2+) release dependent upon NAADP, and accordingly, we report that fertilization also alters organellar pH in a spatio-temporally complex manner. Upon sperm fusion, vesicles deep in the egg center slowly acidify, whereas cortical vesicles undergo a rapid alkalinization. The cortical vesicle alkalinization is independent of exocytosis and cytosolic pH but coincides with the NAADP-dependent fertilization Ca(2+) wave. Microinjection of NAADP mimicked the fertilization cortical response, suggesting that it occurred within NAADP-sensitive acidic Ca(2+) stores. Our data show that NAADP and physiological stimuli alter the pH within intracellular organelles and suggest that NAADP signals through pH as well as Ca(2+).

Morphology of gonads, maturity and spawning season of Loricariichthys spixii (Siluriformes, Loricariidae) in a Subtropical Reservoir

The gonad morphology and spawning season of Loricariichthys spixii in Lajes reservoir were described based on 175 males and 613 females. Cells from the spermatogenic lineage were divided in four phases: spermatogonia (primary and secondary), spermatocytes (primary and secondary), spermatids and spermatozoa, and the cells from the ovocitarian lineage were divided in four phases: primary oocytes (O1), previtellogenic oocytes (O2), cortical vesicle oocytes (O3) and yolk globules or vitellogenic (O4). Five gonadal stages were described for the males/females according to oocytes and spermatogenic lineage cells distribution: resting (1); initial maturation (2a); advanced maturation (2b); partially spent/spawned (4a); totally spent/spawned (4b). Spawning was iteroparous, occurring throughout the year. Resting/recover occurred in July/August for the females coinciding with lower temperature and rainfall and decreasing water level. The wide spawning period was the part of the strategy developed to withstand environmental pressure and to get success in this oligotrophic and poorly structured environment.

Taxonomic characterization of two marine peritrichous ciliates, Epicarchesium corlissi n. sp. and Pseudovorticella jiangi n. sp. (Ciliophora: Peritrichia), from northern China

Two new marine peritrich ciliates, Epicarchesium corlissi n. sp. and Pseudovorticella jiangi n. sp., were discovered in mariculture waters on the coast of northern China near Qingdao. Their morphology, infraciliature and silverline system were investigated based on both living and silver-impregnated specimens. E. corlissi is characterized as follows: marine Epicarchesium with dichotomously branched stalk; zooids elongate, approximately 60–70×25–35 μm in vivo; peristomial collar double-folded; macronucleus J-shaped; single, small contractile vacuole ventrally positioned; more than 60 striations between peristome and aboral trochal band, 13–18 from aboral trochal band to scopula; abstomal end of row 1 of infundibular polykinety 3 terminating at same level as rows 2 and 3 of infundibular polykinety 3; rows 2 and 3 of infundibular polykinety 3 much longer than row 1 and converging adstomally with infundibular polykinety 1. The new species P. jiangi is diagnosed as follows: marine Pseudovorticella; zooid inverted bell-shaped, approximately 80×60 μm in vivo and with a broad, flat, thin peristomial collar that measures approximately 90 μm across; pellicle with transparent cortical vesicles; macronucleus J-shaped; number of silverlines between peristome and aboral trochal band 20–24, from aboral trochal band to scopula 9–11; abstomal end of row 1 of infundibular polykinety 3 diverges from the other two rows of this polykinety and ends alongside row 3 of infundibular polykinety 2.

Oogenesis in calanoid copepods

Summary An ultrastructural investigation of ovary structure and oogenesis was conducted on ten species of cold and warm water calanoid copepods from six families with broad distributions ranging from coastal to mesopelagic habitats. They include Calanus finmarchicus, C. hyperboreus, C. glacialis, Euchaeta marina, Centropages furcatus, C. velificatus, Labidocera aestiva, Acartia tonsa, Pleurommama xiphias, and Undinula vulgaris. All ten species show similarities with respect to ovarian morphology and oogenesis. Oogenesis is asynchronous with all stages of oocytes present within each sexually mature female. The oocytes differentiate to diplotene of the first meiotic division and undergo a growth phase within the ovary before initiating vitellogenesis within the diverticulae. Vitellogenesis involves both an autosynthetic (endogenous) and a heterosynthetic (exogenous) process resulting in the deposition of two different types of yolk bodies. Endogenous yolk formation is initiated by the proliferation of vesicular endoplasmic reticulum (ER) through the blebbing of the outer lamina of the nuclear envelope. These ER vesicles contain a variable number of granules that arise before the vesicles detach from the nuclear envelope. Parallel arrays of annulate lamellae (AL) also appear in this region and their swollen, distal cisternae release ER vesicles into the surrounding ooplasm that are indistinguishable from those generated by the nuclear envelope. Golgi complexes proliferate and produce secretory products that fuse with both the ER cisternae and newly formed yolk bodies. Simultaneous biosynthetic activity of the ER, AL, and Golgi results in the formation of Type I yolk bodies, and primary and secondary cortical vesicles that likely participate in the formation of the egg envelope. In the later stages of vitellogenesis, extraoocytic yolk precursors originating in the hemocoel pass through the follicle cells that encompass the oocytes in an endocytotic process that releases precursors into the pervitelline space. Oocytes then incorporate these precursors endocytotically resulting in the gradual assembly of Type II yolk bodies. To our knowledge, this is the first ultrastructural study to describe the biosynthesis of specific products (yolk bodies and cortical granules) by annulate lamellae in any metazoan oocyte. Vitellogenesis in calanoid coperpods is significantly different from that described in any other crustacean group. Further investiation of this process in related crustaceans might provide useful phylogenetic insights.

Mammalian fertilization.

Mammalian fertilization.

'Nectosome': a novel cytoplasmic vesicle containing nectin in the egg of the sea urchin, Temnopleurus hardwickii.

Localization of an extracellular matrix protein, Th-nectin, in the eggs and embryos of the sea urchin Temnopleurus hardwickii was examined by both immunofluorescence and immunoelectron microscopy. The protein is associated with a tubular structure packaged in rod-shaped vesicles that were designated as 'nectosomes'. In unfertilized eggs, nectosomes are distributed uniformly throughout the cytoplasm, but after fertilization, they gradually translocate to the cortical zone where they are arranged perpendicular to the plasma membrane. The migration of the nectosomes was strongly inhibited by cytochalasin B, which suggested that microfilaments play an important role in this process. Immunocytochemical and immunoblotting analyses both ascertained that nectin is secreted into the hyaline layer. Some nectosomes remain in the apical cytoplasm of dermal cells until the gastrula stage. Ultrastructural examination revealed that the accumulation of nectosomes in the oocyte cytoplasm begins quite early in oogenesis, concomitant with the accumulation of cortical vesicles.

Membrane fusion of secretory vesicles of the sea urchin egg in the absence of NSF.

The role of cytosolic ATPases such as N-ethylmaleimide (NEM)-sensitive fusion protein (NSF) in membrane fusion is controversial. We examined the physiology and biochemistry of ATP and NSF in the cortical system of the echinoderm egg to determine if NSF is an essential factor in membrane fusion during Ca(2+)-triggered exocytosis. Neither exocytosis in vitro, nor homotypic cortical vesicle (CV) fusion required soluble proteins or nucleotides, and both occurred in the presence of non-hydrolyzable analogs of ATP. While sensitive to thiol-specific reagents, CV exocytosis is not restored by the addition of cytosolic NSF, and fusion and NSF function are differentially sensitive to thiol-specific agents. To test participation of tightly bound, non-exchangeable NSF in CV-CV fusion, we cloned the sea urchin homolog and developed a species-specific antibody for western blots and physiological analysis. This antibody was without effect on CV exocytosis or homotypic fusion, despite being functionally inhibitory. NSF is detectable in intact cortices, cortices from which CVs had been removed and isolated CVs treated with ATP-gamma-S and egg cytosol to reveal NSF binding sites. In contrast, isolated CVs, though all capable of Ca(2+)-triggered homotypic fusion, contain less than one hexamer of NSF per CV. Thus NSF is not a required component of the CV fusion machinery.

Description of a Marine Peritrichous Ciliate, Pseudovorticella sinensis n. sp. (Ciliophora, Peritrichia) from China

A new marine peritrich ciliate, Pseudovorticella sinensis n. sp. was isolated from a shrimp-farming pond in the littoral area of Qingdao, China. The morphology, infraciliature, and silverline system were studied based on living and silver-impregnated specimens. This species is characterized by (1) an elongated bell-shaped body that measures 50-60 x 35-45 microm in vivo, (2) one large, ventrally located contractile vacuole, and (3) a pellicle covered by a layer of transparent, cortical vesicles. The number of transverse silverlines from the peristomial area to the aboral ciliary wreath is 26-32, and from the aboral ciliary wreath to the scopula is 12-15. The stalk measures about 160-250 microm long x 5-6 microm wide. The spasmoneme has one row of conspicuous thecoplasmic granules, which are about 0.8 microm in diameter.

Differential participation of actin- and tubulin-based vesicle transport systems during secretion in bovine chromaffin cells.

The role of cytoskeletal elements in vesicle transport occurring during exocytosis was examined in adrenal medullary bovine chromaffin cells maintained in culture. Amperometric determination of depolarization-dependent catecholamine release from individual intact cells treated with actin or myosin inhibitors showed alterations in the fast and slow phases of secretion when compared with untreated cells. In contrast, microtubule disassemblers or stabilizers have a moderate effect on secretion, only affecting the release of slow secretory components. In experiments using confocal dynamic microscopy we have observed the drastic effect of actin and myosin inhibitors in abolishing vesicle movement throughout the cytoplasm, and the inhibition of granule mobility in deep perinuclear regions caused by the microtubule stabilizers. Following loss of mobility, vesicles were associated with filaments of F-actin or microtubules. In addition, the mobility of cortical vesicles was affected by actin-myosin inhibitors but not by microtubule inhibitors. The study of cortical cytoskeleton in living cells showed vesicles associated with dense tubular F-actin structures, with microtubules appearing as low density networks. These findings suggest that the distribution and density of both cytoskeletal elements in the cortical region may influence the recruitment of vesicle pools during secretion.