The Structural Changes of the Basal Region of Wheat Proembryo in Relation to Nurture Absorption

There are some cellular fail and degeneration in the parietal area of the basal region of developing wheat proembryo. Electron microscopic studies reveal that the envelopment of peripheral wall to the proembryo is partly ruptured in this area and the disassembled protoplasm of the degenerated cells mixes with the disintegrated constituents of adjacent endosperm cells. Hence, in the limited area a direct communication between the inner surviving proembryo cells and the surrounding medium is established. A number of ectodesma-like plasmodesmata and open channels appear at the boundary wall, various nutrients may enter the proembryo via symplastic pathway or by endocytosis. The surrounding macromolecules (disassembled nuclei, mitochondria, cytoplasmic granules and vesicles packed with fibrils) appear to traverse across the wall continually, and it seems that this is'an important mode of nurture translocation. Also, within the proembryo some of the densely distributed plasmodesmata undergo modification and become fully opened for macromolect, les traversing, which is in favor of re-distribution of cell contents amongst proembryo cells. Presumably, the structural changes occurred in the basal region is a special kind of differentiation which results in function of this local area as apparatus of nurture absorption. Evidently, it would enhance the incorporation of external materials into the proembryo, and then the normal proliferation, development and differentiation of proembryo cells would be ensured.     

Gap junctions with varied permeability properties establish cell-type specific communication pathways in the rat seminiferous epithelium

Dye coupling experiments were performed to determine whether the gap junctions connecting Sertoli cells with other Sertoli cells and different germ cell stages in rats showed functional variations. Chop loading of adult rat seminiferous tubules was conducted using fluorescent dextran controls and a variety of low-molecular-weight tracers (lucifer yellow, biotin-X-cadaverine, biotin cadaverine, and neurobiotin) to evaluate dye coupling in situ, and scrape loading was used to study dye coupling in Sertoli-germ cell cocultures established using prepuberal rats. Sertoli-Sertoli coupling is relatively short range and nonselective in situ, whereas coupling between Sertoli cells and chains of spermatogonia is strongly selective for the positively charged biotin tracers relative to negatively charged lucifer yellow. Coupling between Sertoli cells and spermatogonia was also asymmetric; lucifer yellow in germ cells never diffused into Sertoli cells, and biotinylated tracers only weakly diffused from spermatogonia to Sertoli cells. Asymmetric coupling would facilitate the concentration in germ cells of molecules diffusing through junctions from Sertoli cells. Dye coupling between Sertoli cells and adluminal germ cells was too weak to detect by fluorescence microscopy, suggesting that the junctional communication between these cells may be functionally different from that between Sertoli and basal germ cells. The results show that there are multiple routes of gap junction communication in rat seminiferous tubules that differ in permeability properties and show alternative gating states. Functional diversity of gap junctions may permit regulated communication among the many interacting Sertoli cells and germ cell stages in the seminiferous epithelium.     

A gradient of gap junctional communication along the anterior-posterior axis of the developing chick limb bud.

A modification of the scrape-loading/dye transfer technique was used to study gap junctional communication along the anterior-posterior (A-P) axis of embryonic chick wing buds at an early stage of development (stage 20/21) when positional values along the A-P axis are being specified. Extensive intercellular transfer of the gap junction-permeable dye, lucifer yellow, from scrape-loaded mesenchymal cells to contiguous cells occurs in the posterior mesenchymal tissue of the wing bud adjacent to the zone of polarizing activity, which is thought to be the source of a diffusible morphogen that specifies A-P positional identity according to its local concentration. Considerably less transfer of lucifer yellow dye occurs in scrape-loaded mesenchymal tissue in the middle of the limb bud compared to posterior mesenchymal tissue, and little or no transfer of lucifer yellow is observed in the mesenchymal tissue in the anterior portion of the limb bud. No intercellular transfer of the gap junction-impermeable dye, rhodamine dextran, occurs in any region of the limb bud. These results indicate that there is a gradient of gap junctional communication along the A-P axis of the developing chick wing bud. This gradient of gap junctional communication along the A-P axis might generate a graded distribution of a relatively low molecular weight intracellular regulatory molecule involved in specifying A-P positional identities.     

Lucifer Yellow Uptake in Cells and Protoplasts of Daucas carota Visualized by Laser Scanning Microscopy

The uptake of lucifer yellow CH by suspension-cultured carrotcells and protoplasts has been studied by laser scanning microscopy.This fluorochrome, which does not diffuse across membranes,gradually accumulates in the cell vacuole over a period of hours.In contrast, the central vacuole of protoplasts did not showlucifer yellow fluorescence. The latter was restricted, in protoplasts,to punctate sources in the peripheral cytoplasm. Confocal opticsallowed the complexity of the vacuolar system to be dramaticallydepicted with the laser scanning microscope. Control experimentssupport the contention that lucifer yellow uptake, as in othereukaryotic systems, occurs via endocytosis. Key words: Carrot cells, endocytosis, laser scanning microscopy, lucifer yellow CH, protoplasts, vacuolar apparatus     

ATP4- permeabilizes the plasma membrane of mouse macrophages to fluorescent dyes

Extracellular ATP induces cation fluxes in thioglycolate-elicited mouse peritoneal macrophages and the J774 macrophage cell line apparently due to ligation of a plasma membrane receptor for ATP4-. We report that ATP permeabilizes the plasma membrane of J774 cells to 6-carboxyfluorescein (376 Da), lucifer yellow (457 Da), and fura-2 (831 Da) but not to trypan blue (961 Da), Evans blue (961 Da), or larger dye conjugates. We employed fluorescence microscopy and quantitative fluorimetry to study entry of lucifer yellow into the cytoplasm of J774 cells. Permeabilization to lucifer yellow appears to be mediated by the same ATP4- receptor that induces cation fluxes because it was inhibited by divalent cations and low pH, was mediated by the nonhydrolyzable analog adenosine 5'-(beta, gamma-imido)triphosphate, and because a variant J774 cell line resistant to ATP-induced Rb+ efflux did not take up lucifer yellow when exposed to ATP. ATP permeabilization was reversed within 5 min by removal of ATP or by addition of divalent cations. ATP also caused a transient increase in lucifer yellow uptake by pinocytosis. These data suggest that ATP4- ligates a receptor on macrophages which induces the formation of a channel admitting molecules less than or equal to 831 daltons into the cytoplasmic matrix and that removal of ATP4- from the medium causes rapid channel closure.     

Movement of lucifer yellow in leaves of Coleus blumei Benth

Abstract Individual spongy mesophyll cells in green areas of variegated Coleus blumei leaves were injected with the symplast-mobile dye lucifer yellow and its movement to other cell types was followed with fluorescence microscopy. In 13 trials, the dye remained in the injected cell twice, moved only to other mesophyll cells five times, and moved up to and along minor veins six times. Where extensive movement of the dye occurred, the tissue was fixed with 4% glutaraldehyde, dehydrated, embedded in plastic, sectioned at 3 μm, and examined again with fluorescence microscopy. The dye was found in abaxial bundle-sheath cells for up to 200 μm or more distant from the site of injection near the minor vein, but no convincing evidence was found for its presence in the vascular tissue itself. It thus appears that superficial whole-mount views of lucifer yellow movement along leaf minor veins cannot be taken as certain evidence for symplastic transport of the dye into and along the vascular tissues.     

Spatial-temporal characteristics of intercellular junctions in early zebrafish and loach embryos before and during gastrulation

 Injections of lucifer yellow and fluorescein dyes into loach (Misgurnus fossilis) and zebrafish (Danio rerio) embryos were used to analyse the intercellular communication via gap junctions (GJs) and their role in morphogenetic processes during the period from early blastula to late gastrula. It is shown that the efficiency of dye transfer between the superficial blastomeres increases by the late blastula stage. Blastomeres of the basal layer, on the other hand, become gradually uncoupled from the yolk cell (YC). This process is spatially uneven and finishes by the late gastrula stage. Prior to it, at the early epiboly stage, a local increase in dye transfer is observed in the circular zone of the blastoderm margin. During gastrulation, GJ communication between blastomeres and the YC in this zone and also in the newly-formed germ ring region (the prospective mesoderm domain) persists for a longer period of time (up to the stage of 60–70% epiboly) than in the remaining part of the basal layer (the prospective ectoderm domain). Taking into account the data on changes in the adhesive properties of blastomeres during normal development and observations on embryos with retarded epiboly, we hypothesize that changes in GJ communication between superficial blastomeres, on one hand, and between basal blastomeres and the YC, on the other, are the consequences of the same, more general morphogenetic process of compaction occurring within the blastoderm, which supports epiboly and is probably responsible for the distinction between mesodermal and ectodermal fates of cells differently located within the forming epithelioid sheet. Received: 18 October 1996 / Accepted: 4 April 1997     

Gap junctional communication during limb cartilage differentiation

The onset of cartilage differentiation in the developing limb bud is characterized by a transient cellular condensation process in which prechondrogenic mesenchymal cells become closely apposed to one another prior to initiating cartilage matrix deposition. During this condensation process intimate cell-cell interactions occur which are necessary to trigger chondrogenic differentiation. In the present study, we demonstrate that extensive cell-cell communication via gap junctions as assayed by the intercellular transfer of lucifer yellow dye occurs during condensation and the onset of overt chondrogenesis in high density micromass cultures prepared from the homogeneous population of chondrogenic precursor cells comprising the distal subridge region of stage 25 embryonic chick wing buds. Furthermore, in heterogeneous micromass cultures prepared from the mesodermal cells of whole stage 23/24 limb buds, extensive gap junctional communication is limited to differentiating cartilage cells, while the nonchondrogenic cells of the cultures that are differentiating into the connective tissue lineage exhibit little or no intercellular communication via gap junctions. These results provide a strong incentive for considering and further investigating the possible involvement of cell-cell communication via gap junctions in the regulation of limb cartilage differentiation.     

Osmotic swelling of endocytic compartments induced by internalized sucrose is restricted to mature lysosomes in cultured mammalian cells.

To determine which endocytic compartments are sensitive to sucrose-induced osmotic swelling, CHO and Vero cells were cultured for 1-3 days in media containing 0.03 to 0.05 M sucrose. (Sucrose is internalized but not digested by these cells.) To immunolocalize late endocytic compartments, cells were fixed with formaldehyde and labeled with antibodies against the 215-kDa mannose 6-phosphate receptor (prelysosomal compartment) and LAMP-1 and -2 (mature lysosomes). Early endosomes were labeled by a 2-min uptake of lucifer yellow, mature lysosomes by greater than or equal to 16-h uptake of lucifer yellow followed by a 2-h chase. The data showed that sucrose induced swelling of mature lysosomes only (mannose 6-phosphate receptor negative, LAMP-1 and LAMP-2 positive); early endosomes and the prelysosomal compartment were not affected by the presence of sucrose, i.e., osmotically swollen. Accumulation of lucifer yellow in the swollen compartment was insensitive to cycloheximide. These results suggest, by inference, that the complement of membrane transport proteins that regulate the osmotic properties of endocytic organelles must be discontinuously distributed along the endocytic pathway.     

Transfer Cells in Suspensur and Endosperm during Early Embryogeny of Vigna sinensis

During early embryogeny, structural differentiation of the suspensor and endosperm can be observed with the formation of cells with wall ingrowths. In the early proembryo stage, wall ingrowths are seen only on the boundary walls of the embryo sac around the proembryo and at the chalazal end. Later, ingrowths appear in the outer walls of the basal suspensor cells and some wall ingrowths also begin to develop in the outer walls of cellular endospermic cells adjacent to the nucellar cap and the inner integumentary tissues. The suspensor appears to remain active throughout the differentiation stages. Two regions can be clearly distinguished in the suspensor: a basal region and a neck region. Wall ingrowths appear to form only in the cells of the basal region. During the development of the cellular endospermic sheath, its cell number and size both increase slightly. Later, these cells rapidly become separated from each other. Those endospermic cells that abut directly onto the integumentary tissues also develop wall ingrowths. In the region of the fluid endosperm, wall ingrowths are especially abundant in the boundary walls on the ventral side of the embryo sac. The possible pathway of nutrient flow to the developing embryo is discussed.     

Visualization of exocytotic secretory processes of mast cells by fluorescence techniques

Secretory processes via exocytosis in rat peritoneal mast cells were visualized by two complementary fluorescence techniques; one staining pre-exocytotic granules with a basic probe and the other staining post-exocytotic granules with acidic probes. Granules within mast cells were selectively stained with acridine orange and emitted orange yellow fluorescence. Upon stimulation with compound 48/80, release of acridine orange from granules was observed both in population and single cell measurements. This release was seen in some localized area of mast cells. Opening of pores between plasma membranes and granule membranes was monitored using acidic fluorescence probes such as 6-carboxyfluorescein or lucifer yellow CH. Not only granules located at peripheral region, but also granules near the core region participated in exocytosis. The existence of junctions between these granules was suggested. TMA-DPH, a lipophilic membrane probe, which was localized at plasma membrane before stimulation, diffused into granule membranes after stimulation. This shows that after stimulation, some constituents of plasma and granule membranes were mixed. Even after extensive degranulation, mast cells extruded acidic probes, indicating the plasma membranes still play a role of barrier. Activation of lateral motion of granules preceding to exocytosis was not observed. It was concluded that the visualization of secretory processes by fluorescence and image processing techniques will be useful for the study of molecular mechanisms underlying exocytosis.     

Endocytosis and phosphate transport in OK epithelial cells

Endocytosis was studied in OK epithelial cells, an established cell line from opossum kidney. The presence of fluid-phase endocytosis in these cells was demonstrated by measuring cell uptake of lucifer yellow and horseradish peroxidase. The intracellular distribution of lucifer yellow fluorescence was consistent with uptake by endocytosis. Endocytosis was inhibited in medium made hyperosmolar by addition of sucrose. In hyperosmolar medium the action of parathyroid hormone on Na+/phosphate cotransport was significantly diminished. We suggest that an intact endocytic mechanism is required for the full inhibitory effect of parathyroid hormone on Na+/phosphate cotransport.     

Activation of Protein Kinase C Blocks Astroglial Gap Junction Communication and Inhibits the Spread of Calcium Waves

The following two processes related to astrocytes are thought to depend on intercellular coupling through gap junctions: the spatial buffering of K+o and the spread of calcium waves in the astrocytic syncytium. We have used the following two independent methods to measure the open state of gap junctions: injection of lucifer yellow, and optical calcium imaging of calcium waves in response to probing the cells with a micropipette. The spread of lucifer yellow and calcium waves was inhibited if the cells were treated with either phorbol 12-myristate 13-acetate (PMA) or a synthetic diacylglycerol that activates protein kinase C. Down-regulation of protein kinase C by a 24-h treatment with PMA inhibited the uncoupling effect of PMA, supporting a direct involvement of protein kinase C in the regulation of astroglial gap junctions. Purinergic P2Y receptors, which are coupled to the inositol phospholipid pathway, are expressed by most astroglia in culture. Activation of the P2Y purinergic receptor with the selective agonist 2-methylthio-ATP uncoupled astroglia in a manner similar to the effect of treatment with PMA. Modulation of gap junctional conductance could isolate specific pathways within the astrocytic syncytium to form an extraneuronal information transfer network in brain.     

Polar protein transport between apical and basal cells during tobacco early embryogenesis

Key message

We found that protein trafficking between apical and basal cell can be unidirectional, which reveals the different roles of the two cells in the cell-to-cell communication between them during early embryogenesis.

Abstract

In most angiosperm species, asymmetric zygote division results in an apical cell and a basal cell that have distinct cell fates. Much has been speculated about possible communication between these cell types in relation to their cell fate determination. Here, we report on the use of photoactivatable green fluorescent protein (PA-GFP) in tobacco to trace intercellular communication between apical and basal cells during early embryogenesis. We found that PA-GFP was transported between apical and basal cells of a two-celled proembryo, and that protein trafficking was unidirectional toward the apical cell, highlighting different cell communication roles. Further ultrastructural analysis showed numerous plasmodesmata in the walls connecting the apical and basal cells, which may provide channels for protein trafficking. Our data show a possible unique method of cell-to-cell communication between apical and basal cells during early embryogenesis.     

Monoclonal antibody ECCD-1 inhibits intercellular communication in teratocarcinoma PCC3 cells

The monoclonal antibody ECCD-1 recognizing a certain class of cell surface proteins inhibits the Ca²⁺-dependent cell-to-cell adhesion in teratocarcinoma stem cells. In this paper, we studied the effect of ECCD-1 on cell-to-cell communication in PCC3 cells by measuring the transfer of lucifer yellow between cells. To this aim, PCC3 cells were cultured in the presence of ECCD-1 for various periods, and then the fluorescent dye was injected into a cell located in the center of cell colonies, followed by counting number of cells to which the dye was transferred. The results showed that ECCD-1 inhibits the dye transfer between cells, suggesting that the Ca²⁺-dependent cell-to-cell adhesion system (CDS) is essential for the functions of gap junction.     

Intracellular pathways of native iron in the maternal part of the porcine placenta

In the diffuse epitheliochorial porcine placenta iron is secreted as uteroferrin by the maternal epithelium of the areola-gland subunit of the placenta. To elucidate the intracellular pathways of physiological iron in uterine gland epithelium material from 10 sows at 15 to 111 days of gestation was processed for electron microscopy by different routine methods with or without postfixation in osmium tetroxide. Ferritin particles were identified by their size and shape and the content of iron was confirmed by X-ray energy dispersive microanalysis of accumulated ferritin particles. Distinct ferritin particles were not observed in the extracellular space either basal to or luminal to the epithelial cells. Intracellular ferritin was observed apparently free in the cytoplasm, but in variable amounts. Transfer tubules and dense bodies were located basally in the secretory cells. Both of these organelles contained ferritin particles, showed reaction sites for acid phosphatase and were stained by periodic acid-thiocarbohydrazide-silver proteinate. The ciliated cells differed by having apically located dense bodies containing numerous ferritin particles. Our finding of native ferritin in cells with hormonally regulated iron transport supports the concept that transfer tubules as part of the lysosomal complex are part of the endocytic pathway in secretory cells and indicate that ferritin here is an intracellular transport or storage intermediate.     

Solid-phase Synthesis and Cellular Localization of a C- and/or N-terminal Labelled Peptide

We report the solid-phase synthesis by the Fmoc strategy of a peptide containing a cysteamide group at its C-terminus. This peptide was subject to further modifications including the linkage of fluorophores, namely lucifer yellow and coumarin respectively, at the C- and/or N-terminals. After incubation with living cultured cells these two probes were localized and it is concluded that the post-synthesis modifications can strongly modify the localization of the peptide.     

Morphological and functional changes in cell junctions during secretory stimulation in the perfused rat submandibular gland

To examine the influence of cholinergic and beta-adrenergic agents on paracellular transport, we applied confocal microscopy and freeze-fracture to the isolated, perfused submandibular gland of the rat. By confocal microscopy, perfusion of lucifer yellow through an arterial catheter, revealed a bright fluorescence in the basolateral spaces of acini, but not in the intercellular canaliculi. However, addition of isoproterenol on carbachol stimulation, induced lucifer yellow fluorescence in intercellular canaliculi. This finding indicates that isoproterenol is capable of opening the paracellular route. The tight junction strands surrounding intercellular canaliculi were visualized using freeze replicas. Fixation was carried out both by vascular perfusion with Karnovsky's solution and by metal contact rapid freezing with liquid helium. In the chemically-fixed specimens, the strand particles of tight junctions formed 2-5 lines at the P-face along most of the apical portion at rest. With carbachol/isoproterenol stimulation, the strand particles rearranged with free ends and terminal loops. In the rapidly frozen specimens, the strand particles were arranged more irregularly even in the resting state. The meshwork of strands became more disheveled and interrupted during carbachol/ isoproterenol stimulation. The present findings led us to conclude that: 1) the beta-adrenergic agent, isoproterenol, can open the paracellular transport. 2) in the rapidly frozen specimen, the tight junction strand particles are arranged roughly and become disheveled and interrupted during stimulation by carbachol/isoproterenol. These findings may be related to rearrangement of subcellular structures, especially of the actin filament network.     

Chorioallantoic placenta formation in the rat: I. Luminal epithelial cell death and extracellular matrix modifications in the mesometrial region of implantation chambers.

On days 7 and 8 of pregnancy, mesometrial regions of rat gestation sites were examined by light microscopy and transmission electron microscopy to determine what changes occur before the chorioallantoic placenta forms in that region. By day 7, gestation sites contained a uterine lumen mesometrially and an antimesometrial extension of the uterine lumen, the implantation chamber. The implantation chamber consisted of a mesometrial chamber between the uterine lumen and the conceptus, an antimesometrial chamber that contained the conceptus, and a decidual crypt antimesometrial to the conceptus. Stromal cells that formed the walls of the implantation chamber were closely packed decidual cells, while those that surrounded the uterine lumen were loosely arranged. Late on day 7, a portion of the epithelium lining the mesometrial chamber was degenerating, but this area of initial degeneration was never adjacent to the antimesometrial chamber. By early day 8, most of the epithelial cells lining the mesometrial chamber were degenerating and were being sloughed into the chamber lumen. Although degeneration of these epithelial cells morphologically resembled necrosis, it was precisely controlled, since adjacent epithelial cells lining the uterine lumen remained healthy. The space that separated the denuded luminal surface of the mesometrial chamber from underlying decidual cells became wider and was occupied by an extracellular matrix rich in cross-banded collagen fibrils. Decidual cell processes, that earlier had penetrated the basal lamina beneath healthy epithelial cells, protruded into this matrix and penetrated the basal lamina at the luminal surface. By late day 8, large areas of denuded chamber wall were covered with decidual cell processes, little remained of the basal lamina, and cross-banded collagen fibrils were scarce in the area occupied by decidual cell processes. During the times studied, uterine tissues that formed the walls of the mesometrial chamber were not in direct contact with the conceptus. This study indicates that trophoblast does not play a direct role in epithelial degeneration, basal lamina penetration, or extracellular matrix modifications in the mesometrial region of implantation chambers where part of the chorioallantoic placenta forms, although trophoblast may be required to trigger or modulate some of the changes.     

黄花油点草胚胎发育研究

利用石蜡切片技术对百合科植物黄花油点草[Tricyrtis maculata(D.Don)Machride]双受精、胚及胚乳发育进行了研究,以明确其胚胎发育的特征,为百合科植物的系统研究提供生殖生物学资料。结果表明:(1)黄花油点草为珠孔受精;进入胚囊的2枚精子分别与卵细胞和中央细胞进行正常的双受精,其受精作用属有丝分裂前型。(2)受精后的初生胚乳核立即分裂,其发育方式为核型胚乳;早期的游离胚乳核沿胚囊的边缘分布,胚囊中央部位主要为胚乳细胞质,随着游离胚乳核数量的增加,胚乳核慢慢充满整个胚囊;当发育至球形胚早期阶段,在各胚乳核周围产生胚乳细胞壁,形成完整的胚乳细胞。(3)合子有较长的休眠时间,胚的发育方式为茄型;合子第一次有丝分裂为横裂,分裂后形成基细胞和顶细胞;基细胞经过3次横裂,形成一列胚柄细胞;顶细胞经过分裂形成胚体,依次形成球形胚、棒状胚和盾形胚。(4)种子成熟时胚无器官分化;成熟种子由种皮、胚和胚乳三部分组成。