Endocytosis in maize root cap cells

Summary Primary roots of maize seedlings have been treated with solutions of lanthanum and lead salts in an attempt to demonstrate endocytosis. Subsurface cells in the root cap reveal deposits of these heavy metals in coated pits in the plasma membrane and in coated vesicles. In addition lead deposits were observed in coated evaginations (pits) on large (secretory) vesicles present at the trans-pole of the Golgi apparatus and on small vacuoles. Lead was also found in the peripheral regions of individual cisternae throughout the dictyosomal stack. We interpret our results as providing evidence for coated pit/coated vesicle-mediated endocytosis and for the direct recycling of plasma membrane to the Golgi apparatus.     

Surface multivesicular structures associated with maturing erythrocytes in rats

Summary Surface multivesicular structures associated with the plasmalemma of erythrocytes were observed in the peripheral blood of rats which have a significant number of circulating reticulocytes. These surface structures appear as ovoid evaginations (0.2 to 0.7 in diameter) of the plasma membrane and contain numerous small vesicles ranging from 0.05 to 0.1 in diameter. The structures were present during the final stages of maturation of erythrocytes, after nuclei and mitochondria had been extruded and only a few polysomes and small vesicles remained. They appear quite distinct from the autophagic vacuoles which have been described in association with degeneration and extrusion of mitochondria from erythrocytes. The exact origin of the small internal vesicles of these surface multivesicular structures is unknown; however, similar vesicles have been observed in the cytoplasm of the maturing erythrocyte especially in the vicinity of the Golgi body. These structures suggest a process by which Golgi elements and other small cytoplasmic vesicles are extruded during the late stages of maturation of rat erythrocytes.Supported by U.S. Public Health Service Research Grant AM 12950.The author is indebted to Dr. Edward G. Rennels and Dr. William B. Winborn for their guidance.     

In vitro reconstitution of exocytosis from sea urchin egg plasma membrane and isolated cortical vesicles

We have succeeded in reconstituting an exocytotically active egg cortex fraction by recombining purified cortical vesicles (CVs) with egg plasma membrane (PM). CVs were dislodged from a suspension of egg cortex by gentle homogenization in a dissociative buffer with a pH of 9.1, and purified by two rounds of differential centrifugation. Egg PM was prepared by shearing the cortical vesicles from a cortical lawn preparation with a jet of isotonic buffer. PM lawns produced by this procedure consist of an array of CV-free PM fragments attached via their extracellular surface to a polylysine coated glass slide. When a neutralized suspension of CVs was recombined with a PM lawn, CVs reassociated with the cytoplasmic face of the plasma membrane to form a reconstituted lawn (RL). RLs undergo a morphological change in response to Ca²⁺-containing buffers that is similar to the exocytotic release of CV contents from cortical lawns. In both reactions CV contents are vectorially transferred from the cytoplasmic to the extracytoplasmic face of the egg PM. A quantitative binding assay was developed and used to show that adherence of CVs to a heterologous PM lawn prepared from human red blood cells is minimal.     

Clathrin-Coated Vesicle Subtypes in Mammalian Brain Tissue: Detection of Polypeptide Heterogeneity by Immunoprecipitation with Monoclonal Antibodies

A panel of monoclonal antibodies (mAbs) was developed to identify polypeptides sorted in subtypes of brain coated vesicles (CVs) and to separate these by immunoprecipitation. The corresponding antigen of some of the mAbs elicited by CV components was present also in synaptosomal plasma membrane, synaptic vesicles, or microsomes. On immunoblots the mAbs reacted with constitutive brain CV proteins, with cargo molecules, and with a novel CV component that interacts with the actin cytoskeleton. Analysis of radioiodinated brain CVs immunoprecipitated with a tubulin antibody revealed that all brain CVs contained tubulin. The mAb A-7C11 recognized a 40-kilodalton (kDa) polypeptide on the clathrin coat and immunoprecipitated one-quarter of the total brain CVs. The mAb S-11D9 reacted with a 44-kDa antigen and immunoprecipitated 25% of the CVs. This antigen (44 kDa) was present in synaptic vesicles and synaptosomal membrane as well. Moreover, this mAb (S-11D9) reacted with a polypeptide of 56 kDa detected only in synaptosomal membrane. A mAb (C-10B2) that reacted with one of the clathrin light chains (LCb) immunoprecipitated 90% of the brain CVs. One of the mAbs immunoprecipitated a CV subtype that displayed a reversed ratio of the clathrin LCs (LCa greater than LCb). Each of the mAbs yielded different immunofluorescent staining patterns of vesicles in culture cell types that included nerve growth factor-differentiated PC12 cells, neuroblastoma cells, and Madin Darby bovine kidney cells. The data suggest that in brain tissue there is a heterogeneous population of CVs with different polypeptide compositions and subcellular distributions and that each of these subtypes performs a different role in nerve cells.     

Isolation and partial characterization of clathrin-coated vesicles from pea (Pisum sativum L.) cotyledons

Summary Clathrin-coated vesicles have been isolated from cotyledons of both developing and germinating pea seeds using differential centrifugation, ribonuclease treatment, discontinuous sucrose gradients, and isopycnic centrifugation on a linear D₂O-Ficoll gradient. The yield of coated vesicles from developing pea cotyledons was exceptional, being 1.6 × higher than the yield from hog and bovine brain, 5.3 × higher than the yield from carrot suspension cultures, and 13 × the yield from cotyledons of germinating pea seeds. The pea coated vesicles are similar to other plant coated vesicles in size (approximately 80 nm in diameter) and in having a clathrin heavy chain of 190,000 M_r. The lipid phosphorus to protein ratio, 190–250 nmol P per mg protein, of the coated vesicles from plants is comparable to that reported for highly purified coated vesicles from animals. The nondenatured pea clathrin reacted weakly with an antiserum to bovine brain clathrin, but pea clathrin denatured by sodium dodecyl sulfate did not.Abbreviations CLC Clathrin light chain - CHC clathrin heavy chain - CV coated vesicle - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethyl ether) N,N-tetraacetic acid - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TBS Tris buffered saline     

Organization and dynamics of lipids in bovine brain coated and uncoated vesicles

Three characteristics have been demonstrated by the chemical analysis of bovine brain coated vesicles following removal of the coat proteins: a high protein content, a high cholesterol/lipid ratio and a high percentage of phosphatidylethanolamine amongst the phospholipids.The study of lipid bilayer organization and dynamics has been performed using the fluorescent probes pyrene and parinaric acid (cis and trans). This has allowed the study of both lateral mobility and rotational motion in the lipid bilayer of the coated and uncoated vesicles.Lateral mobility in the fluid phase of the lipid is slightly reduced by the presence of the clathrin coat, as indicated by the lower diffusion coefficient of pyrene in coated compared with uncoated vesicles.At all temperatures from 6° to 30°C, solid-phase domains, probed by trans parinaric acid, coexist with fluid-phase domains in the lipid bilayer. The temperature dependence of the parinaric acid lifetimes and of their amplitudes strongly suggests that the solid phase domains decrease in size with temperature, both in coated and uncoated vesicles.However, the difference in the value of the anisotropy at long times (r ), between coated and uncoated vesicles (a difference which is more pronounced for cis than for trans parinaric acid), indicates that the presence of the clathrin coat introduces disorder in the surrounding lipids, thus suggesting a possible role of the clathrin in the formation of the pits on the plasma membrane.Abbreviations CVs coated vesicles - UVs uncoated vesicles - TLC thin layer chromatography - DMSO dimethylsulfoxide - DPPC dipalmitoylphosphatidylcholine - cis Pna cis parinaric acid - (9,11,13,15-cis-trans-trans-cis) octadecatetraenoic acid - Trans Pna Trans parinaric acid - (9,11,13,15-all-trans) octadecatetraenoic acid     

An electron microscopical study of absorbing cells in the posterior caput epididymidis of rabbits

Summary The columnar cells in regions 3 and 4 of the ductus epididymidis in rabbits display ultrastructural features characteristic of absorbing cells. The stereocilia show basal anastomoses and often a fibrillar core continuous with a fibrillar web in the apical cytoplasm. Numerous invaginations of the slightly downy apical cell membrane and many thick-walled apical vesicles and vacuoles contain an opaque substance similar to that seen in the lumen. The vacuoles often contain small vesicles or bodies, probably formed from the vacuolar wall by budding. Numerous bodies or vacuoles with moderately dense contents are seen in the Golgi area and in the supranuclear and intranuclear cytoplasm in region 3. In region 4 they are denser and mainly seen above the nucleus. A high acid phosphatase activity was demonstrated in most dense and some light bodies. India ink introduced by way of the rete testis was taken up from the lumen into apical invaginations, vesicles and vacuoles and slowly transferred to denser bodies below the Golgi apparatus.These observations are interpreted as evidence for a resorption of substances from the lumen by a pinocytotic process, and for their storage and perhaps digestion in the dense bodies, which appear to have a lysosomal character. The Golgi apparatus is large with many vesicles of two types and empty cisternae but few typical Golgi vacuoles. The partly granular endoplasmic reticulum is very well developed and has opaque contents. Microtubules run from the terminal bar region into the Golgi area. Thick-walled vesicles occur throughout the cytoplasm, sometimes in continuity with the cell membrane. The basal parts of the cell borders often interdigitate.Supported by a grant from the Swedish State Medical Research Council.     

In vivo biosynthesis of clathrin and other coated vesicle proteins from rat liver

A biosynthetic study of rat liver coated vesicle (CV) proteins was undertaken by using in vivo labeling with L-[35S]methionine. CVs were isolated and purified by using standard procedures and characterized by electron microscopy, sedimentation, and sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by fluorography, or by gel slicing and liquid scintillation counting. After 5 1/2 min of labeling (the earliest time examined), incorporation of radioactive clathrin heavy-chain (180-kD (kilodalton] subunits as well as a 90-kD CV-associated protein into purified CVs was demonstrated. The level of labeled 180-kD clathrin in coated vesicles increased rapidly during the first 2 hr of labeling and then continued to rise at a slower rate between 4 and 16 hr. This slow accumulation of labeled clathrin heavy chains in the CV pool may reflect early compartmental sequestration of a fraction of newly synthesized clathrin with delayed assembly into free CVs. By 16 hr of labeling, clathrin 180-kD chains and the 90-kD CV-associated protein accounted for approximately 48 and 26%, respectively, of the radioactivity in all CV proteins. Two proteins of MWa 68 kD and 53 kD showed marked declines in cpm/unit protein between 30 min and 4 hr, raising the possibility that these species may be transferred out of CVs during or after transport without loss of the other CV proteins. The possibility is also raised that clathrin heavy chains may be recycled during CV formation. Possible heterogeneity within individual CV preparations with respect to protein composition and derivation from both plasma membrane and Golgi regions are proposed.     

The uptake of horseradish peroxidase by cortical synapses in rat brain

Summary Horseradish peroxidase (HRP) was introduced directly into the cerebral cortex of adult rats, which were allowed to survive for 60 min before perfusion fixation. After the tissue had been incubated to demonstrate HRP at the LM and EM levels, blocks of cortical tissue were taken at varying distances from the injection site. These eight blocks of tissue constituted a time sequence for HRP diffusion.Qualitative examination of the presynaptic terminals showed that the most commonly encountered profiles are the plain synaptic vesicles, many of which accumulate tracer. In some terminals labelled vesicles are lined-up in tubular fashion. Other profiles commonly labelled are coated vesicles, tubular and vacuolar cisternae, and plain and coated pinocytotic vesicles.Quantitative analyses based on the number of terminals containing labelled profiles demonstrate an early rise in the rate of labelling of both plain synaptic vesicles and coated vesicles, after which synaptic vesicle labelling rises slowly towards a plateau. By contrast, there is a late parallel increase in the rate of labelling of coated vesicles and cisternae. A more detailed analysis, based on the actual numbers of labelled and total profiles within each presynaptic terminal, highlight early and late periods of rapid labelling for plain synaptic vesicles, coated vesicles and cisternae. A further aspect of HRP incorporation studied, concerns its uptake into four delineated regions of the presynaptic terminal.Our data indicate that membrane uptake into the presynaptic terminal is accomplished mainly via coated vesicles, although plain synaptic vesicles may also be involved. Coated vesicles, in turn, appear to give rise directly to plain synaptic vesicles, with some coalescing to produce vacuolar cisternae. The latter are involved in a two-way interchange of membrane with tubular cisternae, plain synaptic vesicles and coated vesicles. An additional source of plain synaptic vesicles are the tubular cisternae. Exocytosis of plain synaptic vesicles constitutes the mechanism by which transmitter is released from the presynaptic terminal.Supported by the Nuffield Foundation. We are grateful to Mr. M. Austin for help with the photography     

In vitro reconstitution of exocytosis from plasma membrane and isolated secretory vesicles

We describe the reconstitution of exocytotic function through recombination of purified cortical secretory vesicles (CVs) and plasma membrane from sea urchin eggs. CVs were dislodged from a cell surface complex preparation by gentle homogenization in an isotonic dissociation buffer, and purified by differential centrifugation. CV-free plasma membrane fragments were obtained by mechanically dislodging CVs from cortical lawn (CL) preparations with a jet of CL isolation buffer. This procedure produced a "plasma membrane lawn" preparation, consisting of plasma membrane fragments attached via their vitelline layer (an extracellular glycocalyx) to a polylysine-coated microscope slide. When freshly prepared CVs were incubated with plasma membrane lawns, CVs reassociated with the cytoplasmic face of the plasma membrane, forming an exocytotically competent, reconstituted cortical lawn (RL). Exocytosis in RLs was monitored by phase-contrast microscopy, and quantitated with a sensitive microphotometric assay. Half-maximal exocytosis in RLs occurred at 18.5 microM free Ca2+; half-maximal exocytosis in control lawns occurred at 5.7 microM free Ca2+. Greater than 90% of the purified CVs that were not attached to a plasma membrane lawn remained intact when bathed in a buffer containing millimolar Ca2+. This result excluded the possibility that Ca2+-triggered CV lysis was responsible for our observations, and confirmed that the association of CVs with the plasma membrane was required for exocytosis in RLs. Evidence that the Ca2+-stimulated release of CV contents in CLs and RLs is the in vitro equivalent of exocytosis was obtained with an immunofluorescence-based vectorial transport assay, using an antiserum directed against a CV content protein: stimulation of RLs or partially CV-depleted CLs with Ca2+ resulted in fusion of the CV and plasma membranes, and the vectorial transport of CV contents from the cytoplasmic to the extracytoplasmic face of the egg plasma membrane.     

Reassociation of cortical secretory vesicles with sea urchin egg plasma membrane: Assessment of binding specificity

Summary An assay has been developed for quantitating the reassociation of cortical secretory vesicles (CVs) with fragments of sea urchin egg plasma membrane attached to glass slides (PM lawns). Binding ofS. pupuratus CVs to homologous PM lawns increased with time and CV concentration. The observation that CV binding was blocked by chymotrypsin digestion of the PM fragments suggested that a PM protein(s) is required for reassociation. The possibility that the extent of CV lysis that occurred during CV preparation (15.4±3.8% as assessed by ovoperoxidase assay) influenced reassociation was investigated by determining the effect of CV content proteins (isolated as fertilization product) on binding. Various concentrations of fertilization product (up to equivalent amounts of fertilization product and CV protein) had no effect on CV binding. The specificity of binding was investigated by assessing the ability of CVs to bind to PM lawns prepared from human red blood cells, and by determining the ability of heterologous vesicles to bind to egg PM fragments. PM lawns from HRBCs did not support CV binding; however, PM lawns prepared from the eggs of several species of sea urchin did bindS. pupuratus CVs. Vesicles from a partially purified preparation of yolk platelets bound to egg PM lawns with low efficiency (1/7 that of CVs), but immunofluorescence analysis with an anti-hyalin monoclonal antibody demonstrated that 74±9% of the bound vesicles were CVs that contaminated the yolk platelet preparation. Dioleoylphosphatidyl choline liposomes were also unable to bind to egg PM lawns. These results are consistent with hypothesis that CV binding to egg PM lawns is a specific, protein-mediated event.     

Cytoplasmic vacuoles and bodies of the osteoclast

Summary Cytoplasmic vacuoles and bodies in the osteoclast (rat) were studied by electron microscopy. The vacuole-like structures (0.03–5 in diameter) may be classed as a) vacuoles b) coated vacuoles and c) invaginations. The cytoplasmic bodies vary in size from 0.02–3 in diameter and these may similarly be classed as a) light cytoplasmic bodies, b) dense cytoplasmic bodies, c) coated cytoplasmic bodies and d) cytoplasmic bodies containing inclusions. Both the cytoplasmic vacuoles and the bodies are limited by a triple layered membrane of about 91 Å in thickness. Their relationship to the lysosomal system and the role of this system in the osteoclast is discussed.This research was supported by the Danish Research Council. Grant no. 512–727 and 512–819.     

Structure and behavior of contractile vacuoles inChlamydomonas reinhardtii

Summary The contractile vacuole (CV) cycle ofChlamydomonas reinhardtii has been investigated by videomicroscopy and electron microscopy. Correlation of the two kinds of observation indicates that the total cycle (15 s under the hypo-osmotic conditions used for videomicroscopy) can be divided into early, middle, and late stages. In the early stage (early diastole, about 3 s long) numerous small vesicles about 70–120 nm in diameter are present. In the middle stage (mid-diastole, about 6 s long), the vesicles appear to fuse with one another to form the contractile vacuole proper. In the late stage (late diastole, also about 6 s long), the CV increases in diameter by the continued fusion of small vesicles with the vacuole, and makes contact with the plasma membrane. The CV then rapidly decreases in size (systole, about 0.2 s). In isosmotic media, CVs do not appear to be functioning; under these conditions, the CV regions contain numerous small vesicles typical of the earliest stage of diastole. Fine structure observations have provided no evidence for a two-component CV system such as has been observed in some other cell types. Electron microscopy of cryofixed and freeze-substituted cells suggests that the irregularity of the profiles of larger vesicles and vacuoles and some other morphological details seen in conventionally fixed cells may be shrinkage artefacts. This study thus defines some of the membrane events in the normal contractile vacuole cycle ofChlamydomonas, and provides a morphological and temporal basis for the study of membrane fusion and fluid transport across membranes in a cell favorable for genetic analysis.Abbrevations CV contractile vacuole - PM plasma membrane     

Helical structure in the apical tubules of several absorbing epithelia

Summary Unique and highly ordered structures were discovered in the so-called apical tubules of several absorbing epithelia (kidney proximal tubule, visceral yolk sac and ductuli efferentes) fixed in situ with a mixture of formaldehyde, glutaraldehyde and osmium tetroxide. The apical tubules were especially numerous in the apical cytoplasm, in addition to the invaginations of the apical plasma membrane, newly formed endocytic vesicles and large endocytic vacuoles. They showed a cylindrical structure (80 nm in diameter) limited by a smooth membrane. Helically wound parallel rows of particles (11 nm in diameter) were found in the apical tubules in close proximity to their limiting membrane. The structure of the helix was determined by following the rows through serial sections and semithin sections, and was found to be a left-handed quadruple helix. These particles surround an electron-lucent cylinder (35 nm in diameter), containing at its center a single row of particles (9 nm in diameter). The apical tubules with the luminal specializations were not seen in continuity with the apical plasma membrane, but were frequently connected with the large endocytic vacuoles, which were present in the deeper levels of the apical cytoplasm. From these observations, it is suggested that the apical tubules are not derivatives of the apical plasma membrane; rather, they represent an intracellular compartment, which is morphologically related to the large endocytic vacuoles.     

Morphological changes in cultured myotubes treated with agents that interfere with lysosomal function

Summary Treatment of cultured muscle cells with the inhibitors of lysosomal function, leupeptin, and chloroquine, decrease the degradation of acetylcholine receptors (AChR) and causes accumulation of undegraded receptors intracellularly. Under these conditions the number of cytoplasmic coated vesicles, i.e. structures that appear to transport this receptor within the cultured muscle cell, increases in parallel. This study investigates the effects of leupeptin and chloroquine on the morphology of cultured myotubes in order to learn more about the turnover of acetylcholine (ACh) receptors and the origin of the coated vesicles. Chloroquine causes involution of the plasma membrane, disorganization in the arrangement of sarcomeres, vacuolization, and enlargement of dense lysosome-like bodies in myotubes. The diameter of dense bodies in untreated myotubes is 0.36 ± 0.01 m (mean ± SEM) compared with 2 ± 0.12 m after 48 h of incubation with chloroquine. Leupeptin does not disrupt the normal architecture of sarcomeres and does not cause vacuolization of the myotubes. However, leupeptin does enlarge the dense bodies, although to a lesser extent than chloroquine (average diameter after 48 h treatment, 1.0 ± 0.06 m, p < 0.01). Untreated myotubes appear to contain equal numbers of large and small coated vesicles. After chloroquine treatment 95% of coated vesicles are large (80–120 nm in diameter), whereas after leupeptin treatment the majority of coated vesicles are small (40–70 nm in diameter). After incubation with horseradish peroxidase (HRP) 62% ± 9 of coated vesicles in chloroquinetreated cells contain the tracer, whereas in control cells only 11% ± 4 of coated vesicles contain HRP reaction product. These observations indicate that chloroquine causes accumulation of coated vesicles and interferes with degradation of AChR by preventing fusion of lysosomes with coated vesicles originating by endocytosis.     

Immunocytochemical colocalization of adhesive proteins with clathrin in human blood platelets: further evidence for coated vesicle-mediated transport of von Willebrand factor,fibrinogen and fibronectin

Coated membranes and vesicles play an important role in receptor-mediated endocytosis and intracellular trafficking in various cell types, and are also present in blood platelets. Platelets take up certain proteins from the blood plasma, such as von Willebrand factor and fibrinogen, and these substances are transferred to storage granules. The receptors for these plasma proteins on the platelet plasma membrane have been well characterized, but morphological evidence for their transport to the storage granules is not yet available. In an attempt to clarify this aspect, we employed postembedding immunocytochemistry on platelets embedded in the acrylic resin LR White. Clathrin as the major coat component of coated vesicles was localized in the cytoplasm, on the plasmic faces of -granules and the open canalicular system, and on the plasmic face of the plasma membrane. Colocalizations of the adhesive proteins, von Willebrand factor, fibrinogen and fibronectin, with clathrin could be observed at the same typical locations as coated vesicles were seen in Araldite-embedded material. These colocalizations have not been reported to date and furnish further evidence for a coated vesicle-mediated transport of blood plasma-derived adhesive proteins from their receptors on the outer plasma membrane to the -granules.     

Ultrastructural changes in adrenaline- and SGC-cells after morphine coincide with alterations of adrenaline and dopamine levels

Summary The effects of morphine on chromaffin cell ultrastructure and catecholamine contents were studied using the adrenal glands from male mice (ICR strain). After 2 h adrenaline was increased 25% from 8.1 to 11.6 mol/g tissue, followed by a 50% decrease to 5.2 mol/g between 8–24 h and low values persisting at 72 h. Dopamine increased initially, reaching peak values of 0.5 mol/g between 8–24 h, but had returned towards control values of 0.29 mol/g by 72 h. Noradrenaline remained unchanged at 2.5 mol/gram. Naloxone prevented alterations in adrenaline and dopamine levels.Ultrastructural examination revealed several types of catecholamine-storing cells. Of these the adrenaline and small-granule chromaffin (SGC) cells were more affected by morphine than noradrenaline cells. While the initial elevation of adrenaline 2 h after morphine was not accompanied by significant ultrastructural changes, the decrease after 8 and 24 h was paralleled by a significant (p<0.001) loss of adrenaline granules. Signs of active membrane turnover included an increase in the number of vacuoles, and the appearance of numerous coated omega profiles and coated (77.7±0.6 nm) vesicles. Clusters of synaptic-like vesicles (59.8±8.2 nm), slightly larger than neuronal vesicles (45.4±6.4), increased in the SGC-cells. After 72 h, the chromaffin granules in adrenaline cells remained low in numbers and were heterogeneous in electron density. Many synapticlike vesicles were aligned along the SGC-cell membranes where only few chromaffin granules (109.3±20 nm) remained. Thus, continuous morphine exposure for 8–72 h increases the turnover of storage granules in adrenaline and SGC-cells with less effect on the noradrenaline cells which maintain catecholamine levels as indicated by biochemical analysis.     

Polar cytoplasmic evaginations in dividing spermatocytes of the firebug,Pyrrhocoris apterus (Pyrrhocoridae,Hemiptera)

Summary The present fine structure and anti-tubulin immunofluorescence study deals with evaginations from the cell surface in metaphase I spermatocytes of the firebugPyrrhocoris apterus (Pyrrhocoridae, Hemiptera). The surface of spermatogonia and prophase spermatocytes was smooth throughout. Only in metaphase I and anaphase I, cytoplasmic threads projected from polar portions of the spermatocytes. In contrast, equatorial portions of these cells possessed a smooth surface. By mid to late telophase, the evaginations were no longer detectable in spermatocytes. Three ideas are at hand to explain the development of polar cytoplasmic evaginations. First, they could represent a membrane reserve used up during spindle elongation in telophase of meiosis. In order to test this idea, spindle structure was analyzed in meiosis I using simultaneously antibodies to -tubulin and -tubulin. -Tubulin represents a tubulin isoform prevalent in centrosomes. The observations showed that spindle elongation was not very prominent in meiosis of the bug. Although it cannot be ruled out, the formation of a polar membrane reserve prior to spindle elongation is not a likely explanation for the evaginations from the cell surface. Second, the development of surface extensions could bring about increased exchange of metabolites during a particularly active stage of meiosis. Third, the polar evaginations could be an inadvertent product of the aster microtubules protruding towards the plasma membrane.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamidino-2-phenylindole.2 HCl - EGTA ethylene glycol-bis (-aminoethyl ether)-N,N-tetraacetic acid - FITC fluorescein-isothiocyanate - PBS phosphate-buffered saline - PIPES piperazine-N,N bis (2-ethane sulfonic acid) - MT microtubule - MTOC microtubule-organizing centre     

An ultrastructural study of phagocytosis and shrinkage in nutritive phagocytes of the sea urchin <Emphasis Type="Italic">Anthocidaris crassispina</Emphasis>

The ultrastructural mechanisms of waste-sperm phagocytosis and postspawning shrinkage were studied for accessory cells (nutritive phagocytes; NPs) of the sea urchin Anthocidaris crassispina. Sperm cells were phagocytosed by NPs; they penetrated into the cytoplasm of the NPs inside heterophagosomes formed by an invagination of the cell membrane. Single-sperm-containing heterophagosomes aggregated to form large multisperm heterophagosomes that were accompanied by cytoplasmic vesicles and lipids. Two types of vesicle, viz., Golgi-complex-derived electron-dense vesicles (zymogen granules) and smooth-endoplasmic-reticulum-derived electron-lucent vesicles, were incorporated within multisperm heterophagosomes. Completed multisperm heterophagosomes were transformed into electron-dense remnant bodies, the content of which underwent destruction, resulting in empty vacuoles inside the remnant body. The empty vacuoles were then compressed by the surrounding cytoplasm. Shrinkage of NPs occurred upon completion of sperm degeneration in gonad tubules. This process was undertaken by structures termed cell-size-reducing autolysosomes, which performed two types of autolysis, and resulted in the formation of cheese-hole-like vacuoles in the cytoplasm of NPs. Subsequent cytoplasmic compression of these vacuoles was required for the reduction in size of NPs, an essential event for remodeling the cell for the next gametogenetic cycle.We are grateful to the Research Committee, City University of Hong Kong, for the award of a Strategic Grant (no. 7000989). This work also was supported by a grant from Minpromnayka NS 1219.2003.4     

Inhibition of contractile vacuole function by brefeldin A

Brefeldin A (BFA) causes a block in the secretory system of eukaryotic cells. In the scaly green flagellate Scherffelia dubia, BFA also interfered with the function of the contractile vacuoles (CVs). The CV is an osmoregulatory organelle which periodically expels fluid from the cell in many freshwater protists. Fusion of the CV membrane with the plasma membrane is apparently blocked by BFA in S. dubia. The two CVs of S. dubia swell and finally form large central vacuoles (LCVs). BFA-induced formation of LCVs depends on V-ATPase activity, and can be reversed by hypertonic media, suggesting that water accumulation in the LCVs is driven by osmosis. We suggest that the BFA-induced formation of LCVs represents a prolonged diastole phase. A normal diastole phase takes about 20 s and is difficult to investigate. Therefore, BFA-induced formation of LCVs in S. dubia represents a unique model system to investigate the diastole phase of the CV cycle.