scholarly journals Detection of gap junctions between the progeny of a canine macrophage colony-forming cell in vitro.

1979 ◽  
Vol 82 (2) ◽  
pp. 555-564 ◽  
Author(s):  
M Porvaznik ◽  
T J MacVittie
Keyword(s):  
Gap Junctions ◽  
Freeze Fracture ◽  
Growth Conditions ◽  
Structural Basis ◽  
Fracture Plane ◽  
Tracer Techniques ◽  
Lanthanum Tracer ◽  
Colony Forming Cell ◽  
Macrophage Colony

An in vitro monocyte-macrophage colony-forming cell (M-CFC) has been detected in canine bone marrow (BM). The colonies derived from these progenitor cells were similar to murine-derived M-CFC (MacVittie and Porvaznik, 1978, J. Cell Physiol. 97:305--314) colonies, since they showed a singular macrophage line of differentiation, a lag of 14--16 days before initiating colony formation, and they survived significantly longer in culture in the absence of colony-stimulating factor (CSF) than granulocyte-macrophage colony-forming cells (GM-CFC). Endotoxin (Salmonella typhosa lipopolysaccharide W)-stimulated dog serum was used as the CSF (7% vol/vol). Canine-derived M-CFC progeny were identified as macrophages on the basis of morphology, phagocytosis, and the presence of Fc receptors for IgG. Gap junctions were observed only in canine BM, M-CFC-derived colonies using freeze-fracture and lanthanum tracer techniques. They were not observed in any GM-CFC-derived colonies. The number of gap junctions observed in freeze-fracture replicas of BM, M-CFC-derived colonies (21 colonies from three different dogs) showed a significantly positive correlation (Kendall's tau = 0.70, P less than 0.001) with the size of the colony fracture plane area. Gap junctions were observed displaying hexagonal lattices of 9.3 nm +/- 0.08 (SE) particles with a center-to-center spacing of 10.4 nm +/- 1.0 (SE) on membrane P-fracture faces. On membrane E-fracture faces, highly ordered arrays of pits with 8.7 nm +/- 0.12 (SE) center-to-center spacing were observed. Arrays of both particles and pits were also observed in fracture-face breakthroughs within a gap junction. Thus, gap junctions can form in vitro between the cells of macrophage progeny of a canine M-CFC under appropriate growth conditions. The significance of this observation is that there may be a structural basis for cell-to-cell collaboration between BM macrophages and other capable cells that either pass into the tissue for modification or develop there into mature cell forms.

Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

1978 ◽  
Vol 36 (2) ◽  
pp. 650-651
Author(s):  
Raul I. Garcia ◽  
Evelyn A. Flynn ◽  
George Szabo
Keyword(s):  
Direct Injection ◽  
Skin Pigmentation ◽  
Freeze Fracture ◽  
Pigment Granule ◽  
Time Lapse ◽  
Ultrastructural Level ◽  
Lanthanum Tracer ◽  
A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

The relationship between erythropoietin-dependent cellular differentiation and colony-forming ability in prenatal haemopoietic tissues

Development ◽  
1975 ◽  
Vol 34 (3) ◽  
pp. 575-588
Author(s):  
R. J. Cole ◽  
T. Regan ◽  
S. L. White ◽  
E. M. Cheek
Keyword(s):  
Cellular Differentiation ◽  
Cell Numbers ◽  
In Vitro Sensitivity ◽  
Foetal Liver ◽  
Rapid Production ◽  
Colony Forming Cell ◽  
Macrophage Colony ◽  
The Relationship

Levels of haem synthesis achieved by foetal liver erythroblasts responding to erythropoietin in vitro are similar in dissociated cell cultures and in cultures of organized tissues. Erythroid colony-forming cells reach maximum numbers on the sixteenth day of gestation. Their presence in foetal liver is associated with the period of most rapid production of erythrocytes, and with in vitro sensitivity to erythropoietin measured as enhanced haem synthesis. It is concluded that at least a proportion of erythroid colony-forming cells in the foetal liver are dependent on erythropoietin in situ and that these cells are separated from the earliest recognizable pro-erythroblast by 1–2 cell divisions. Populations of granulocyte-macrophage colony-forming cells change independently of erythroid colony-forming cell numbers.

Possible role of gap junctions in activation of myometrium during parturition

1978 ◽  
Vol 235 (5) ◽  
pp. C168-C179 ◽  
Author(s):  
R. E. Garfield ◽  
S. M. Sims ◽  
M. S. Kannan ◽  
E. E. Daniel
Keyword(s):  
Smooth Muscle ◽  
Gap Junctions ◽  
Freeze Fracture ◽  
Normal Pregnancy ◽  
Pregnant Rats ◽  
Junction Formation ◽  
Synchronous Contraction ◽  
Do So

Gap junctions between smooth muscle cells of the myometrium of pregnant rats were found only immediately prior to, during and immediately after parturition by quantitative thin-section and freeze-fracture microscopy. Ovariectomy of 16- to 17-days-pregnant rats resulted in premature termination of pregnancy and the appearance of gap junctions. Methods that prolonged normal pregnancy in rats or maintained pregnancy in ovariectomized animals (progesterone treatment) prevented the appearance of gap junctions. Gap junctions formed in tissues incubated for 24--96 h in vitro without any hormonal influence. We propose that gap junctions are essential for normal labor and delivery for synchronous contraction of the muscle of the uterus. We present a model for control of parturition that may apply to other animals including humans. The model proposes: 1) the possible roles progesterone, prostaglandins, or estrogens may play in initiating gap-junction formation; 2) that the formation of gap junctions is a necessary step in activation of the myometrium leading to labor; and 3) that agents used to stimulate or inhibit labor may do so by affecting gap junctions.

Freeze-fracture study of mammalian skin: melanocyte-keratinocyte interactions

1978 ◽  
Vol 36 (2) ◽  
pp. 652-653
Author(s):  
Raul I. Garcia ◽  
Evelyn A. Flynn ◽  
George Szabo
Keyword(s):  
Skin Color ◽  
Freeze Fracture ◽  
Culture Studies ◽  
Mammalian Skin ◽  
Lanthanum Tracer ◽  
Fracture Study ◽  
Thin Section Analysis ◽  
Section Analysis

The interactions of epidermal melanocytes(M) and keratinocytes(K) involve the unique process of melanosome transfer, from the melanin-synthesizing cell to the recipient, desquamative K, and are responsible for the skin color of man and other mammals. M-K interactions have been well studied in mammalian skin using thin section analysis, and there have been several freeze-fracture studies of skin. This study represents the first application of freeze-fracture to the study of M-K interactions and melanosome transfer. Several mechanisms of melanosome transfer have been proposed to explain the varied observations made in vivo and in vitro: melanosomes migrate from the M perikaryon peripherally into M dendrites, followed by cytophagocytosis by the K of the M dendrite tip containing melanosomes, formation of intercellular communicating channels between M and K through localized fusion of cell membranes with melanosome entry into K through this passage, release of melanosomes from M into the extracellular space(ECS) by exocytosis with subsequent phagocytosis by the K. Our own EM work using lanthanum tracer in skin in vivo has produced evidence supporting the second mechanism while cell culture studies suggest that a different mechanism may operate in vitro.

Intercellular communication between epithelial and fiber cells of the eye lens

1994 ◽  
Vol 107 (4) ◽  
pp. 799-811 ◽  
Author(s):  
S. Bassnett ◽  
J.R. Kuszak ◽  
L. Reinisch ◽  
H.G. Brown ◽  
D.C. Beebe
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Freeze Fracture ◽  
Cell Types ◽  
Lens Epithelium ◽  
Fiber Cell ◽  
Fracture Plane ◽  
Metabolic Cooperation ◽  
Fiber Cells ◽  
Resistance Pathway

Results of electrical, dye-coupling and morphological studies have previously suggested that gap junctions mediate communication between the anterior epithelium of the lens and the underlying lens fiber cells. This connection is believed to permit ‘metabolic cooperation’ between these dissimilar cell types and may be of particular importance to the fiber cells, which are thought incapable of autonomous ionic homeostasis. We reinvestigated the nature of the connection between epithelial and fiber cells of the embryonic chicken lens using fluorescence confocal microscopy and freeze-fracture analysis. In contrast to earlier studies, our data provided no support for gap-junction-mediated transport from the lens epithelium to the fibers. Fluorescent dyes loaded biochemically into the lens epithelium were retained there for more than one hour. There was a decrease in epithelial fluorescence over this period, but this was not accompanied by an increase in fiber cell fluorescence. Diffusional modeling suggested that these data were inconsistent with the presence of extensive epithelium-fiber cell coupling, even if the observed decrease in epithelial fluorescence was attributed exclusively to the diffusion of dye into the fiber mass via gap junctions. Furthermore, the rate of loss of fluorescence from isolated epithelia was indistinguishable from that measured in whole lenses, suggesting that decreased epithelial fluorescence resulted from photobleaching and leakage of dye rather than diffusion, via gap junctions, into the fibers. Analysis of freeze-fracture replicas of plasma membranes at the epithelial-fiber cell interface failed to reveal evidence of gap-junction plaques, although evidence of endocytosis was abundant. These studies were done under conditions where the location of the fracture plane was unambiguous and where gap junctions could be observed in the lateral membranes of neighboring epithelial and fiber cells. Paradoxically, tracer molecules injected into the fiber mass were able to pass into the epithelium via a pathway that was not blocked by incubation at 4 degrees C or by treatment with octanol and which excluded large (approximately 10 kDa) molecular mass tracers. Together with previous measurements of electrical coupling between fiber cells and epithelial cells, these data indicate the presence of a low-resistance pathway connecting these cell types that is not mediated by classical gap junctions.

scholarly journals The permeability barrier in mammalian epidermis.

1975 ◽  
Vol 65 (1) ◽  
pp. 180-191 ◽  
Author(s):  
P M Elias ◽  
D S Friend
Keyword(s):  
Plasma Membrane ◽  
Stratum Corneum ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Water Soluble ◽  
Structural Basis ◽  
Fracture Plane ◽  
Permeability Barrier ◽  
Stratum Granulosum ◽  
Lamellar Bodies

The structural basis of the permeability barrier in mammalian epidermis was examined by tracer and freeze-fracture techniques. Water-soluble tracers (horesradish peroxidase, lanthanum, ferritin) were injected into neonatal mice or into isolated upper epidermal sheets obtained with staphylococcal exfoliatin. Tracers percolated through the intercellular spaces to the upper stratum granulosum, where further egress was impeded by extruded contents of lamellar bodies. The lamellar contents initially remain segregated in pockets, then fuse to form broad sheets which fill intercellular regions of the stratum corneum, obscuring the outer leaflet of the plasma membrane. These striated intercellular regions are interrupted by periodic bulbous dilatations. When adequately preserved, the interstices of the stratum corneum are wider, by a factor of 5-10 times that previously appreciated. Freeze-fracture replicas of granular cell membranes revealed desmosomes, sparse plasma membrane particles, and accumulating intercellular lamellae, but no tight junctions. Fractured stratum corneum displayed large, smooth, multilaminated fracture faces. By freeze-substitution, proof was obtained that the fracture plane had diverted from the usual intramembranous route in the stratum granulosum to the intercellular space in the stratum corneum. We conclude that: (a) the primary barrier to water loss is formed in the stratum granulosum and is subserved by intercellular deposition of lamellar bodies, rather than occluding zonules; (b) a novel, intercellular freeze-fracture plane occurs within the stratum corneum; (c) intercellular regions of the stratum corneum comprise an expanded, structurally complex, presumably lipid-rich region which may play an important role in percutaneous transport.

scholarly journals Increase of gap junctions between pancreatic B-cells during stimulation of insulin secretion.

1979 ◽  
Vol 82 (2) ◽  
pp. 441-448 ◽  
Author(s):  
P Meda ◽  
A Perrelet ◽  
L Orci
Keyword(s):  
Insulin Secretion ◽  
B Cells ◽  
Gap Junctions ◽  
Freeze Fracture ◽  
Secretory Activity ◽  
Isolated Rat Islets ◽  
Pancreatic B Cells ◽  
Stimulation Of

The development of gap junctions between pancreatic B-cells was quantitatively assessed in freeze-fracture replicas of isolated rat islets under different conditions of insulin secretion. The results show that in resting B-cells, gap junctions are small and scarce but that these junctions increase when insulin secretion is stimulated. Both a short (90 min) stimulation by glucose in vitro and a prolonged (2.5 d) stimulation by glibenclamide in vivo raise the number of gap junctions; in addition, the glibenclamide stimulation causes an increase in the size of individual gap junctions. As a consequence, the total area occupied by gap junctions on the B-cell membrane and the ratio of this area to the cell volume were found significantly increased in the latter condition. The slight increase of these values observed after the glucose stimulation did not reach significance. These data indicate a change of gap junctions during the secretory activity of the pancreatic B-cells. The possibility that the coupling of the cells is affected by the treatment is discussed.

Fetal murine hemopoiesis following in utero low-dose irradiation

Development ◽  
1981 ◽  
Vol 62 (1) ◽  
pp. 37-46
Author(s):  
Sheila R. Weinberg ◽  
Emmeline G. McCarthy ◽  
Thomas J. MacVittie
Keyword(s):  
Low Dose ◽  
Fetal Liver ◽  
Soft Agar ◽  
In Utero ◽  
In Vitro Assays ◽  
Plasma Clot ◽  
Erythroid Progenitor ◽  
Colony Forming Cell ◽  
Macrophage Colony

The influence of in utero low-dose ionizing radiation exposure on murine hemopoietic embryogenesis was investigated. In vitro assays such as micro plasma-clot cultures and double-layer soft agar cultures served as sensitive biodosimeters to determine erythropoietic and granulopoietic injuries. Day-10·5, HA/ICR, pregnant mice were irradiated with 0, 50, 100, 150,200, or 300 rads, and day-14·5 fetal livers were studied for colony-forming unit-erythroid (CFU-E), buist-forming unit-erythroid (BFU-E), granulocyte-macrophage colony-forming cell (GM-CFC), and macrophage-colony-forming cell (M-CFC) activity. Fetuses subjected to doses of 200 rads or higher on day 10·5 of gestation responded with a decrease in day-14·5 liver cellularity, reflecting injury to the developing organ and its inability to recover to the nonirradiated values. Difference in response between erythropoietin(EPO)-dependent and EPO-independent CFU-E strongly suggests existence of two populations of erythroid progenitor cells with different radiosensitivities. A dose of 200 rads markedly reduced CFUE recovery, and a dose of 100 rads was sufficient to reduce BFU-E recovery to almost 10% of 0-rad values. Nonirradiated day-14·5 fetal liver had more GM-CFC compared to any of the irradiated fetuses, and a dramatically reduced M-CFC recovery occurred with each increase in dose following 150 rads. Our results showed that (1) fetal liver granulopoiesis is more sensitive to radiation injury compared to erythropoiesis, and (2) fetal liver has a greater potential for erythropoiesis recovery.

scholarly journals Vitamin-A-induced mucous metaplasia. An in vitro system for modulating tight and gap junction differentiation.

1976 ◽  
Vol 68 (2) ◽  
pp. 173-188 ◽  
Author(s):  
P M Elias ◽  
D S Friend
Keyword(s):  
Vitamin A ◽  
Gap Junctions ◽  
Gap Junction ◽  
De Novo ◽  
Freeze Fracture ◽  
Controlled Study ◽  
Thin Sections ◽  
Tight Junction Formation ◽  
Mucous Metaplasia

Stratified squamous epithelia from 14-day chick embryo shank skin contain rare tight-junctional strands and only small gap junctions. Exposure of this tissue to retinoic acid (vitamin-A) (20 U/ml) in organ culture, however, induces mucous metaplasia, accompanied by tight-junction formation and gap-junction growth; untreated specimens continue to keratinize. To investigate sequential stages of junctional assembly and growth, we examined thin sections and freeze-fracture replicas at daily intervals for 3 days. During the metaplastic process, tight junctions assemble in midepidermal and upper regions, beginning on day 1 and becoming maximal on day 3. Two tight-junctional patterns could be tentatively identified as contributing to the emergence of fully formed zonulae occludentes: (a) the formation of individual ridges along the margins of gap junctions; (b) de novo generation of continuous ramifying strands by fusion of short strand segments and linear particulate aggregates near cellular apices. Gap junction enlargement, already maximal at day 1, occurs primarily three to four cell layers deep. Growth appears to occur by annexation of islands of 20-40 8.5-nm particles into larger lattices of islands separated by particle-free aisles. Eventually, a single gap junction may occupy much of the exposed membrane face in freeze-fractured tissue, but during apical migration of the cells such junctions disappear. The vitamin- A chick-skin system is presented as a responsive model for the controlled study of junction assembly.

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