THE MECHANISM OF THE NEPHRIDIAL APPARATUS OF PARAMECIUM MULTIMICRONUCLEATUM : II. The Filling of the Vesicle by Action of the Ampullae

Our recent analysis of the nephridial apparatus of Paramecium multimicronucleatum by high-speed cinematography (300 fps at X 250) indicates that before the water expulsion vesicle ("contractile vacuole") is completely voided of fluid during expulsion, the ampullae surrounding and confluent with the vesicle swell with fluid entering from their respective nephridial tubules. Once the membranes of the excretory pore at the base of the excretory canal (leading from the vesicle proper to the outside) have constricted and resealed the excretory pore, the up till then constricted injection tubules of the ampullae which conduct fluid to the vesicle open as waves of contraction along the coacervate gel around the ampulla and proceed along each ampulla from distal to proximal end. The coacervate gel around any one ampulla does not necessarily contract in phase with that of any other ampulla. Each ampulla acts independently. The fluid from the ampullae is thus pumped sequentially, but not in predetermined order, into the water expulsion vesicle, refilling and distending it. Our previous studies (Organ et al., 1968a) suggest that an actomyosinoid ATP-using mechanism may be functional in the ampullary contractions.     

THE MECHANISM OF THE NEPHRIDIAL APPARATUS OF PARAMECIUM MUL TIMICRONUCLEATUM : I. Expulsion of Water from the Vesicle

Recent analysis of the mechanism of the nephridial apparatus of Paramecium multimicronucleatum by high-speed cinematography (300 fps at x 250) confirms the observations by electron microscopy (Schneider, 1960) that once the pore is opened, the vesicle is invaginated by adjacent cytoplasm and is emptied by collapsing under pressure from that cytoplasm, aided perhaps by pressure of the fibrils which anchor the ampullae to the excretory canal. There is no indication of active contraction of the vesicle or its membrane. There is no permanent pore to the vesicle. The vesicle is closed by a sealing of the ruptured membrane where it is in contact with the pellicular excretory canal. At onset of expulsion of vesicular fluid the membrane across the basal opening of the excretory canal is ripped along one semicircular portion of the excretory pore and is driven up against the opposite wall as a flap while the water rushes out. A constriction of the vesicular and cell membranes at the base of the excretory canal reseals the opening.     

THE ULTRASTRUCTURE OF DERBESIA TENUISSIMA (DE NOTARIS) CROUAN. I. ORGANIZATION OF THE GAMETOPHYTE PROTOPLAST,GAMETANGIUM, AND GAMETANGIAL PORE1,2,3

The fine structure of vegetative and reproductive gametophytes of Derbesia tenuissima is described. Development of the gametangium and release of the gametes progress as follows: (1) In initial stages of gametangium formation, prior to 24 hr before gamete release, there is an accumulation and proliferation of nuclei, chloroplasts, and other organelles. (2) This is followed by separation of the gametangium from the rest of the plant by a gametangial membrane; segregation of organelles into gametes has begun by 12 hr before release and the process is completed by 2.5 hr before release. (3) Enzymatic wall dissolution of the pore area occurs between 2.5 and, 12 hr before normal lights-on time. (4) The release mechanism appears to be an instantaneous light-induced increase in lurgor pressure rupturing the weakened pore area, of the wall and causing a forcible expulsion of the gametes. (5) Following release, the pore is sealed by organellar debris and the gametangial membrane. Additional wall layers are presumed to be laid down internal to the plugged pore by the vegetative protoplasm which migrates into the area.     

Nuclear membrane disassembly and rupture

The nuclear envelope consists of two membranes traversed by nuclear pore complexes. The outer membrane is continuous with the endoplasmic reticulum. At mitosis nuclear pore complexes are dismantled and membranes disperse. The mechanism of dispersal is controversial: one view is that membranes feed into the endoplasmic reticulum, another is that they vesiculate. Using Xenopus egg extracts, nuclei have been assembled and then induced to breakdown by addition of metaphase extract. Field emission scanning electron microscopy was used to study disassembly. Strikingly, endoplasmic reticulum-like membrane tubules form from the nuclear surface after the addition of metaphase extracts, but vesicles were also observed. Microtubule inhibitors slowed but did not prevent membrane removal, whereas Brefeldin A, which inhibits vesicle formation, stops membrane disassembly, suggesting that vesiculation is necessary. Structures that looked like coated buds were observed and buds were labelled for beta-COP. We show that nuclear pore complexes are dismantled and the pore closed prior to membrane rupturing, suggesting that rupturing is an active process rather than a result of enlargement of nuclear pores.     

EXOCYTOSIS OF LATEX BEADS DURING THE ENCYSTMENT OF ACANTHAMOEBA

Cells of Acanthamoeba castellanii (Neff) are known to form mature cysts characterized by a cellulose-containing cell wall when transferred to a nonnutrient medium. Amebas which engulfed latex beads before encystment formed mature cysts essentially devoid of bead material. The encystment of bead-containing cells appeared to be similar to that of control cells since no important differences between the two were observed with respect to cellular levels of glycogen or protein, cellulose synthetase activity, the amount of cyst wall polysaccharide formed, or the percentage of cysts formed. Actinomycin D and cycloheximide inhibited encystment as well as bead expulsion. Ultrastructural analysis revealed that the beads, which initially were contained in phagocytic vesicles, were released from the cell by fusion of vesicular membranes with the plasma membrane. Exocytosis was observed in cells after 3 hr of encystment, with most of the beads being lost before cyst wall formation. Each bead-containing vesicle involved in expulsion was conspicuously demarcated by an area of concentrated cytoplasm, which was more homogeneously granular than the surrounding cytoplasm. Beads were not observed in the cytoplasm of mature cysts but were occasionally found in the cyst wall.     

FINE STRUCTURE OF THE SPERMATOZOON OF HYDROIDES HEXAGONUS (ANNELIDA), WITH SPECIAL REFERENCE TO THE ACROSOMAL REGION

This paper describes in some detail the structure of the acrosomal region of the spermatozoon of Hydroides as a basis for subsequent papers which will deal with the structural changes which this region undergoes during fertilization. The material was osmium-fixed and mild centrifugation was used to aggregate the spermatozoa from collection to final embedding. The studies concern also the acrosomal regions of frozen-thawed sperm prepared by a method which previously had yielded extracts with egg membrane lytic activity. The plasma membrane closely envelops four readily recognizable regions of the spermatozoon: acrosomal, nuclear, mitochondrial, and flagellar. The acrosome consists of an acrosomal vesicle which is bounded by a single continuous membrane, and its periphery is distinguishable into inner, intermediate, and outer zones. The inner and intermediate zones form a pocket into which the narrowed apex of the nucleus intrudes. Granular material adjoins the inner surface of the acrosomal membrane, and this material is characteristically different for each zone. Centrally, the acrosomal vesicle is spanned by an acrosomal granule: its base is at the inner zone and its apex at the outer zone. The apex of the acrosomal granule flares out and touches the acrosomal membrane over a limited area. In this limited area the adjoining granular material of the outer zone is lacking. The acrosomal membrane of the inner zone is invaginated into about fifteen short tubules. The acrosomal membrane of the outer zone is closely surrounded by the plasma membrane. At the apex of the acrosomal region a small apical vesicle is sandwiched between the plasma membrane and the acrosomal membrane. Numerous frozen-thawed specimens and occasional specimens not so treated show acrosomal regions at the apex of which there is a well defined opening or orifice. Around the rim or lip of this orifice plasma and acrosomal membranes may even be fused into a continuum. The evidence indicates that the apical vesicle and the parts of the plasma and acrosomal membranes which surround it constitute a lid, and the rim of this lid constitutes a natural "fracture line" or rim of dehiscence. Should fracture occur, the lid would be removed and the acrosomal vesicle would be open to the exterior.     

Special cutaneous receptor organs of fish. IV. Ampullary organs of the nonelectric catfish, Kryptopterus

Ampullary organs of the transparent catfish, Kryptopterus bicirrhus, are present in large numbers on the head and in a regular pattern of lines on the body and fins. The organs lie in the epidermis, and have a pore that opens to the surface. Flattened cells form a roof and walls. On the floor of the organ there are a “sensory hillock,” composed of spherical receptor cells and columnar supporting cells, and a “secretory hillock” composed of columnar secretory cells. The receptor cells are nonciliated and have only afferent innervation. The organ cavity is filled with jelly. The organs are compared with ampullary organs of the weakly electric fish Eigenmannia, ampullae of Lorenzini of Raja, and small pit organs of Amiurus. Structural characteristics of the ampullary organs of Kryptopterus make them especially suitable for electrophysiological studies.     

THE ACROSOME REACTION IN MYTILUS EDULIS : I. Fine Structure of the Intact Acrosome

The intact acrosome of the Mytilus edulis spermatozoon consists of a conical vesicle, the basal side of which is deeply invaginated so that the whole vesicle forms a sheath around a very slender axial rod, about 2.7 µ long, inserted in a tube passing through the nucleus. The annular base of the acrosomal vesical is filled with a homogeneous substance; the outer wall of the vesicle is lined with a somewhat irregular layer of a particulate substance interspersed with very fine tubular elements, and its lumen is nearly filled by a strand of material which extends from the inner tip of the invagination to the apex of the acrosome. The lumen of the invagination appears empty except for the rod and a delicate sleeve-like structure which surrounds it. The plasma membrane of the sperm cell lies in immediate contact with the acrosomal membrane over its whole outer surface. In its general organization, this molluscan acrosome shows a rather close homology with that of the annelid Hydroides.     

Permeability and the hidden area of lipid bilayers

The passive water permeability of a lipid vesicle membrane was studied, related to the hydrostatic (not osmotic) pressure difference between the inner and the outer side of the vesicle in a water environment without additives. Each pressure difference was created by sucking a vesicle into a micropipette at a given sucking pressure. The part of the membrane sucked into the micropipette (the projection length) was measured as a function of time. The time dependence can be divided into two intervals. We put forward the idea that smoothing of membrane defects, accompanied by an increase of the membrane area, takes place during the initial time interval, which results in a faster increase of the projection length. In the second time interval the volume of the vesicle decreases due to the permeability of its membrane and the increase of the projection length is slower. The hidden area and the water permeability of a typical lipid bilayer were estimated. The measured permeability, conjugated to the hydrostatic pressure difference, is an order of magnitude higher than the known value of the permeability, conjugated to the osmotic pressure difference. A hypothesis, based on pore formation, is proposed as an explanation of this experimental result.     

THE ISOLATION OF A CELL MEMBRANE FRACTION FROM RAT LIVER

A procedure is described for isolating cell membranes from rat liver homogenates. 20 gm. of rat liver was homogenized in a Dounce homogenizer in ice cold water buffered to pH 7.5 with NaHCO₃, rupturing all of the cells and most nuclei. The diluted homogenate was filtered through cheesecloth to remove precipitated nucleoprotein and centrifuged at 1500 g, 10 minutes, to sediment a crude membrane fraction. The membrane containing sediment was recentrifuged 3 times in conical tubes (1220 g, 10 minutes), the top layer of the 2-layered sediment being retained. Flotation in a sucrose solution d = 1.22 freed the preparation from contaminating cell fragments and nuclear membranes not previously disintegrated. The floating material ~0.4 ml. was quite homogeneous and consisted of thin amorphous membranes. Electron micrographs revealed numerous double profiles similar in shape and dimensions to apposed liver cell membranes in intact tissue.     

THE FINE STRUCTURE OF ACANTHAMOEBA CASTELLANII : I. The Trophozoite

The fine structure of the trophozoite of Acanthamoeba castellanii (Neff strain) has been studied. Locomotor pseudopods, spikelike "acanthopodia," and microprojections from the cell surface are all formed by hyaline cytoplasm, which excludes formed elements of the cell and contains a fine fibrillar material. Golgi complex, smooth and rough forms of endoplasmic reticulum, digestive vacuoles, mitochondria, and the water-expulsion vesicle (contractile vacuole) are described. A canicular system opening into the water-expulsion vesicle contains tubules about 600 A in diameter that are lined with a filamentous material. The tubules are continuous with unlined vesicles or ampullae of larger diameter. Centrioles were not observed, but cytoplasmic microtubules radiate from a dense material similar to centriolar satellites and are frequently centered in the Golgi complex. Cytoplasmic reserve materials include both lipid and glycogen, each of which amounts to about 10% of the dry weight.     

Concentration-dependent staining of lactotroph vesicles by FM 4-64

Hormones are released from neuroendocrine cells by passing through an exocytotic pore that forms after vesicle and plasma membrane fusion. An elegant way to study this process at the single-vesicle level is to use styryl dyes, which stain not only the membrane, but also the matrix of individual vesicles in some neuroendocrine cells. However, the mechanism by which the vesicle matrix is stained is not completely clear. One possibility is that molecules of the styryl dye in the bath solution dissolve first in the plasma membrane and are then transported into the vesicle by lateral diffusion in the plane of the membrane, and finally the vesicle matrix is stained from the vesicle membrane. On the other hand, these molecules may enter the vesicle lumen and reach the vesicle matrix by permeation through an open aqueous fusion pore. To address these questions, we exposed pituitary lactotrophs to different concentrations of FM 4-64 to monitor the fluorescence increase of single vesicles by confocal microscopy after the stimulation of cells by high K(+). The results show that the membrane and the vesicle matrix exhibit different concentration-dependent properties: the plasma membrane staining by FM 4-64 has a higher affinity in comparison to the vesicle matrix. Moreover, the kinetics of vesicle loading by FM 4-64 exhibited a concentration-dependent process, which indicates that FM 4-64 molecules stain the vesicle matrix by aqueous permeation through an open fusion pore.     

A theory of osmotic lysis of lipid vesicles

Osmotic lysis of vesicles is shown to begin when the membrane expansion due to osmotic pressure exceeds its critical value, delta S, at which a membrane ruptures to form a pore. The dependence of delta S on the vesicle radius and respective osmotic pressures are obtained. It is found that osmotic pressure necessary for small (100 A) vesicles to rupture should exceed 30 atm, for large (10 000 A) vesicles it being as small as 10(-3) atm. In the case of large (greater than or approximately 1000 A) vesicles the value of relative expansion of the membrane at which its rupture occurs in a reasonable time only depends slightly on the vesicle radius. For instance, for 10 000 A vesicles it amounts to 3%. The tension of membrane rupture is about 8 dyn/cm for large vesicles. Membrane tension, although it decreases considerably as a result of rupture and pore formation, does not vanish completely. It supports the residual intravesicular pressure causing the efflux of vesicle (cell) contents. Simultaneously, osmotic influx of water through the membrane occurs that results in either complete rupture of the membrane with the efflux of the whole of the contents, or its gradual washout in either of two, quasi-steady or pulse-wise regimes. In the first case a pore is steadily open, whereas in the second case it alternately opens and closes, ejecting about 5% of internal solution each time. Lysis kinetics is analyzed. Pulse-wise regime of lysis is shown to be the most likely one.     

Loss of sensory elements in the apical sensory organ during metamorphosis in the nudibranch Phestilla sibogae

Larvae of the nudibranch Phestilla sibogae are induced to metamorphose by a water-borne chemical cue released by the adult nudibranch's prey, the coral Porites compressa. In competent larvae, the apical sensory organ (ASO) includes five serotonergic parampullary neurons; five ampullary neurons, the ampullae of which are filled with sensory cilia; and a basal neuropil. After sensing the coral cue, the ASO undergoes radical morphological changes: a deterioration of sensory elements in the ASO and serotonergic axons originating from them to innervate the velum. Three hours after metamorphic induction, the velar lobes are lost, the serotonergic axons begin to break apart, the five parampullary neurons begin to degenerate, and the five ampullary neurons retract away from the epidermal surface. The extent of deterioration evident by this time suggests that the parampullary and ampullary components of the ASO are no longer functional. By 10 h after metamorphic induction, labeling of the ciliary bundles in the ampullary neurons has disappeared, and it is likely that these cells have degenerated. The results presented here provide evidence that the sensory neurons of the ASO and probably the entire organ are solely larval structures that do not persist into the adult sensory-nervous system in P. sibogae.     

THE DEVELOPMENT OF BASAL BODIES AND FLAGELLA IN ALLOMYCES ARBUSCULUS

The development of basal bodies and flagella in the water mold Allomyces arbusculus has been studied with the electron microscope. A small pre-existing centriole, about 160 mµ in length, was found in an inpocketing of the nuclear membrane in the vegetative hypha. Thus, formation of a basal body does not occur de novo. When the hyphal tip started to differentiate into gametangia, the centrioles were found to exist in pairs. One of the members of the pair then grew distally to more than three times its original length, whereas the other remained the same size. The larger centriole would correspond to the basal body of a future gamete. Gametogenesis was usually induced by transferring a "ripe" culture to distilled water. Shortly after this was done, a few vesicles were pinched off from the cell membrane of the gametangium and came in contact with the basal body. Apparently, they fused and formed a large primary vesicle. The flagellum then started to grow by invaginating into it. Flagellar fibers were evident from the very beginning. As the flagellum grew so did the vesicle by fusion with secondary vesicles, thus coming to form the flagellar sheath. The different stages of flagellar morphogenesis are described and the possible interrelationships with other processes are discussed.     

Peristomial tube feet and plates of regular echinoids

Summary Peristomial tube feet, ampullae and plates are described in 16 species of regular echinoids. Two basic arrangements are recognised. In cidaroids and echinothurioids there are many tube feet and ampullae per column and the radial water vessel extends on to the peristomial membrane. Tube feet terminate in a small sensory pad. Ampullae are small and flattened. In other echinoids there are only ten peristomial tube feet and the radial water vessel does not extend on to the peristomial membrane. Tube feet terminate in a broad disc and ampullae are cylindrical tubes. Plate structure and pore morphology also vary and are correlated with tube foot structure. Echinothurioids are considered to be derived from a cidaroid ancestor.     

Kiss-and-coat and compartment mixing: coupling exocytosis to signal generation and local actin assembly

Regulated exocytosis is thought to occur either by "full fusion," where the secretory vesicle fuses with the plasma membrane (PM) via a fusion pore that then dilates until the secretory vesicle collapses into the PM; or by "kiss-and-run," where the fusion pore does not dilate and instead rapidly reseals such that the secretory vesicle is retrieved almost fully intact. Here, we describe growing evidence for a third form of exocytosis, dubbed "kiss-and-coat," which is characteristic of a broad variety of cell types that undergo regulated exocytosis. Kiss-and-coat exocytosis entails prolonged maintenance of a dilated fusion pore and assembly of actin filament (F-actin) coats around the exocytosing secretory vesicles followed by direct retrieval of some fraction of the emptied vesicle membrane. We propose that assembly of the actin coats results from the union of the secretory vesicle membrane and PM and that this compartment mixing represents a general mechanism for generating local signals via directed membrane fusion.     

Discovery of the Porosome: revealing the molecular mechanism of secretion and membrane fusion in cells

Secretion and membrane fusion are fundamental cellular processes involved in the physiology of health and disease. Studies within the past decade reveal the molecular mechanism of secretion and membrane fusion in cells. Studies reveal that membrane-bound secretory vesicles dock and fuse at porosomes, which are specialized plasma membrane structures. Swelling of secretory vesicles result in a build-up of intravesicular pressure, which allows expulsion of vesicular contents. The discovery of the porosome, its isolation, its structure and dynamics at nm resolution and in real time, its biochemical composition and functional reconstitution, are discussed. The molecular mechanism of secretory vesicle fusion at the base of porosomes, and vesicle swelling, have been resolved. With these findings a new understanding of cell secretion has emerged and confirmed by a number of laboratories.     

THE EFFECT OF SALT CONCENTRATION OF THE MEDIUM ON THE RATE OF OSMOSIS OF WATER THROUGH THE MEMBRANE OF LIVING CELLS

1. Using the unfertilized egg of the sea urchin, Arbacia, as osmometer, it was found that the rate with which water enters or leaves the cell depends on the osmotic pressure of the medium: the velocity constant of the diffusion process is higher when the cell is in concentrated sea water, and lower when the sea water medium is diluted with distilled water. Differences of more than tenfold in the value of the velocity constant were obtained in this way. When velocity constants are plotted against concentration of medium, a sigmoid curve is obtained. 2. These results are believed to indicate that cells are more permeable to water when the osmotic pressure of the medium is high than when it is low. This relation would be accounted for if water should diffuse through pores in a partially hydrated gel, constituting the cell membrane. In a medium of high osmotic pressure, the gel is conceived to give up water, to shrink, and therefore to allow widening of its pores with more ready diffusion of water through them. Conversely, in solutions of lower osmotic pressure, the gel would take up water and its pores become narrow.