The role of asymmetric cell division in pteridophyte cell differentiation. I. Localized metal accumulation and differentiation inVittaria gemmae andOnoclea prothallia

Summary In gemmae ofVittaria graminifolia and prothallia ofOnoclea sensibilis, cell differentiation is initiated by nuclear migration and geometrically asymmetric cell division. The small daughter cells inVittaria develop into antheridia in the presence of gibberellic acid or into rhizoids or new prothallia in its absence. Antheridial differentiation from asymmetric division is induced inOnoclea byPteridium antheridiogen, whereas rhizoid or vegetative cell formation occurs in its absence. Although asymmetric cytokinesis initiates differentiation, it does not in itself determine the developmental fate of the smaller cell. Several histochemical techniques demonstrate that prior to nuclear migration and cell division, Ca²⁺ accumulates in the cytoplasm and wall of the cell at the site where asymmetric division will occur, regardless of the developmental fate of the small cell. The cytoplasmic localization of Ca²⁺ appears to reflect a mobilization of Ca²⁺ from within the cell that eventually moves into the cell wall. We propose that this internal accumulation of Ca²⁺ leads to a localized decrease in cytosolic [Ca²⁺] which in turn may regulate developmental events such as nuclear migration.Publishing prior to 1984 as Alix R. Bassel.     

Structure and possible function(s) of charasomes; complex plasmalemma-cell wall elaborations present in some characean species

Summary Charasomes, complex membrane structures, were found along the longitudinal walls of internodal and lateral branch cells ofChara corallina andC. braunii, but not along their transverse walls or in other cell types. Charasome-complexes were larger and more numerous in the lateral branch cells than in internodal cells. InC. corallina, a dioecious species, especially large elaboration of charasome material occurs in the lateral branch cells of the female plant, sometimes reaching a cross-sectional width which is as great as that of the adjacent cell wall. Chara internodes transport hydroxyl (OH^–) out of the cell and bicarbonate (HCO₃ ^–) into the cell. Spatial distribution of charasomes along the cell was examined with respect to these transport phenomena, which occur at specific identifiable regions along the cell. Charasome-complexes were always found in regions in which HCO₃ ^– transport occurs but were often fewer, reduced in size or absent in areas of OH^– efflux.Nitella flexilis exhibited similar patterns of OH^– and HCO₃ ^– transport along the cell; however, there was a complete absence of charasomes. Ultrastructural examinations onNitella translucens indicated that charasomes were also absent in this species. The observation that charasomes are present in both transport regions ofChara but are totally lacking in the twoNitella spp. indicates that the charasome-complex is not involved in transport of either substance. Other possible functions for the charasomes, including a role in osmoregulation, are discussed.Charasome substructure is the same in bothChara species, consisting of a mass of short (50 nm average length) anastomosing tubules (30 nm average diameter) derived from the plasmalemma. The interior of the tubules is open to the cytoplasm while the area surrounding the tubules is ultimately open to the wall and thus can be considered to be wall space. Charasomes are quite variable in size and shape, but are roughly globular, with the bulk of the structure projecting into the cell cytoplasm. Tubular components of the charasome were sometimes seen to extend into the microfibrillar wall matrix. A three dimensional model of the charasome-complex presented details the great complexity of this membrane system.     

Adenosine triphosphatase in yeast phaseParacoccidioides brasiliensis

We have previously detected various enzymatic activities inP. brasiliensis. In the present study we have examined Adenosine Triphosphatase (ATPase) activity in yeast phase cultures ofP. brasiliensis of increasing age. We employed Wachstein and Meisel's method and electron microscopy and found specific electron-dense deposits indicating ATPase activity to be present in the cytoplasm around vacuoles. Their distribution varied according to age. Deposits decreased or became absent in old cultures. We assume that inP. brasiliensis, ATPase is involved (as in other systems) with transport of Na⁺ and K⁺.     

Gravity-dependent polarity of cytoplasmic streaming inNitellopsis

Summary The internodal cells of the characean algaNitellopsis obtusa were chosen to investigate the effect of gravity on cytoplasmic streaming. Horizontal cells exhibit streaming with equal velocities in both directions, whereas in vertically oriented cells, the downwardstreaming cytoplasm flows ca. 10% faster than the upward-streaming cytoplasm. These results are independent of the orientation of the morphological top and bottom of the cell. We define the ratio of the velocity of the downward- to the upward-streaming cytoplasm as the polar ratio (PR). The normal polarity of a cell can be reversed (PR<1) by treatment with neutral red (NR). The NR effect may be the result of membrane hyperpolarization, caused by the opening of K⁺ channels. The K⁺ channel blocker TEA Cl^– inhibits the NR effect.External Ca²⁺ is required for normal graviresponsivness. The [Ca²⁺] of the medium determines the polarity of cytoplasmic streaming. Less than 1 M Ca²⁺ resulted in a PR<1 while greater than 1 M Ca²⁺ resulted in the normal gravity response. The voltage-dependent Ca²⁺ -channel blocker, nifedipine, inhibited the gravity response in a reversible manner, while treatment with LaCl₃ resulted in a PR<1, indicating the presence of two types of Ca²⁺ channels. A new model for graviperception is presented in which the whole cell acts as the gravity sensor, and the plasma membrane acts as the gravireceptor. This is supported by ligation and UV irradiation experiments which indicate that the membranes at both ends of the cell are required for graviperception. The density of the external medium also affects the PR ofNitellopsis. Calculations are presented that indicate that the weight of the protoplasm may provide enough potential energy to open ion channels.     

Pinocytosis and locomotion in amoebae

Summary Different methods were used to demonstrate the existence of Ca⁺⁺-binding sites (Ca⁺⁺-bs) at the plasma membrane ofAmoeba proteus. In pinocytoting animals the number (indicated by the average distanced in nm) and size (average longitudinal axiss in nm) of Ca⁺⁺-bs at the cytoplasmic surface of the cell membrane were significantly increased (d=162±15;n=41 ands=93±5;n=47) in comparison to controls (d=208 ±21;n=37 ands=59±8;n=45). The ratio of P: Ca obtained by X-ray microanalysis was in the range of 1.5. The differences observed in the two experimental groups of amoebae are explained by conformational changes in the molecular structure and an increased Ca⁺⁺-permeability of the plasma membrane during induced pinocytosis.Microplasmodia of the acellular slime moldPhysarum polycephalum investigated for comparison were found to have no Ca⁺⁺-bs at the interior cell surface.     

Membrane control of cytoplasmic streaming in characean cells

When a characean cell generates an action potential, cytoplasmic streaming transiently stops and then recovers gradually. Calcium ion is one of the most important factors mediating between membrane excitation and cessation of cytoplasmic streaming. When an internode ofNitella flexilis is subjected to transcellular osmosis, both membrane depolarization and cessation of streaming take place at the endoosmotic part of the cell. It was also found that Ca²⁺ plays a key role in mediating between osmosis induced hydration of the cytoplasm and the cessation of cytoplasmic streaming. The present article reviews how Ca²⁺ acts as a second messenger in intracellular signal transduction in controlling the cytoplasmic streaming.     

Rapid plant movements triggered by action potentials

Rapid bendings of the pulvinus inMimosa pudica, of the trap lobes inDionaea muscipula andAldrovanda vesiculosa, and of the tentacle in Drosera are triggered by action potentials in their motor cells. The action potential ofMimosa may be a C1⁻-spike, and that ofDionaea andAldrovanda may be a Ca²⁺-spike. Propagation of action potentials in the petiole or motor organ is thought to be electrotonie, cell-to-cell, transmission. The Ca 2+ release from unidentified organelles in the pulvinus or the Ca²⁺ influx of the cells in the trap with the action current and activation of contractile fibrillar network having ATPase activity in the cytoplasm must be involved in the rapid bending. Contractions of fibrils may open pores in the membrane of the motor cells upon activation. Outward bulk flow of the vacuolar sap through these pores, due to the pressure inside the cell, must result in turgor loss of the motor cells and then the bending of the organ.     

Ca2+ reversibly inhibits active rotation of chloroplasts in isolated cytoplasmic droplets ofChara

Summary The effects of Ca²⁺ and other cations on chloroplast rotation in isolated cytoplasmic droplets ofChara were investigated by iontophoretically injecting them. Chloroplast rotation stopped immediately after Ca²⁺ injection and recovered with time, suggesting the existence of a Ca²⁺-sequestering system in the cytoplasm. The Ca²⁺ concentration necessary for the stoppage was estimated to be >10^–4M. Sr²⁺ had the same effect as Ca²⁺. Mn²⁺ and Cd²⁺ induced a gradual decrease in the rotation rate with low reversibility. K⁺ and Mg²⁺ had no effects. Ba²⁺ had effects sometimes similar to Ca²⁺ or Sr²⁺ and sometimes similar to Mn²⁺ or Cd²⁺.Reversible inhibition by Ca²⁺, together with its specificity, strongly supports the hypothesis that a transient increase in the Ca²⁺ concentration in the cytoplasm upon membrane excitation directly stops the cytoplasmic streaming inCharaceae internodes (Hayama et al. 1979).     

X-ray microanalysis of elements in frozen-hydrated sections of an electrogenic K+ transport system: The posterior midgut of tobacco hornworm (Manduca sexta) in vivo andin vitro

Summary The lepidopteran midgut is a model for the oxygendependent, electrogenic K⁺ transport found in both alimentary and sensory tissues of many economically important insects. Structural and biochemical evidence places the K⁺ pump on the portasome-studded apical plasma membrane which borders the extracellular goblet cavity. However, electrochemical evidence implies that the goblet cell K⁺ concentration is less than 50mm. We used electron probe X-ray microanalysis of frozenhydrated cryosections to measure the concentration of Na, Mg, P, S, Cl, K, Ca and H₂O in several subcellular sites in the larval midgut ofManduca sexta under several experimental regimes. Na is undetectable at any site. K is at least 100mm in the cytoplasm of all cells. Typicalin vivo values (mm) for K were: blood, 25; goblet and columnar cytoplasm, 120; goblet cavity, 190; and gut lumen, 180. The high K concentration in the apically located goblet cavity declined by 100mm under anoxia. Both cavity and gut fluid are Cl deficient, but fixed negative charges may be present in the cavity. We conclude that the K⁺ pump is sited on the goblet cell apical membrane and that K⁺ follows a nonmixing pathway via only part of the goblet cell cytoplasm. The cavity appears to be electrically isolated in alimentary tissues, as it is in sensory sensilla, thereby allowing a PD exceeding 180 mV (lumen positive) to develop across the apical plasma membrane. This PD appears to couple K⁺ pump energy to nutrient absorption and pH regulation.     

Simultaneous recording of cytosolic Ca2+ levels inDidinium andParamecium during aDidinium attack onParamecium

Summary The carnivorous ciliateDidinium nasutum captures prey such asParamecium by discharging extrusomes, known as toxicysts, while the attackedParamecium defensively discharges trichocysts. Several authors have suggested that both discharges, the toxicysts ofDidinium and the trichocysts ofParamecium, are evoked by the rise in cytosolic Ca²⁺ level in each cell. However, these putative increases in cytosolic Ca²⁺ levels have not as yet been recorded simultaneously in these cells during aDidinium attack onParamecium. We injected the fluorescent Ca²⁺ indicator Ca-Green 1 dextran into bothDidinium andParamecium, and simultaneously observed the cytosolic Ca²⁺ levels in these cells asDidinium attackedParamecium. When aParamecium came into contact with theDidinium proboscis, theDidinium showed a significant rise in cytosolic Ca²⁺ in the basal portion of the proboscis. One video frame (33 ms) after the onset of the Ca²⁺ rise inDidinium, theParamecium also showed an increase in cytosolic Ca²⁺. This is the first simultaneous recording of changes in the Ca²⁺ level during a predator-prey interaction in ciliates. The possible roles of these Ca²⁺ increases are discussed in relation to the discharge of toxicysts during theDidinium attack and of trichocysts as a defensive behavior ofParamecium.Abbreviations AED aminoethyldextran - P_i inorganic phosphate - FITC fluorescein isothiocyanate     

Injection of wild-type cytoplasm and poly(A)+ RNA provokes phenotype rescue in spätzle mutant Drosophila embryos

Summary spätzle (spz), a maternal effect gene of Drosophila, is involved in the establishment of the dorso-ventral axis during embryogenesis. Eggs from females lacking the spz gene product develop into completely dorsalized embryos, i.e. the ventral and lateral pattern elements fail to develop. Upon injection of either cytoplasm or poly(A)⁺ RNA from early wild-type embryos, spz embryos develop lateral pattern elements represented by Filzkörper and in the case of injected cytoplasm additional ventral pattern elements represented by ventral setae. Wild-type cytoplasm retains the rescuing activity longer than the poly(A)⁺ RNA fraction does, and cytoplasm is always more effective in provoking the rescue than poly(A)⁺ RNA. Mosaic females containing spz germ cells surrounded by spz ⁺ tissues were generated by pole cell transplantations; a mutant genotype in the germ cells is sufficient to produce all aspects of the spz mutant phenotype, suggesting that the maternal source of spz gene product is the germ line.     

Introduction of a plant intron into the luciferase gene ofPhotinus pyralis

We describe a new luciferase reporter gene,luc ^INT, for early detection of luciferase activity inAgrobacterium transformation studies, and present improved techniques for the extraction of luciferase that decrease the time needed to quantitate luciferase activity. Theluc ^INT reporter gene combines the PIV2 intron fromGUS ^INT withluc ^*, the modified luciferase gene.luc ^INTis expressed in plant cells but not inAgrobacterium, allowing earlier detection of gene expression in the presence ofAgrobacterium during transformations in tobacco leaf discs. Stable expression levels ofluc ^INT andluc ^* in tobacco suspension cultures are compared for two different promoters. The nucleotide sequence data for the gene will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number U84006.     

The Na<Superscript>+</Superscript>-translocating ATPase in the plasma membrane of the marine microalga<Emphasis Type="Italic"> Tetraselmis viridis</Emphasis> catalyzes Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange

Our previous investigations have established that Na⁺ translocation across the Tetraselmis viridis plasma membrane (PM) mediated by the primary ATP-driven Na⁺-pump, Na⁺-ATPase, is accompanied by H⁺ counter-transport [Y.V. Balnokin et al. (1999) FEBS Lett 462:402–406]. The hypothesis that the Na⁺-ATPase of T. viridis operates as an Na⁺/H⁺ exchanger is tested in the present work. The study of Na⁺ and H⁺ transport in PM vesicles isolated from T. viridis demonstrated that the membrane-permeant anion NO₃^– caused (i) an increase in ATP-driven Na⁺ uptake by the vesicles, (ii) an increase in (Na⁺+ATP)-dependent vesicle lumen alkalization resulting from H⁺ efflux out of the vesicles and (iii) dissipation of electrical potential, , generated across the vesicle membrane by the Na⁺-ATPase. The (Na⁺+ATP)-dependent lumen alkalization was not significantly affected by valinomycin, addition of which in the presence of K⁺ abolished at the vesicle membrane. The fact that the Na⁺-ATPase-mediated alkalization of the vesicle lumen is sustained in the absence of the transmembrane is consistent with a primary role of the Na⁺-ATPase in driving H⁺ outside the vesicles. The findings allowed us to conclude that the Na⁺-ATPase of T. viridis directly performs an exchange of Na⁺ for H⁺. Since the Na⁺-ATPase generates electric potential across the vesicle membrane, the transport stoichiometry is mNa⁺/nH⁺, where m>n.Abbreviations BTP Bis-Tris-Propane, 1,3-bis[tris(hydroxymethyl)methylamino]-propane - CCCP Carbonyl cyanide m-chlorophenylhydrazone - DTT Dithiothreitol - NCDC 2-Nitro-4-carboxyphenyl N,N-diphenylcarbamate - PMSF Phenylmethylsulfonyl fluoride - PM Plasma membrane     

The effect of temperature onshibire ts cell clones in the compound eye ofDrosophila melanogaster

Summary We have analysed the effect of temperature on both developing and adult eye cell clones homozygous forshi ^ST139, a temperature-sensitive mutant ofDrosophila melanogaster. The mutant gene, autonomous in its cellular expression, causes structural modifications of ommatidial cells when adult clones of cells are exposed to the restrictive temperature (29°C) for several days. However, the mutant phenotype reverses to normal within 4 days at the permissive temperature (20°C). The results of pulse, shift-up and shift-down experiments show that the temperaturesensitive period for developing compound eye cells is from the late second instar up to the early pupa. Cytodifferentiation of compound eye cells is blocked by restrictive temperature treatment during this period, whereas cell proliferation does not seem to be directly affected. These results are discussed with regard to the other known aspects of the phenotype observed in mutant individuals.     

Nuclear-accumulation kinetics of p9 CksHs1 and p9 CksHs2 in live plant cells correlate with immunochemical characteristics

Summary The two human homologues of the fission yeast cell cycle protein p13 ^suc1 displayed structural characteristics consistent with their existing in solution as differently folded monomers despite 81% identity with respect to their primary structures and both being capable of fulfilling the functions of their homologues in fission and budding yeasts. Carboxyfluorescein-labelled p9 ^CksHs1 and p9 ^CksHs2 retained their native structures. When microinjected into live stamen hair cells ofTradescantia virginiana, the labelled proteins accumulated in the nuclei of the cells. Markedly different nuclearaccumulation kinetics indicated that the human proteins interact differently with other cellular constituents, which supports the proposition that they may have different roles in cellular regulation.Abbreviations Cdk cyclin-dependent kinase - tris tris(hydroxymethyl)aminomethane - Hepes N-(2-hydroxyethyl)piperazine-N-(3-ethanesulphonic acid) - CF 5(6)-carboxyfluorescein-N-hydroxysuccinamide ester - SDS-PAGE sodium dodecyl sulphatepolyacrylamide gel electrophoresis - IEF isoelectric focusing - DEAE Sephacel diethylaminoethyl Sephacel - ELISA enzyme-linked immunosorbent assay - IgG immunoglobulin     

Effect of<Emphasis Type="Italic">Trichoderma</Emphasis> species on growth of Fusarium<Emphasis Type="Italic">proliferatiom</Emphasis> and production of fumonisins,fusaproliferin and beauvericin

Trichoderma species has been suggested as potential biocontrol agent forFusarium verticillioides on maize. In this cereal,F. verticillioides and F. proliferatum contributed to fumonisin accumulation. In addition,F. proliferatum could produce beauvericin and fusaproliferin. The aim of this work was to evaluate the effect ofTrichoderma spp. on growth and fumonisin B¹ fusaproliferin and beauvericin production byF. proliferatum. Dual cultures of F.proliferatum andT. harzianum ITEM 3636 andT. longibrachiatum ITEM 3635 on maize meal agar at 0.995 aw were done. The effect ofTrichoderma spp. on the lineal growth ofF. proliferatum was determined. The effect ofTrichoderma species on fumonisin B¹, fusaproliferin and beauvericin production byF. proliferatum was determined on co-inoculated maize kernels by HPLC.T. harzianum suppressedF. proliferatum growth once contact between the colonies occurred.T. longibrachiatum showed a less antagonistic effect againstF. proliferatum. A reduction on fumonisin B¹ production of 98% and 88% was observed in the co-incubation ofF. proliferatum withT. harzianum andT. longibrachiatum, respectively. The decrease of FB¹ production was significant even in maize kernels on whichF. proliferatum had been growing 7 days prior to the addition ofTrichoderma spp. The concentration of beauvericin and fusaproliferin produced during 30 days coincubation ofF. proliferatum with bothTrichoderma spp. did not differ to those produced byF. proliferatum alone. These mycotoxins might enter the food chain causing so far unknown consequences to the health of domestic animals and humans. For this reason it is important, when a potential biocontrol agent is under study, to test the effect on the fungal growth and on the putative mycotoxin produced. Part of the information was presented at the Mycotoxin Prevention Cluster Dissemination Day and Mycoglobe Launch Conference, Brussels, Belgium, Oct 20–21, 2004 Financial support: Agenda Córdoba Ciencia, grant N^o 0279–000431/00     

Ultrastructural study ofCryptococcus neoformans by quick-freezing and deep-etching method

The three-dimensional ultrastructure ofCryptococcus neoformans was studied by quick-freezing and deep-etching (QF-DE) method.C. neoformans, strain CDC551, was cultured on agar. The viable yeast cells (10⁷ cells) were inoculated into each mouse from the tail vein. Three weeks after the inoculation, the brains of the mice were perfused with fixatives, quickly frozen, freeze-fractured, deeply etched and rotary shadowed with platinum and carbon. In addition, the viable cells ofC. neoformans on agar were picked up and quickly frozen, and replica membranes were prepared as described above. The ultrastructure ofC. neoformans was three-dimensionally demonstrated by the QF-DE method. The capsule was composed of fine meshworks of microfibrils (10–13 nm in diameter), which were directly attached to the cell walls. The capsule of the in vivo yeasts (yeast cells in the brain lesion) was thicker than that of the in vitro yeasts (yeast cells on agar culture). At the outer part of the cell wall, a particle-accumulating layer was observed. This layer in vivo was thicker than that in vitro. Occasionally, the yeast cells were ingested by phagocytes in the mouse brain. Although the cytoplasm of such yeast cells was destroyed, the capsular meshworks were well preserved. The ultrastructure of the capsule was the same both in cultured and phagocytized yeasts in the cystic lesions of the brains. This lack of morphological changes of the capsular meshworks suggests that they are resistant to the digestion by phagocytes. This stability of capsular structures may provide one of the important pathogenic factors in cystic lesions byC. neoformans.     

Bicuculline-insensitive GABA-gated Cl<Superscript>−</Superscript> channels in the larval nervous system of the moth<Emphasis Type="Italic"> Manduca sexta</Emphasis>

GABA-gated Cl^– channels were studied in the nervous system of the larval tobacco hawk moth, Manduca sexta, using electrophysiology, ³⁶Cl^– uptake into membrane microsacs and immunocytochemistry. A GABA-induced increase in Cl^– conductance was recorded from a visually identifiable neurone (fg1) in the desheathed frontal ganglion. The response was insensitive to the vertebrate GABA_A receptor antagonist, bicuculline, but was blocked by picrotoxinin. Bicuculline-insensitive, picrotoxinin-sensitive, GABA-stimulated ³⁶Cl^– uptake was also detected in membrane microsacs prepared from the isolated larval M. sexta nervous system. Such receptors appear to be the major type of GABA receptor in larval nervous system membrane microsac preparations. An antibody raised against a 17 amino acid peptide, based on the predicted C-terminus of the Drosophila GABA receptor subunit (RDL), stained not only cell bodies, including that of fg1, but also the neuropile in the frontal ganglion, indicating the existence of RDL-like GABA receptor subunits in neurones of this ganglion. Thus, bicuculline-insensitive GABA-gated Cl^– channels are present in the larval nervous system of M. sexta.     

The isolation and characterization of a mutant allele at a new X-linked locus,mex, affecting NADP+-dependent enzymes inDrosophila melanogaster

The isolation and characterization of mutant alleles in a regulatory gene affecting NADP⁺-dependent enzymes are described. The locus,mex, is at position 26.5 ± 0.74 on the X chromosome ofDrosophila melanogaster. The newly isolated mutant allele,mex ¹, is recessive to either themex allele found in Oregon-R wild-type individuals or that found in thecm v parental stock in which the new mutants were induced. Themex ¹ mutant allele is associated with statistically significant decreases in malic enzyme (ME) specific activity and ME specific immunologically cross-reacting material (ME-CRM) in newly emerged adult males. During this same developmental stage in males, the NADP⁺-dependent isocitrate dehydrogenase specific activity increases to statistically significant levels. Females of themex ¹ mutant strain show statistically significant elevated levels of the pentose phosphate shunt enzymes, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Isoelectric focusing and thermolability comparisons of the active ME from mutant and control organisms indicate that the enzyme is the same. Developmental profiles ofmex ¹ and control strains indicate that this mutant allele differentially modulates the levels of ME enzymatic activity and ME-CRM during development. This work was supported by an Operating Grant from the Natural Sciences and Engineering Research Council of Canada to M.M.B.     

Local accumulation of membrane-associated calcium according to cell pattern formation inMicrasterias denticulata,visualized by chlorotetracycline fluorescence

Summary Developing cells ofMicrasterias denticulata Bréb. show a characteristic fluorescence of the plasma membrane (or cortical protoplasm) after treatment with chlorotetracycline (CTC), which is known to be an indicator for membrane-bound Ca²⁺. Depending on the stage of development the fluorescing sites of the young half cell are distributed in a specific pattern which corresponds to cell pattern formation. Therefore growth and thus cytomorphogenesis inMicrasterias seem to be mediated by a patterned accumulation of Ca²⁺ at the periphery of the differentiating cell. Participation of Ca²⁺ in a membrane-recognition process responsible for local vesicle incorporation is discussed.